Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M506070200 on September 28, 2005

J. Biol. Chem., Vol. 281, Issue 1, 528-542, January 6, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/1/528    most recent
M506070200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sandrin, V.
Right arrow Articles by Cosset, F.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sandrin, V.
Right arrow Articles by Cosset, F.-L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Intracellular Versus Cell Surface Assembly of Retroviral Pseudotypes Is Determined by the Cellular Localization of the Viral Glycoprotein, Its Capacity to Interact with Gag, and the Expression of the Nef Protein*

Virginie Sandrin1 and François-Loïc Cosset2

From the INSERM U412, Lyon Ecole Normale Supérieure de Lyon, and IFR128 BioSciences Lyon-Gerland, Lyon, F-69007 France

Received for publication, June 3, 2005 , and in revised form, September 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Retroviral Gag and Env glycoproteins (GPs) are expressed from distinct cellular areas and need to encounter to interact and assemble infectious particles. Retroviral particles may also incorporate GPs derived from other enveloped viruses via active or passive mechanisms, a process known as "pseudotyping." To further understand the mechanisms of pseudotyping, we have investigated the capacity of murine leukemia virus (MLV) or lentivirus core particles to recruit GPs derived from different virus families: the G protein of vesicular stomatitis virus (VSV-G), the hemagglutinin from an influenza virus, the E1E2 glycoproteins of hepatitis C virus (HCV-E1E2), and the retroviral Env glycoproteins of MLV and RD114 cat endogenous virus. The parameters that influenced the incorporation of viral GPs onto retroviral core particles were (i) the intrinsic cell localization properties of both viral GP and retroviral core proteins, (ii) the ability of the viral GP to interact with the retroviral core, and (iii) the expression of the lentiviral Nef protein. Whereas the hemagglutinin and VSV-G glycoproteins were recruited by MLV and lentivirus core proteins at the cell surface, the HCV and MLV GPs were most likely recruited in late endosomes. In addition, whereas these glycoproteins could be passively incorporated on either retrovirus type, the MLV GP was also actively recruited by MLV core proteins, which, through interactions with the cytoplasmic tail of the latter GP, induced its localization to late endosomal vesicles. Finally, the expression of Nef proteins specifically enhanced the incorporation of the retroviral GPs by increasing their localization in late endosomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Retroviral particles consist of viral envelope glycoproteins (Env GP),3 core proteins (Gag), and genomic RNAs that are expressed from distinct cellular areas but need to encounter to interact and assemble infectious particles. The Env GPs are synthesized in the endoplasmic reticulum, whereas Gag proteins are synthesized by ribosomes of the cytosol. The intracellular trafficking of Gag proteins and Env GPs modulates their ability to encounter each other. The viral assembly process has long been thought to occur exclusively at the plasma membrane. However, it is becoming clear that the retroviral assembly and budding sites depend on the type of producer cell. Indeed, in T lymphocytes, assembly of human immunodeficiency virus (HIV) and viral budding is thought to occur at the plasma membrane (1, 2), whereas in macrophages, these events may occur intracellularly, into multivesicular bodies (MVBs) (3-8). Recent studies suggest that the lipid composition of intracellular versus plasma membranes is critical for the targeting of Gag to either site (9, 10).

During the assembly process, retroviral particles incorporate both viral and cellular proteins via active or passive mechanisms. The relative abundance of some cellular proteins in viral particles and in intracellular and plasma membranes has been compared. Some proteins are enriched in viral particles (or actively included), diluted (or excluded), or constant (or passively incorporated) (5, 6, 11, 12). Passive incorporation of membrane proteins onto budding viral particles occurs in the absence of specific interactions with the viral core proteins, provided they are present at the budding site and are not sterically incompatible with the viral core. In contrast, active incorporation may require interactions between the Env GP and the viral Gag proteins. These interactions can be direct or indirect, via a cellular factor. Early evidence for Gag/Env interactions indicated that whereas Gag buds from any pole of polarized cells in the absence of Env GP, budding is relocated to the basolateral side upon co-expression with basolateral-located Env GP (13, 14). Other evidence further support Gag/Env interactions, at least for lentiviruses and onco-retroviruses (15-20). Some motifs implicated in Gag/Env interactions have been identified for HIV-1 Env GP and reside in the {alpha}-helix 2 of the gp41 cytoplasmic tail (16).

Retroviral particles harboring GPs derived from heterologous enveloped viruses are called "pseudotypes" and are particularly useful to optimize gene transfer into specific cell types (for a review, see Ref. 21). However, the co-expression of retroviral cores proteins and heterologous GPs does not necessarily lead to highly infectious particles, and restrictions in pseudotype formation have been reported (22-26). The Gag/GP combinations that lead to assembly of retroviral pseudotypes are rather unpredictable, in large part because of our insufficient knowledge of the mechanisms that dictate the assembly and budding of retroviral particles.

To further understand the mechanisms of pseudotype formation, here we have investigated the recruitment by murine leukemia virus (MLV) or simian immunodeficiency virus (SIV) core particles of envelope glycoproteins derived from different virus families: the G protein of vesicular stomatitis virus (VSV-G), the hemagglutinin (HA) from an influenza virus-fowl plague virus (FPV-HA), the E1E2 glycoproteins of hepatitis C virus (HCV-E1E2), and the retroviral Env glycoproteins of MLV or of the RD114 cat endogenous virus. Through a combination of infection assays, biochemical methods, and confocal microscopy, we have determined some viral and cellular parameters that influenced the incorporation of viral GPs onto retroviral core particles. Our results point to distinct mechanisms operating during pseudotype formation: (i) the intrinsic cell localization properties of both viral GP and retroviral core proteins, (ii) the ability of the viral GP to interact with the retroviral core, and (iii) the expression of the lentiviral Nef protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells—The 293T human embryo kidney (ATCC CRL-1573), the COS-7 African green monkey kidney (ATCC CRL-1651), the TE671 human rhabdomyosarcoma (ATCC CRL-8805), and the Huh-7 hepatocellular carcinoma (27) cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics. 293T cells and COS-7 cells were used as producer cells for the assembly of pseudotyped viral particles (19). TE671 cells and Huh-7 cells were used as target cells for infection assays (19, 25, 28).

Viral Glycoprotein Expression Constructs—The phCMV-G plasmid, encoding the VSV-G protein (29), was used as an expression vector construct for the surface glycoproteins derived from the RD114 cat endogenous virus (GenBank® accession X87829 [GenBank] ) (30), the 4070A strain of amphotropic MLV (31), an HCV of genotype 1a (28), and the influenza H7 strain (HA) (25). The phCMV-RD114 and phCMV-4070A expression vectors, expressing the RD114 and MLV virus envelope glycoproteins, were further modified to generate chimeric glycoproteins that harbor the cytoplasmic tails of the MLV and RD114 glycoproteins, respectively. The resulting chimeric glycoproteins were called RD/TRMLV (19) and MLV/TRRD (Fig. 1) (details of cloning strategy and sequences of oligonucleotides are available upon request). The Nef gene of SIVmac251 was cloned into the pSI expression vector.

Production of Virions—The production of a pseudotyped SIV-based vector was described in a previous report (25). Briefly, 293T cells plated in 10-cm diameter plates were transfected by 8.1 µg of the pR4SA green fluorescent protein (GFP) vector construct (32), 8.1 µg of the pSIV-3+ packaging construct (33), and 2.7 µg of the construct expressing the viral glycoprotein. Pseudotyped MLV-derived vectors were generated in a similar manner by DNA transfection of the pTG5349 MLV packaging construct (33), the pTG13077 MLV GFP vector construct (33), and the glycoprotein-expressing construct. A calcium-phosphate transfection kit (Clontech) was used according to the manufacturer's instructions. The medium (8 ml/plate) was replaced 16 h after transfection, and the supernatant was harvested 24 h later and filtered through 0.45-µm pore size membranes. COS-7 cells were transfected with FuGENE 6 according to the manufacturer's instructions (Roche Diagnosis).

Infection Assays—To determine the infectious virus titers of the different pseudotyped vectors, serial dilutions of vector preparations were added to 2.5 x 105 target cells in the presence of 8 µg of Polybrene/ml for 4 h at 37°C. The medium was then replaced with normal culture medium for 72 h at 37 °C. The transduction efficiency, determined as the percentage of GFP-positive cells, was then measured by fluorescence-activated cell sorter analysis, as previously described (25). The infectious titers, provided as infectious units (i.u.)/ml, were calculated by using the formula: Titer = %inf x (2.5 x 105/100) x d, where "d" is the dilution factor of the viral supernatant and "%inf" is the percentage of GFP-positive cells as determined by fluorescence-activated cell sorter analysis using dilutions of the viral supernatant that transduce between 5 and 10% of GFP-positive cells.

Antibodies—The RD114 SU goat antiserum (ViroMed Biosafety Labs) raised against the RD114 gp70 envelope surface protein (SU) and the RD114 TM (transmembrane protein) rabbit antiserum (Eurogentec, Seraing, Belgium) were used, respectively, at 1/5,000 and 1/500 dilution for Western blotting. The gp70 Rausher leukemia virus goat antiserum (ViroMed Biosafety Labs) and the 42/114 rat monoclonal antibody (kind gift of A. Pinter) were used at 1/2,000 and 1/2 dilutions to detect the SU and TM proteins of MLV by Western blotting. The 83A25 rat monoclonal antibody (kind gift of L. Evans), directed against MLV SU, was used in immunofluorescence (IF) studies at 1/500. The FPV-HA Kp/Ro rabbit antiserum (kind gift of W. Garten) was used diluted at 1/500 to visualize the different forms of HA glycoprotein. The P5D4 monoclonal antibody (Sigma), directed against VSV-G, was used diluted 1/10,000 for Western blotting and 1/2,000 for IF experiments. The HCV-E2 glycoproteins were detected with the H52 mouse monoclonal antibody at 1/1,000 for Western blotting and with undiluted supernatants of the H53 hybridoma for IF studies (34). The 2F12 mouse monoclonal antibody, directed against the SIV capsid (CA) protein (National Institutes of Health AIDS Research and Reference Reagent Program), was used at a 1/500 dilution for Western blotting. A mouse antiserum against the SIV matrix (MA) protein (National Institutes of Health AIDS Research and Reference Reagent Program) was used at 1/1,000 dilution to visualize SIV Gag IF studies. The Rausher leukemia virus CA goat antiserum (ViroMed Biosafety Labs) and rabbit antiserum (kind gift of A. Rein) were both used at 1/10,000 dilution for Western blotting IF studies.

Detection of Viral Glycoproteins—Forty hours post-transfection, virus producer cells were chilled on ice, washed twice with cold phosphate-buffered saline2+ (PBS2+) (PBS, pH 8.0, supplemented with 0.7 mM CaCl2 and 0.25 mM MgSO4) and incubated with 0.5 mg/ml of sulfo-NHS-LC-LC-biotin (Pierce) for 30 min at 4 °C. Biotinylation was stopped by incubating the cells with 1 M glycine in PBS2+ for 5 min at 4 °C. The cells were then washed with PBS, 0.1 M glycine, lysed with MacDougal buffer (20 mM Tris-HCl, pH 8.0, 120 mM NaCl, 200 µM EGTA, 0.2 µM NaF, 0.2% sodium deoxycholate, 0.5% Nonidet P-40) containing a protease inhibitor mixture (Complete Mini; Roche Diagnostic) and 0.1 M glycine, and centrifuged at 13,000 x g for 30 min; 80% of the cell lysates were incubated overnight at 4 °C with streptavidin-Sepharose beads (Pierce). The beads were then washed with MacDougal glycine buffer, resuspended in a denaturing buffer (1% {beta}-mercaptoethanol, 0.5% SDS), and boiled for 5 min. This fraction allowed the detection of cell surface-expressed proteins. The remaining 20% suspension was used to detect the total proteins in crude cell lysates.

Purified virion samples were obtained by ultracentrifugation of viral supernatants through a 1.5-ml 20% sucrose cushion in a Beckman SW41 rotor (25,000 rpm, 2.5 h, 4 °C) and suspended in PBS. All samples were mixed 5/1 (v/v) in a loading buffer (375 mM Tris-HCl (pH 6.8) containing 6% SDS, 30% {beta}-mercaptoethanol, 10% glycerol, and 0.06% bromphenol blue), boiled for 5 min, and then run on SDS-10% polyacrylamide gel electrophoresis.

Western blotting was performed using standard procedures. The Super Signal West Pico Chemiluminescent substrate was used to reveal proteins (Pierce). Membrane scanning was performed with the Storm 860 device and signal densities were calculated with the ImageQuant program (Amersham Biosciences).

IF and Confocal Microscopy Imaging—FuGENE 6-transfected 293T or COS-7 cells were grown on 35-mm diameter coverglass dishes coated with D-lysine (Mattek Corp., Ashland, MA) or on uncoated 14-mm diameter glass coverslips, respectively. IF staining was performed 40 h post-transfection at room temperature. The cells were washed with PBS, fixed with 3% paraformaldehyde in PBS for 15 min, quenched with 50 mM NH4Cl, and permeabilized by 0.2% Triton X-100 for 8 min. The fixed cells were incubated for 1 h with the primary antibody in 1% bovine serum albumin/PBS, washed, and stained for 1 h with the corresponding fluorescent Alexa-conjugated secondary antibody (at 0.5 µg/ml) in 1% bovine serum albumin/PBS. The cells were then washed several times with PBS and mounted on microscope slides with the antifading agent Prolong (Molecular Probes) prior to image acquisition. Images were acquired with an LSM 510 confocal microscope equipped with an Axiovert 100M microscope (Carl Zeiss Inc., Thornwood, NY) and a 63 x 1.3 numerical aperture Apocromat objective. Alexa 488 was excited with an argon laser line at 488 nm, and emissions were collected with a band pass filter (BP505-550). Alexa 546 or 555 were excited, independently of Alexa 488, with an HeNe laser line at 543 nm, and emissions were collected with a long-pass filter (LP560). The secondary antibodies for Alexa were purchased from Molecular Probes, Inc. Expression vectors encoding specific endosomal marker proteins, cellubrevin-GFP and TI-VAMP-GFP (35) (kind gifts from T. Galli), were cotransfected with GP and Gag vectors.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 1.
Schematic representation of envelope glycoproteins. Domain organization of glycoproteins and retroviral Env chimeras. The ectodomain, transmembrane, and cytoplasmic tail of the different GPs used in this study are represented. The amino acid sequences located at the boundaries of each domain are indicated. The cytoplasmic tail of the MLV/TRRD chimeric GP was replaced with that of the MLV-A GP. The chimera called RD/TRMLV contains the ectodomain and the transmembrane domain of the RD114 Env glycoprotein and harbors the cytoplasmic tail of the MLV Env GP.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Retroviral Cores Are Efficiently Pseudotyped by Several Heterologous GPs—We studied the surface GP derived from several enveloped viruses (Fig. 1) for their ability to pseudotype MLV core particles: the VSV-G, FPV-HA, HCV-E1E2, and the retroviral Env glycoproteins from the amphotropic MLV or, as control, of the cat endogenous virus RD114 (19). The infectivity of pseudotyped viral particles harboring a GFP marker gene was determined on appropriate target cells, depending on the cell surface receptors required for cell entry by each vector pseudotype (25, 28). As reported previously, the infectious titers of the pseudotyped vectors varied greatly, depending on the type of glycoprotein, and ranged between 1 x 104 and 1 x 107 i.u./ml as determined on the most permissive target cells (Fig. 2B). Such variations of the infectious titers could be dependent on several cellular factors such as receptor density, receptor binding, and cell entry route of the virions as well as the inherent pseudotyping capacity of the glycoproteins. However, following a screening of a wide array of cell lines and primary cells, we could not identify alternative cell types that were more susceptible to infection than the indicator cells displayed in Fig. 2 (data not shown and discussed in Refs. 25 and 28). Finally, similar relative differences of infectivity were detected for retroviral pseudotypes produced from either 293T versus COS-7 cells, although the infectious titers were generally lower for viral particles assembled in the latter cells (data not shown and Fig. 2). Thus, the levels of infectivity reported here reflected the intrinsic functionality of either heterologous glycoprotein upon loading on MLV core particles.

In a previous report (19), we found that the RD114 Env GP is not readily incorporated on lentiviral core particles, whereas a RD114 Env GP mutant harboring the cytoplasmic tail (cyt) of the MLV Env GP (RD/TRMLV chimera) restored incorporation and infectivity of pseudotyped lentiviral core particles (Fig. 2). Thus, to further investigate the potential role of the cyt of GPs in retroviral pseudotyping, we constructed a cytoplasmic tail mutant of the MLV Env GP harboring the cyt of the RD114 Env GP (MLV/TRRD Env chimera; Fig. 1). We then compared the capacity of all retroviral and non-retroviral GPs to pseudotype either MLV or SIV core particle (Fig. 2, A and B). We did not observe significant differences in the capacity of the GPs to pseudotype either retroviral core, except for the MLV/TRRD and RD114 Env glycoproteins. Indeed, whereas the MLV/TRRD and RD114 GPs could efficiently pseudotype MLV particles, reaching infectious titers over 2 x 106 i.u./ml, they were poorly assembled on SIV cores, resulting in titers of up to 3 x 104 i.u./ml with the latter core particles. This extended our previous results (19) that suggested that the cytoplasmic tail of the MLV Env GP promotes efficient glycoprotein incorporation on lentiviral core particles as compared with that of the RD114 Env GP. Again, the same relative differences of infectivity were detected between pseudotyped SIV viral particles produced in either 293T or COS-7 cells (data not shown and see Figs. 9 versus 2). Altogether, these data indicated that both the type of retroviral core and glycoprotein influenced pseudotype formation. Because the cyt modulates both intracellular localization (19, 65, 66) and pseudotyping properties (Fig. 2) of a glycoprotein, we further investigated the cellular pathways of GP recruitment by onco-retroviral and lentiviral Gag proteins, by means of biochemical and confocal microscopy analyses.

Variable Incorporation of Pseudotyping GPs on Viral Particles—We then characterized the expression of Gag proteins and envelope GPs by Western blot analysis of purified virions, crude cell lysates, and biotinylated cell surfaces of virion-producer cells (Fig. 3 and Table 1). The analysis of the GP levels in these different compartments raised different interesting observations.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Quantification of expression of the viral glycoproteins in producer cells and pseudotyped retroviruses

 



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 2.
Results of infection assays. Target cells (TE671 for viruses pseudotyped with FPV-HA, VSV-G and retroviral Env GPs; Huh-7 cells for viruses pseudotyped with HCV E1E2 GPs) were infected with diluted supernatants harvested from 293T producer cells expressing GFP expressing SIV or MLV vectors, SIV or MLV Gag-Pol proteins, and the indicated viral glycoproteins. The transduction efficiency, expressed as the percentage of GFP-positive cells, was measured by fluorescence-activated cell sorter analysis 72 h post-infection and was used to determine the number of infectious viral particles present in the producer cell supernatants. The infectious titers are deduced as the number of GFP infectious units per milliliter of viral supernatant (i.u./ml). The values are the mean ± S.D. of up to five independent experiments. Similar results were obtained when using COS-7 as producer cells; yet the absolute infectious titers were lower from the latter cells as compared with 293T cells (not shown).

 
First, we noticed that the efficiency of virion release was independent of the type of pseudotyping GP. Indeed, the ratios of Gag signals detected in purified viral particles versus in total lysates of producer cells did not vary, whatever the GP (Fig. 3A (SIV particles), compare ratios of, e.g. lanes a/c versus d/f versus g/i versus j/l versus m/o and likewise in Fig. 3B (MLV particles)).

Second, we found that the incorporation levels of the GPs onto viral particles seemed to correlate with the infectious titers. Indeed, despite the lack of an antibody common to all GP that would allow a direct comparison, we estimated the relative levels of GP incorporation on virions by comparing the ratios of GP signals detected in purified viral particles (GPV) versus in total lysates of virion producer cells (GPL) (Fig. 3 and Table 1). No significant differences of these ratios were noticed for the MLV Env, FPV-HA, and VSV-G GPs, whatever the type of viral core, because the GPV/GPL ratios were in the range of 200-300% (Table 1). These results were in agreement to the maximal infectious titers of the resulting pseudotyped virions that ranged between 106 and 107 i.u./ml (Fig. 2). Compared with these latter glycoproteins, and despite strong expression levels in producer cells, the HCV GPs were less efficiently incorporated on viral particles irrespective of the viral core type (Fig. 3), with GPV/GPL ratios in the range of 10-15% (Table 1), consistent with poorer infectious titers (Fig. 2). Finally, despite expression levels in producer cell lysates similar to those of MLV Env GP (Fig. 3, compare lanes o' versus l' and o versus l), the MLV/TRRD GP was poorly incorporated onto SIV core particles as compared with the MLV Env GP (Fig. 3A, compare lanes m versus j and m' versus j'; Table 1), as detected using the same antibody. This was consistent with the 40-50-fold difference of infectivity between virions pseudotyped with either GP (Fig. 2).

Third, we found that the viral incorporation of some retroviral GPs was strongly influenced by the type of core particle. Indeed, whereas the MLV/TRRD and RD114 GPs were efficiently recruited by MLV core particles, in agreement with the high infectious titers of the resulting viral particles (Fig. 2), they could poorly pseudotype SIV core particles (compare SU and TM protein levels in lanes m versus m' of Fig. 3 for MLV/TRRD GP and in Ref. 19 for RD114 GP; Table 1, GPV/GPL ratios of 200 and 15% for the MLV/TRRD GP on MLV and SIV particles, respectively). This difference of GP incorporation levels between MLV and SIV core particles was not because of a difference of budding/release of either virion type because similar levels of viral incorporation were noticed for the other glycoproteins, i.e. FPV-HA, VSV-G, HCV-E2, and MLV Env (Fig. 3, compare lanes a, d, g, j versus a', d', g', j'). Thus, these results expanded our previous results (19) that suggested that the RD114 cytoplasmic tail restricts pseudotype formation with lentiviral particles.

The Detection of Different GP Forms on Virions Versus Cell Surface Predicts Alternative Sites for GP Recruitment—Depending on their glycosylation and/or maturation status (i.e. cleavage of GP precursors), several forms were detected for most glycoproteins and the ratios of these forms for each GP varied in the different compartments analyzed (i.e. purified viral particles (GPV), cell surface (GPS), and lysates of virion producer cells (GPL)). The analysis of the variations of these ratios raised several points.



View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 3.
Characterization of envelope glycoproteins and pseudotypes. Detection of the different envelope glycoproteins and viral core proteins was performed by Western blotting of crude cell lysate (L), cell surface-biotinylated proteins (S), and viral particles purified by ultracentrifugation of the cell supernatants on sucrose cushions (V). The different GPs were expressed in 293T cells individually (panel C), in the presence of Gag proteins derived from SIV (panel A), MLV (panel B), or of a myristoylation-defective MLV G2A-Gag mutant (panel D). The viral proteins were revealed with appropriate antibodies against the GP or Gag capsid proteins (CA). Several forms could be detected for most GPs. FPV-HA was synthesized as HA0 precursor proteins that were cleaved by cellular furins into HA1 and HA2 GPs (HA2 was not revealed in these blots). MLV and MLV/TRRD Env GPs were synthesized as immature precursor proteins (Pr) that were further cleaved into mature surface (SU) and transmembrane (TM) subunits. Several glycosylated forms were revealed for HCV-E2. Deglycosylation of the samples by PNGase F treatment raised a single band of about 40 kDa (76). Scanning of Western blot membranes was performed with the Storm 860 device and signal densities were calculated with the ImageQuant program. Similar patterns of GP expression and maturation were obtained when using COS-7 as the producer cells (not shown).

 
Relative to total cell expression, the VSV-G and FPV-HA GPs, which were efficiently incorporated on virions, were abundantly expressed at the cell surface (Table 1, GPS/GPL ratios over 200% and Fig. 3, compare lanes b versus c and a; e versus f and d, respectively). Furthermore, the FPV-HA GP was detected as an uncleaved HA0 precursor and a mature, cleaved, HA1 GP. The ratios of either form detected in purified virions versus at the cell surface were similar (Fig. 3, lanes a versus b; a' versus b'; Table 1, see HA0/HA1 ratios in parentheses). In contrast, lower HA1 levels in the HA1/HA0 ratios were detected in cell lysates as compared with those determined at the cell surface or in purified virions (Fig. 3, lanes a versus c; a' versus c'; Table 1). This suggested that recruitment of FPV-HA GP by retroviral Gag proteins occurs at the cell surface, a possibility that was corroborated by in situ analysis using confocal microscopy (see below).



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 4.
Detection of retroviral core protein and glycoprotein expressed individually. COS-7 cells separately expressing the MLV or SIV core (Gag) proteins (A) or the indicated viral glycoproteins (B) were grown on glass coverslips, fixed in parformaldehyde, permeabilized, stained with the appropriate antibodies against the Gag proteins or the GPs, and imaged by confocal microscopy. Similar patterns of expression were obtained with 293T cells (not shown). The dotted lines correspond to the plasma membranes of cells expressing HCV and MLV GPs, as drawn from phase-contrast micrographs (not shown). The plasma membranes of cells expressing FPV-HA and VSV-G are readily stained by GP antibodies and thus are not highlighted. N, nucleus. The scale bars are indicated. The arrows point to areas of interest, as described in the text. Note that in some pictures (e.g. panels b and d in B), the nuclei display considerable GP staining. This apparent staining of the nucleus is in fact because of staining of GP in the endoplasmic reticulum, where GP expression is abundant and usually surrounds the nucleus. Depending on the focal plan of the observation, the nucleus will then appear as "empty" or "full" of GP.

 
In contrast to VSV-G and FPV-HA, the HCV GPs were poorly detected at the cell surface as compared with their expression in lysates of producer cells (Fig. 3, compare e.g. lanes h/i (HCV-E2) versus e/f (VSV-G) versus b/c (FPV-HA); Table 1, GPS/GPL ratios of <20, >225, and >250%, respectively). Moreover, the HCV-E2 protein was detected as a fully glycosylated and heterogeneous form on viral particles, whereas its cell surface-associated form was mostly expressed as a poorly glycosylated form (Fig. 3, compare lanes g versus h; g' versus h' and i'). This suggested that the HCV GPs are not recruited at the cell surface by retroviral core proteins, but rather in some intracellular compartments where fully glycosylated HCV GPs may accumulate, as confirmed below by confocal microscopy analysis.

A similar deduction was inferred for the MLV and MLV/TRRD Env glycoproteins. Indeed, only mature GP forms (i.e. SU and TM) were found on MLV and SIV virions despite an abundant expression of both mature SU and precursor (Pr) GPs at the surface of producer cells (Fig. 3, compare lanes m versus n; m' versus n'; j versus k; j' versus k' and see the difference of Pr/SU ratios in Table 1 (in parentheses)). That only mature SU and no Pr forms were incorporated on viral particles was confirmed by enhancing the separation between either protein after removal of N-linked carbohydrates by PNGase F treatment or on gels of greater resolution (data not shown). Furthermore, we found that both the cyt and retroviral core types influenced the maturation of the MLV and MLV/TRRD Env glycoproteins, as deduced from the Western blot analysis of the total lysates and the surface of the producer cells. First, in either compartment, whereas only a small fraction of Env Pr could be detected for the MLV Env GP co-expressed with SIV Gag proteins (Table 1, Pr/SU ratio of less than 15/85 in cell lysates), the MLV/TRRD was mainly found as precursor proteins (Table 1, Pr/SU ratio over 94/6; Fig. 3A, compare lanes k and l versus n and o). Second, the maturation of the MLV Env GP was significantly augmented in either compartment upon co-expression with MLV Gag proteins (Fig. 3B) as compared with when these GPs were expressed alone (Fig. 3C) or co-expressed with SIV Gag proteins (Fig. 3A). Whereas the Pr/SU ratio in cell lysates was 15/85 upon co-expression with SIV Gag proteins or without co-expressed Gag proteins, this ratio was changed to 55/45 for MLV Env GP co-expressed with MLV Gag proteins (Table 1, Pr/SU ratios in parentheses; Fig. 3, compare lanes l and r versus l'). This alteration of GP processing was specifically because of the expression of membrane-bound MLV Gag proteins because the Pr/SU ratio was identical whether the MLV Env glycoproteins were expressed alone (Fig. 3C) or co-expressed with either SIV Gag proteins (Fig. 3A) or myristoylation-defective MLV Gag proteins, which prevent Gag attachment to cell membranes (Fig. 3D) (Table 1, compare Pr/SU ratios in parentheses). This suggested the possibility of an interaction, of direct or indirect nature, between MLV Env GP and MLV Gag proteins bound to membranes. Altogether, these results extended our previous findings obtained with the RD114 GP (19) that indicated that the MLV Env cyt promotes more efficient GP maturation than that of RD114 Env GP and that MLV Gag influences GP processing and localization.




View larger version (136K):
[in this window]
[in a new window]
  		FIGURE 5.
Detection of viral glycoproteins and core proteins by confocal microscopy. COS-7 cells expressing the indicated GP and either the SIV (A) or MLV (B) core proteins (Gag), as indicated, were grown on glass coverslips, fixed in paraformaldehyde, permeabilized, stained at room temperature, and imaged by confocal microscopy. GP and Gag expression are shown in the red (GP detection) and green (Gag detection) channels, respectively. Co-localization of both viral components was assessed by confocal microscopy analysis (Merge). Similar co-localization patterns were detected in 293T cells (not shown). The dotted lines correspond to the plasma membranes of cells expressing HCV and MLV GPs, as drawn from phase-contrast micrographs (not shown). The plasma membranes of cells expressing FPV-HA and VSV-G are readily stained by GP antibodies and thus are not highlighted. N, nucleus. The scale bars are indicated. The arrows point to areas of interest, as described in the text.

 
The Viral GP Determines the Site of Gag/GP Co-localization—To investigate the site of viral assembly, we analyzed the cellular localization of GP and Gag by confocal microscopy. Protein markers were used to identify the different intracellular compartments where the viral components localize (19): TI-VAMP for late endosomes and MVBs (35), cellubrevin for recycling endosomes (36), and calreticulin for the endoplasmic reticulum (ER) (37). Expression of SIV and MLV Gag proteins was detected as punctuate patterns, dispersed underneath the plasma membrane (green arrows) and throughout the cell cytoplasm (Fig. 4A), as previously described (7, 8), hence suggesting different possible sites where the Gag proteins could recruit the viral glycoproteins.

The different GPs were abundantly expressed in the ER and/or in the Golgi apparatus, consistent with their synthesis through the secretory pathways, yet they also exhibited additional cellular localizations (Fig. 4B). Indeed, the FPV-HA and VSV-G glycoproteins were readily detected at the cell surface (Fig. 4B, red arrows in panels a and b), in agreement with the results of immunoblots (Fig. 3), and, for FPV-HA, in some intracellular vesicles. Interestingly, whereas co-expression with either MLV or SIV Gag proteins was not found to influence the localization of FPV-HA and VSV-G (compare Fig. 4B versus Fig. 5, A and B), the localization of the MLV and SIV Gag proteins at the plasma membrane was reinforced upon co-expression with either glycoprotein, as detected by patches of continuous staining lining the cell surface (compare Fig. 4A versus Fig. 5, green arrows in panels a, b, a', and b'). Intensive co-localization of Gag proteins with the FPV-HA and VSV-G glycoproteins was detected at these cellular locations (Fig. 5, yellow arrows in panels a, b, a', and b'). Thus, these results suggested that the distribution of the MLV or SIV Gag proteins was altered upon FPV-HA or VSV-G expression and corroborated the notion of immunoblot analysis (Fig. 3) that the plasma membrane was the cellular site where the glycoproteins are recruited during envelope assembly of these viral pseudotypes.

In contrast to VSV-G and FPV-HA, the HCV, MLV, and MLV/TRRD glycoproteins were not detected at the cell surface (Figs. 4 and 5, see the outlines of the cell surface as white lines in panels c, d, e, c', d', and e'), consistent with the results of immunoblots that indicated poorer expression at this site (Fig. 3). However, the MLV Env GP was frequently detected in endosomal vesicles (Figs. 4 and 5, red arrows in panels d and d'), as shown by using specific protein markers (Fig. 6 and data not shown). Likewise, MLV or SIV Gag proteins could not or could hardly be detected under the plasma membrane upon co-expression with the MLV glycoprotein (Fig. 4A versus Fig. 5, A and B, panels d and d': see the outline of the cell surface in white lines), but were predominantly localized in endosomal vesicles. A strong co-localization between MLV GP and either SIV or MLV Gag proteins was observed in these vesicles (Fig. 5, A and B, yellow arrows in panels d and d') that were further characterized as late endosomes using a TI-VAMP protein marker (Fig. 6, panel b). This strong GP/Gag co-localization was consistent with the high infectious titers of the resulting retroviral pseudotypes (Fig. 2). In contrast to the MLV GP, the HCV and MLV/TRRD glycoproteins were rarely detected in vesicles of the endocytic pathway (Fig. 4B, red arrows in panels c and e) and were predominantly found in the ER. Such a localization pattern was unchanged whether these glycoproteins were expressed alone (Fig. 4B) or co-expressed in the presence of SIV Gag proteins (Fig. 5A). Co-localization of the HCV GPs with SIV or MLV Gag proteins could not be detected in the ER but exclusively in the few endosomal vesicles where these glycoproteins were also found (Fig. 5, A and B, yellow arrows in panels c and c'), which were further identified as late endosomes (Fig. 6, panel a). Likewise, co-localization between the SIV Gag proteins and the MLV/TRRD GPs was only observed in the rare endosomal vesicles where this GP was detected (Fig. 5A, yellow arrow in panel e). Altogether, the low level of co-localization in late endosomes of retroviral Gag with HCV GP and of lentiviral Gag with MLV/TRRD GP seemed in good agreement with the low viral incorporation levels of these glycoproteins (Fig. 3) and with the poor infectious titers of the resulting viral pseudotypes (Fig. 2).



View larger version (66K):
[in this window]
[in a new window]
  		FIGURE 6.
Co-localization of viral glycoproteins and cellular marker of the late endosomes. COS-7 cells co-expressing a GFP-tagged TI-VAMP marker protein (TI-VAMP), MLV Gag proteins and HCV-E1E2 (panel a), MLV Env (panel b), or MLV/TRRD Env (panel c) glycoproteins were imaged by confocal microscopy analysis. Co-localization of both GP (red channel) and TI-VAMP (green channel) is shown (Merge). The arrows indicate the localization of GPs in late endosomes. The dotted lines correspond to the plasma membranes, as drawn from phase-contrast micrographs (not shown). N, nucleus. The scale bars are indicated. Similar results were obtained with SIV cores, except for the MLV/TRRD Env GP, which was localized in the ER, as shown in Fig. 5.

 
Interestingly, whereas the MLV/TRRD GP expressed alone (Fig. 4B) or in the presence of SIV Gag proteins (Fig. 5A) was poorly detected in endosomal vesicles, its intracellular localization was clearly altered upon co-expression with MLV Gag proteins, leading to frequent detection into endosomal vesicles (Figs. 4 and 5, A versus B, red arrows, compare panels e versus e'). Additionally, strong co-localization of this GP with MLV Gag proteins could be observed in such vesicles (Fig. 5B, yellow arrows in panel e') that were further characterized as late endosomes (Fig. 6, panel c). This indicated that MLV Gag proteins could specifically, directly or indirectly, interact with the MLV/TRRD GP and induce its intracellular re-localization. This was correlated to the strong increase of incorporation of the MLV/TRRD GP on MLV core particles as compared with SIV core particles (Fig. 3, compare lanes m' versus m) and to the increased infectivity of these former pseudotypes as compared with later ones (Fig. 2).

Altogether, these results pointed to distinct mechanisms operating during pseudotype formation. The first mechanism depends on intrinsic properties of the pseudotyping glycoprotein that dictates the site of co-localization between GP and Gag proteins at the cell surface (i.e. FPV-HA and VSV-G) or, alternatively, in late endosomes (i.e. HCV and MLV GPs). The second mechanism depends on the possibility of interactions between either viral component (i.e. MLV and MLV/TRRD GPs with MLV Gag) that could induce their re-localization in an appropriate assembly compartment (i.e. the late endosome). For either mechanism, the level of co-localization between Gag and GP detected by confocal microscopy was consistent with the level of viral incorporation of the glycoprotein and with the infectious titers of the resulting retroviral pseudotypes.

Nef Selectively Influences Pseudotype Formation with Retroviral GPs—The Nef accessory protein was previously found to influence the infectivity of pseudotyped lentiviruses (38, 39). To further investigate its influence on pseudotype formation, we compared the expression/assembly of the Gag/GP viral components and the infectivity of lentiviral particles in the presence or absence of SIV Nef proteins. Correct expression of the Nef protein was demonstrated by immunoblotting of cell lysates and confocal microscopy (data not shown).

Interestingly, we detected an increase of GP viral incorporation, ranging from to 2- to 20-fold depending on the type of glycoprotein, when the virions were generated in the presence of Nef proteins (Fig. 7; Table 2). This effect was induced, in part, by the 2-fold increase of the expression of Gag proteins and virion release (Fig. 7, compare lanes g versus h and e versus f; Table 2), which appeared to be independent of the GP type. This result was consistent with the notion that the Nef protein acts at different cellular levels to increase the efficiency of the viral assembly process directly via stimulating Gag processing and release (40) or, indirectly, via increasing the biogenesis of viral assembly platforms, such as raft and MVBs (41, 42). To determine the influence of Nef in specific viral incorporation of the GP, independently of the increase of Gag release, we calculated the GP/Gag ratios using the signals acquired from Western blot analysis (Table 2). Remarkably, Nef expression selectively modified the viral incorporation of the retroviral glycoproteins (Fig. 7). Indeed, whereas the GP/Gag ratios detected for the FPV-HA, VSV-G, and HCV GPs were not modified whether or not Nef was expressed (Fig. 7, for example, compare lanes a versus b or e versus f or i versus j; Table 2), Nef expression increased the GP/Gag ratios of the MLV and MLV/TRRD GPs, as well as of the previously described RD114 and RD/TRMLV GPs (19), by about 3-10-fold (Fig. 7, for example, compare lanes e' versus f' or m' versus n'; Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2
Influence of Nef expression on virion incorporation of viral glycoproteins

 



View larger version (75K):
[in this window]
[in a new window]
  		FIGURE 7.
Effect of Nef protein expression in pseudotype formation. Detection of the different envelope glycoproteins and viral core proteins was performed by Western blotting of crude cell lysate (L) and lentiviral particles purified by ultracentrifugation of the cell supernatants on sucrose cushions (V). These experiments were performed in COS-7 cells, from African green monkey, to optimize the interactions between SIV Nef and its cellular adaptor proteins. Note that although the infectious titers of the viral pseudotypes produced in COS-7 cells were lower than those obtained with 293T cells, the relative differences of infectivity observed with the pseudotyped SIV retroviral core particles, as well as the patterns of expression/incorporation and cellular localization of the Gag/GP viral components, were maintained (compare Figs. 2 with 9 and see Ref. 19). The GPs and core viral components were expressed in the presence (+) or absence (-)of SIV Nef proteins. The detection of indicated GPs and SIV Gag proteins was carried out as described in the legend to Fig. 3. The expression of the Nef protein was confirmed by Western blotting (data not shown). Scanning of Western blot membranes was performed with the Storm 860 device and signal densities were calculated with the Image-Quant program.

 
Additionally, although they were predominantly detected in the ER in the absence of Nef (Fig. 8A, panels a and c), the MLV/TRRD and RD114 glycoproteins were re-localized in intracellular vesicles upon Nef co-expression (Fig. 8A, red arrows in panels b and d), which appeared to be late endosomes, as demonstrated using the TI-VAMP marker (Fig. 8B). Of note, such an alteration of the localization of these retroviral glycoproteins was only achieved via GP/Nef interactions, direct or indirect, and was independent of the presence of lentiviral Gag proteins (data not shown). Furthermore, the late endosomal re-localization of MLV/TRRD and RD114 GPs detected upon Nef expression was associated with co-localization between SIV Gag and either glycoprotein (Fig. 8A, yellow arrows in panels b and d). No obvious effect of Nef could be detected for the MLV and RD/TRMLV GPs (data not shown) that (Figs. 4, 5, 6 and Ref. 19), perhaps because further accumulation of either GPs that were already efficiently targeted to late endosomal vesicles in the absence of Nef cannot be detected by confocal microscopy. Because we found that the co-localization of retroviral GPs and lentiviral Gag proteins was critical for pseudotype formation (Fig. 5), these results suggested that the increased viral incorporation of the retroviral GPs (Fig. 7 and Table 2) was because of their Nef-induced augmented accumulation in late endosomes and co-localization with SIV Gag proteins (Fig. 8). Consistently, the infectivity of lentiviral vectors pseudotyped with the MLV/TRRD and RD114 glycoproteins was increased by up to 12-fold (Fig. 9), most likely as a result of strongly augmented viral incorporation. The influence of Nef on the infectivity of vectors pseudotyped with the MLV and RD/TRMLV GPs was less marked than for the RD114 and MLV/TRRD glycoproteins (Fig. 9), despite significant increase of viral incorporation of all these retroviral GPs (Fig. 7). Perhaps these former GPs were already incorporated in the absence of Nef over a threshold of density sufficient to promote optimal levels of infectivity. Indeed, note that the incorporation levels of the MLV and RD/TRMLV GPs were already high as compared with those of the MLV/TRRD and the RD114 GPs in the absence of Nef (Fig. 7, panels b' versus f' and n' versus j') and were similar to those of the latter glycoproteins in the presence of Nef (Fig. 7, panels b' versus e' and n' versus i'). Thus, an increased GP incorporation over a certain threshold may not necessarily result in augmented infectivity, as reported previously for MLV or lentiviral vectors pseudotyped with Eco-, ampho-MLV, or HIV glycoproteins (43). Altogether, these results indicated that Nef expression optimizes co-localization of retroviral GPs with lentiviral Gag proteins in late endosomes and hence viral incorporation, with a particularly marked effect for those retroviral GPs that inherently do not accumulate in the endosomal pathway.




View larger version (168K):
[in this window]
[in a new window]
  		FIGURE 8.
Effect of Nef protein expression on cellular localization of retroviral glycoproteins. COS-7 cells co-expressing the indicated GP and SIV core proteins (Gag), as indicated, were grown on glass coverslips, fixed in paraformaldehyde, permeabilized, stained at room temperature, and imaged by confocal microscopy analysis. A, the SIV core proteins (green channel) and RD114 (panels a and b) or MLV/TRRD (panels c and d) Env GPs (red channel) were expressed in the presence (panels b and d) or absence (panels a and c) of SIV Nef proteins. The arrows point to intracytoplasmic vesicles where Gag/GP co-localization can be detected. B, the viral GPs (red channel) and SIV components (Gag, Nef, as indicated) were co-expressed with a GFP-tagged TI-VAMP marker protein (TI-VAMP). The arrows point to late endosomal vesicles where TI-VAMP/GP co-localization can be detected. The dotted lines correspond to the plasma membranes, as drawn from phase-contrast micrographs (not shown). N, nucleus. The scale bars are indicated. The detection of Nef was also performed and revealed a punctuate pattern in the cytoplasm and under the plasma membrane (data not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To gain insights in the process of envelope assembly of retroviral pseudotypes, here we have investigated the capacity of envelope GPs derived from different virus families to interact and/or coat onco-retroviral and lentiviral core particles. The parameters that were found to influence the incorporation of viral GPs onto retroviral core particles were (i) the intrinsic cell localization properties of both the viral GP and the retroviral core proteins, (ii) the ability of the viral GP to interact, directly or not, with the retroviral core, and (iii) the expression of the lentiviral Nef protein. These different parameters determined the ability of Gag and GP to co-localize in a common cellular compartment and, ultimately, to lead to assembly of infectious viral particles. Whereas the data reported here were obtained from transiently transfected 293T or COS-7 cells, it is possible that the mechanisms that govern pseudotype formation also operate in envelope assembly of wild-type retroviruses in naturally infected cells.

Envelope Assembly of Pseudotyped Retroviruses Is Determined by GP Localization at the Cell Surface or in Late Endosomes—The assembly of the envelopes of retroviruses and pseudotyped retroviruses, and the subsequent budding of viral particles has long been thought to occur at the plasma membrane of infected cells (44), a pathway supported by electron microscopy data (reviewed in Refs. 45 and 46) and by plasma membrane co-localization of the two types of viral components in glycolipid-enriched microdomains (47-50). In agreement with these previous findings, our results suggest that the VSV-G and FPV-HA GPs were most likely incorporated on retroviral particles from the cell surface. Compared with other GPs such as those derived from HCV and MLV, we found that expression of VSV-G and FPV-HA GPs was strongly enriched at the cell surface and was not affected by co-expression of Gag proteins. Furthermore, co-localization between VSV-G or FPV-HA GPs and Gag proteins was predominantly detected at the cell surface but only poorly into vesicles of the endosomal and secretory pathways. This is consistent with the available evidence from the literature that indicate the efficient export of VSV-G from the endoplasmic reticulum (52) and the association of FPV-HA and VSV-G GPs in raft microdomains of the cell surface (50, 51). Additionally, the envelope assembly on VSV or FPV virions is thought to occur at the cell surface (53, 54). Finally, although the VSV-M matrix protein directly interacts with the cytoplasmic tail of VSV-G (55, 56), the localization of VSV-G into membrane microdomains was reported to occur independently of the homologous viral core components (54). Altogether, these previous results are congruent with our finding that cell surface localization of VSV-G and FPV-HA GPs was independent of the co-expression of the retroviral core proteins and suggest that the viral incorporation of these GPs on retroviral core particles occurs passively, via concentration of both types of components in coincident cell locations. These considerations are not coherent, however, with the finding that the localization of the Gag proteins under the cell surface seemed intensified upon co-expression with either GP. Indeed, no direct interactions are expected to occur between the retroviral Gag proteins and the cytoplasmic tails of FPV-HA or VSV-G, which are derived from very different viruses. Results of others have indicated that the modification of the subcellular distribution of the phospholipid phosphatidylinositol (4,5)-bisphosphate (9) and of the essential raft-component cholesterol (57) could influence the targeting of Gag proteins to either the plasma membrane or the MVBs. Thus, one possibility to explain that FPV-HA or VSV-G seem to influence Gag localization is that overexpression of either GP induces a perturbation of the cellular lipids repartition leading to redistribution of Gag proteins and, indirectly, to an efficient co-localization of both GP and Gag proteins.

In contrast to these two GPs, the HCV GPs were predominantly detected in the ER but very poorly at the cell surface. Several studies revealed that the HCV GPs harbor ER retention signals within their transmembrane domains and that no canonical endocytic trafficking signal can be found in their very short cytoplasmic tails (58). Furthermore, HCV assembly is thought to occur intracellularly, in the ER or into poorly characterized intracellular membranous formations (59-61). Additionally, cellular localization and processing of HCV glycoproteins is dependant on the available expression systems (62). In addition to ER localization, low level cell surface expression (28, 62), as well as localization of the HCV GPs in endosomal vesicles (Fig. 3), can also be detected when using appropriate expression systems, in a manner independent of the co-expression of retroviral core proteins. Finally, co-localization of HCV GPs and retroviral Gag proteins could be detected in late endosomes, but neither at the cell surface nor in the ER.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 9.
Effect of Nef expression in infectivity of pseudotyped lentiviral particles. SIV viral particles were produced in the presence or absence of the SIV Nef protein and were titrated on appropriate target cells, as described in the legend to Fig. 2. The histograms show the ratios of the infectious titers of virions produced in the presence versus in the absence of Nef. The infectious titers reported in the table below the histograms are the mean ± S.D. of up to seven independent experiments.

 
Altogether, the results obtained with FPV-HA, VSV-G, and HCV GPs suggest that the choice between two possible sites of envelope assembly of retrovirus pseudotypes, i.e. the cell surface versus the late endosome, is largely determined by intrinsic properties of the heterologous viral glycoproteins and that their recruitment on retroviral core particles occurs passively. This is consistent with the current notion that different sites of assembly of retroviral core particles and, potentially, of viral egress, may co-exist and/or depend on the virion producer cell type (63, 64). In HeLa and T cells, the majority of assembling HIV-1 particles are detected at the plasma membrane. In contrast, results from several groups indicate that the assembly process of MLV and lentiviruses occurred in late endosomes or in MVBs in macrophages, COS and 293T cells (5, 7, 8, 20), in agreement with this report.

Pseudotype Formation Is Influenced by Interactions between Gag and the Retroviral GP—The mechanisms of recruitment of retroviral assembly of the retroviral envelopes remain poorly characterized. There is evidence that the cytoplasmic tails of the retroviral Env GPs harbor several determinants that allow their trafficking between the secretory vesicles, the cell surface, and the endosomal vesicles (65-69) and that determine the site of virion budding in polarized cells (13, 70). The punctuated staining detected for the MLV GP as well as its co-localization with intracellular markers indicates its abundant presence in endosomal vesicles, particularly in late endosomes and MVBs, but a much weaker expression at the plasma membrane that could only be detected using cell surface biotinylation. It is likely that these vesicles are sites where Gag recruits the retroviral Env GPs, at least in 293T and COS-7 cells that were used in this work. Indeed, alterations of the MLV GP cyt, as in the MLV/TRRD chimera, which reduce its accumulation in late endosomes (19), prevent co-localization with SIV Gag proteins and hence recruitment by lentiviral core particles. This is consistent with our previous findings (19) that introducing in the cyt of an ER-retained retroviral glycoprotein (e.g. the RD114 Env GP) the appropriate motifs that stimulate intracellular trafficking leads to GP re-localization to late endosomes and incorporation on lentivirus particles. Thus, the correlation between co-localization of these different onco-retroviral GPs with SIV Gag proteins in late endosomes and their levels of incorporation onto SIV particles suggested that they are passively recruited during envelope assembly of lentiviral pseudotypes. In contrast, indirect evidence suggested that specific interactions occurred between the cytoplasmic tails of the MLV Gag proteins and the MLV or RD114 GPs. Indeed, in the presence of MLV core proteins, (i) the maturation of both MLV and MLV/TRRD GPs was modified, (ii) the MLV/TRRD GP was re-localized into MVBs, and (iii) the infectious titers of the MLV/TRRD pseudotypes were significantly increased. These results indicated that whereas the cytoplasmic tails of the RD114 and MLV GPs induce differential intracellular traffic and localization, they share conserved determinants mediating direct or indirect interactions with MLV Gag proteins. Such interactions are likely to modify the trafficking pathway of these GPs (71), perhaps via competition between the MLV Gag proteins and the intracellular trafficking effectors that interact with their cytoplasmic tails. For example, MLV Gag may prevent the interaction of the Env cytoplasmic tail with a cellular factor involved in GP recycling from the endosomal vesicles to the trans Golgi network or the cell surface. This could explain the accumulation of onco-retroviral GPs in late endosomes and their strong co-localization with MLV Gag proteins. Thus, the cytoplasmic tail of a retroviral GP could differentially influence pseudotype formation on onco-retroviral or lentiviral core particles by allowing direct or indirect interactions with Gag proteins, hence providing an active mechanism of envelope assembly (e.g. MLV or RD114 GPs with MLV core proteins) or, in a nonexclusive manner, by allowing concentration of Env GPs at the envelope assembly sites, hence explaining passive mechanism of GP incorporation (e.g. MLV GP with heterologous SIV cores proteins).

Nef Stimulates Pseudotype Formation with Retroviral GPs—Our results indicate that the lentiviral Nef protein selectively stimulates pseudotype formation with retroviral GPs but not with the other GPs. This is partially in agreement with previous reports that indicated that the Nef protein differentially influenced the infectivity of pseudotyped lentiviruses, depending on the cell entry route determined by the pseudotyping GP (38, 39). These previous studies suggested that Nef acts at a post-entry step of infection and that virions pseudotyped with pH-dependant GPs do not require Nef for optimal infectivity, perhaps because the viral cores of virions pseudotyped with such GPs are directly released into a intracellular compartment that facilitate post-fusion events. Our results extend these findings and confirm that, in contrast to retroviral GPs, the FPV-HA, VSV-G, and HCV GPs, which are pH-dependant (72), are not sensitive to Nef. Furthermore, our data suggest that the phenotype of sensitivity to Nef also relies on mechanisms of envelope assembly, thus providing novel insights on how Nef may influence viral infectivity. Indeed, we found that Nef increased endosomal accumulation and viral incorporation of GPs harboring determinants in their cytoplasmic tail that allow intracellular trafficking through the endocytic pathway and whose assembly site are MVBs (i.e. RD114, RD/TRMLV, MLV, and MLV/TRRD Env GPs). This is reminiscent of the pleiotropic effects of Nef on early/recycling endosomal compartments and on protein trafficking within the endosomal system (42, 73, 74). In contrast, no Nef effect was detected for the GPs recruited at the cell surface (VSV-G and FPV-HA GPs) or that do not harbor in their cytoplasmic tail determinants of endocytic trafficking (HCV GPs). Thus, in addition to a role of Nef at a post-entry stage, our results suggest an additional mechanism to the wide array of activities attributed to Nef and stimulation of infection (75).


   FOOTNOTES
 
* This work was supported in part by "Agence Nationale pour la Recherche sur le SIDA et les Hépatites Virales" (ANRS), La Ligue Nationale Contre le Cancer, the European Community (contract LSHB-CT-2004-005242 "CONSERT"), and Région Rhône-Alpes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a fellowship of Vaincre la Mucoviscidose. Back

2 To whom correspondence should be addressed: LVRTG, ENS de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France. Tel.: 33-472-72-87-32; Fax: 33-472-72-80-80; E-mail: flcosset{at}ens-lyon.fr.

3 The abbreviations used are: Env GP, envelope glycoprotein; HIV, human immunodeficiency virus; MLV, murine leukemia virus; SIV, simian immunodeficiency virus; VSV-G, G protein of vesicular stomatitis virus; HA, hemagglutinin; FPV, fowl plague virus; HCV-E1E2, E1E2 glycoproteins of hepatitis C virus; GFP, green fluorescent protein; i.u., infectious units; IF, immunofluorescence; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; cyt, cytoplasmic tail; TM, transmembrane; VAMP, vesicle-associated membrane protein. Back


   ACKNOWLEDGMENTS
 
We thank Edouard Bertrand and Thierry Galli for the gift of TI-VAMP-GFP and cellubrevin-GFP vectors, Didier Nègre for the gift of the SIV Nef expression vector, Alan Rein, A. Pinter, W. Garten, L. Evans, M. Schweizer, and the National Institutes of Health for the gift of antibodies, and Fabienne Simian-Lermé and Claire Lionnet of the PLATIM platform for technical assistance in confocal microscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gelderblom, H. R. (1991) Aids 5, 617-637[Medline] [Order article via Infotrieve]
  2. Gelderblom, H. R., Ozel, M., and Pauli, G. (1989) Arch. Virol. 106, 1-13[CrossRef][Medline] [Order article via Infotrieve]
  3. Orenstein, J. M., Meltzer, M. S., Phipps, T., and Gendelman, H. E. (1988) J. Virol. 62, 2578-2586[Abstract/Free Full Text]
  4. Raposo, G., Moore, M., Innes, D., Leijendekker, R., Leigh-Brown, A., Benaroch, P., and Geuze, H. (2002) Traffic 3, 718-729[CrossRef][Medline] [Order article via Infotrieve]
  5. Pelchen-Matthews, A., Kramer, B., and Marsh, M. (2003) J. Cell. Biol. 162, 443-455[Abstract/Free Full Text]
  6. Nguyen, D. G., Booth, A., Gould, S. J., and Hildreth, J. E. (2003) J. Biol. Chem. 278, 52347-52354[Abstract/Free Full Text]
  7. Sherer, N. M., Lehmann, M. J., Jimenez-Soto, L. F., Ingmundson, A., Horner, S. M., Cicchetti, G., Allen, P. G., Pypaert, M., Cunningham, J. M., and Mothes, W. (2003) Traffic 4, 785-801[CrossRef][Medline] [Order article via Infotrieve]
  8. Nydegger, S., Foti, M., Derdowski, A., Spearman, P., and Thali, M. (2003) Traffic 4, 902-910[CrossRef][Medline] [Order article via Infotrieve]
  9. Ono, A., Ablan, S. D., Lockett, S. J., Nagashima, K., and Freed, E. O. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 14889-14894[Abstract/Free Full Text]
  10. Resh, M. D. (2004) Subcell. Biochem. 37, 217-232[Medline] [Order article via Infotrieve]
  11. Hammarstedt, M., and Garoff, H. (2004) J. Virol. 78, 5686-5697[Abstract/Free Full Text]
  12. Gould, S. J., Booth, A. M., and Hildreth, J. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10592-10597[Abstract/Free Full Text]
  13. Lodge, R., Delamarre, L., Lalonde, J. P., Alvarado, J., Sanders, D. A., Dokhelar, M. C., Cohen, E. A., and Lemay, G. (1997) J. Virol. 71, 5696-5702[Abstract]
  14. Lodge, R., Gottlinger, H., Gabuzda, D., Cohen, E., and Lemay, G. (1994) J. Virol. 68, 4857-4561[Abstract/Free Full Text]
  15. Cosson, P. (1996) EMBO J. 15, 5783-5788[Medline] [Order article via Infotrieve]
  16. Murakami, T., and Freed, E. O. (2000) J. Virol. 74, 3548-3554[Abstract/Free Full Text]
  17. Vincent, M. J., Melsen, L. R., Martin, A. S., and Compans, R. W. (1999) J. Virol. 73, 8138-8144[Abstract/Free Full Text]
  18. Wyma, D. J., Kotov, A., and Aiken, C. (2000) J. Virol. 74, 9381-9387[Abstract/Free Full Text]
  19. Sandrin, V., Muriaux, D., Darlix, J. L., and Cosset, F. L. (2004) J. Virol. 78, 7153-7164[Abstract/Free Full Text]
  20. Basyuk, E., Galli, T., Mougel, M., Blanchard, J. M., Sitbon, M., and Bertrand, E. (2003) Dev. Cell 5, 161-174[CrossRef][Medline] [Order article via Infotrieve]
  21. Sandrin, V., and Cosset, F. L. (2003) Curr. Top. Microbiol. Immunol. 100, 137-178
  22. Christodoulopoulos, I., and Cannon, P. (2001) J. Virol. 75, 4129-4138[Abstract/Free Full Text]
  23. Lindemann, D., Bock, M., Schweizer, M., and Rethwilm, A. (1997) J. Virol. 71, 4815-4820[Abstract]
  24. Mammano, F., Salvatori, F., Indraccolo, S., de Rossi, A., Chieco-Bianchi, L., and Göttlinger, H. G. (1997) J. Virol. 71, 3341-3345[Abstract]
  25. Sandrin, V., Boson, B., Salmon, P., Gay, W., Nègre, D., LeGrand, R., Trono, D., and Cosset, F.-L. (2002) Blood 100, 823-832[Abstract/Free Full Text]
  26. Takeuchi, Y., Simpson, G., Vile, R., Weiss, R., and Collins, M. (1992) Virology 186, 792-794[CrossRef][Medline] [Order article via Infotrieve]
  27. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T., and Sato, J. (1982) Cancer Res. 42, 3858-3863[Abstract/Free Full Text]
  28. Bartosch, B., Dubuisson, J., and Cosset, F. L. (2003) J. Exp. Med. 197, 633-642[Abstract/Free Full Text]
  29. Yee, J. K., Friedmann, T., and Burns, J. C. (1994) Methods Cell Biol. 43, 99-112[Medline] [Order article via Infotrieve]
  30. Cosset, F.-L., Takeuchi, Y., Battini, J., Weiss, R., and Collins, M. (1995) J. Virol. 69, 7430-7436[Abstract]
  31. Ott, D., Friedrich, R., and Rein, A. (1990) J. Virol. 64, 757-766[Abstract/Free Full Text]
  32. Mangeot, P.-E., Nègre, D., Dubois, B., Winter, A. J., Leissner, P., Methali, M., Kaiserlian, D., Cosset, F.-L., and Darlix, J.-L. (2000) J. Virol. 74, 8307-8315[Abstract/Free Full Text]
  33. Nègre, D., Mangeot, P., Duisit, G., Blanchard, S., Vidalain, P., Leissner, P., Winter, A., Rabourdin-Combe, C., Mehtali, M., Moullier, P., Darlix, J.-L., and Cosset, F.-L. (2000) Gene Ther. 7, 1613-1623[CrossRef][Medline] [Order article via Infotrieve]
  34. Flint, M., Maidens, C., Loomis-Price, L. D., Shotton, C., Dubuisson, J., Monk, P., Higginbottom, A., Levy, S., and McKeating, J. A. (1999) J. Virol. 73, 6235-6244[Abstract/Free Full Text]
  35. Alberts, P., and Galli, T. (2003) Biol. Cell 95, 419-424[CrossRef][Medline] [Order article via Infotrieve]
  36. Galli, T., Chilcote, T., Mundigl, O., Binz, T., Niemann, H., and De Camilli, P. (1994) J. Cell. Biol. 125, 1015-1024[Abstract/Free Full Text]
  37. Smith, M. J., and Koch, G. L. (1989) EMBO J. 8, 3581-3586[Medline] [Order article via Infotrieve]
  38. Aiken, C. (1997) J. Virol. 71, 5871-5877[Abstract]
  39. Chazal, N., Singer, G., Aiken, C., Hammarskjold, M. L., and Rekosh, D. (2001) J. Virol. 75, 4014-4018[Abstract/Free Full Text]
  40. Costa, L. J., Zheng, Y. H., Sabotic, J., Mak, J., Fackler, O. T., and Peterlin, B. M. (2004) J. Virol. 78, 5311-5323[Abstract/Free Full Text]
  41. Zheng, Y. H., Plemenitas, A., Fielding, C. J., and Peterlin, B. M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 8460-8465[Abstract/Free Full Text]
  42. Stumptner-Cuvelette, P., Jouve, M., Helft, J., Dugast, M., Glouzman, A. S., Jooss, K., Raposo, G., and Benaroch, P. (2003) Mol. Biol. Cell 14, 4857-4870[Abstract/Free Full Text]
  43. Bachrach, E., Marin, M., Pelegrin, M., Karavanas, G., and Piechaczyk, M. (2000) J. Virol. 74, 8480-8486[Abstract/Free Full Text]
  44. Swanstrom, R., and Wills, J. W. (1997) in Retroviruses (Coffin, J. M., Hughes, S. H., and Varmus, H. E., eds) pp. 263-334, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  45. Freed, E. O. (1998) Virology 251, 1-15[CrossRef][Medline] [Order article via Infotrieve]
  46. Garoff, H., Hewson, R., and Opstelten, D. J. (1998) Microbiol. Mol. Biol. Rev. 62, 1171-1190[Abstract/Free Full Text]
  47. Briggs, J. A., Wilk, T., and Fuller, S. D. (2003) J. Gen. Virol. 84, 757-768[Abstract/Free Full Text]
  48. Nguyen, D. H., and Hildreth, J. E. (2000) J. Virol. 74, 3264-3272[Abstract/Free Full Text]
  49. Ono, A., and Freed, E. O. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13925-13930[Abstract/Free Full Text]
  50. Pickl, W. F., Pimentel-Muinos, F. X., and Seed, B. (2001) J. Virol. 75, 7175-7183[Abstract/Free Full Text]
  51. Zhang, J., Pekosz, A., and Lamb, R. A. (2000) J. Virol. 74, 4634-4644[Abstract/Free Full Text]
  52. Nishimura, N., Bannykh, S., Slabough, S., Matteson, J., Altschuler, Y., Hahn, K., and Balch, W. E. (1999) J. Biol. Chem. 274, 15937-15946[Abstract/Free Full Text]
  53. Sidorenko, Y., and Reichl, U. (2004) Biotechnol. Bioeng. 88, 1-14[CrossRef][Medline] [Order article via Infotrieve]
  54. Brown, E. L., and Lyles, D. S. (2003) J. Virol. 77, 3985-3992[Abstract/Free Full Text]
  55. Dubovi, E. J., and Wagner, R. R. (1977) J. Virol. 22, 500-509[Abstract/Free Full Text]
  56. Lyles, D. S., McKenzie, M., and Parce, J. W. (1992) J. Virol. 66, 349-358[Abstract/Free Full Text]
  57. Lindwasser, O. W., and Resh, M. D. (2004) J. Virol. 78, 6013-6023[Abstract/Free Full Text]
  58. Dubuisson, J. (2000) Curr. Top. Microbiol. Immunol. 242, 135-148[Medline] [Order article via Infotrieve]
  59. Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D., and Bienz, K. (2002) J. Virol. 76, 5974-5984[Abstract/Free Full Text]
  60. Greive, S. J., Webb, R. I., Mackenzie, J. M., and Gowans, E. J. (2002) J. Viral Hepat. 9, 9-17[CrossRef][Medline] [Order article via Infotrieve]
  61. Shimizu, Y. K., Feinstone, S. M., Kohara, M., Purcell, R. H., and Yoshikura, H. (1996) Hepatology 23, 205-209[Medline] [Order article via Infotrieve]
  62. Dumonceaux, J., Cormier, E. G., Kajumo, F., Donovan, G. P., Roy-Chowdhury, J., Fox, I. J., Gardner, J. P., and Dragic, T. (2003) J. Virol. 77, 13418-13424[Abstract/Free Full Text]
  63. Freed, E. O., and Ross, S. R. (2004) Retrovirology 1, 25[CrossRef][Medline] [Order article via Infotrieve]
  64. Ono, A., and Freed, E. O. (2004) J. Virol. 78, 1552-1563[Abstract/Free Full Text]
  65. Berlioz-Torrent, C., Shacklett, B. L., Erdtmann, L., Delamarre, L., Bouchaert, I., Sonigo, P., Dokhelar, M. C., and Benarous, R. (1999) J. Virol. 73, 1350-1361[Abstract/Free Full Text]
  66. Blot, G., Janvier, K., LePanse, S., Benarous, R., and Berlioz-Torrent, C. (2003) J. Virol. 77, 6931-6945[Abstract/Free Full Text]
  67. Boge, M., Wyss, S., Bonifacino, J. S., and Thali, M. (1998) J. Biol. Chem. 273, 15773-15778[Abstract/Free Full Text]
  68. Ohno, H., Aguilar, R. C., Fournier, M. C., Hennecke, S., Cosson, P., and Bonifacino, J. S. (1997) Virology 238, 305-315[CrossRef][Medline] [Order article via Infotrieve]
  69. Wyss, S., Berlioz-Torrent, C., Boge, M., Blot, G., Honing, S., Benarous, R., and Thali, M. (2001) J. Virol. 75, 2982-2992[Abstract/Free Full Text]
  70. Weclewicz, K., Ekstrom, M., Kristensson, K., and Garoff, H. (1998) J. Virol. 72, 2832-2845[Abstract/Free Full Text]
  71. Egan, M., Carruth, L., Rowell, J., Yu, X., and Siliciano, R. (1996) J. Virol. 70, 6547-6556[Abstract/Free Full Text]
  72. Bartosch, B., Vitelli, A., Granier, C., Goujon, C., Dubuisson, J., Pascale, S., Scarselli, E., Cortese, R., Nicosia, A., and Cosset, F.-L. (2003) J. Biol. Chem. 278, 41624-41630[Abstract/Free Full Text]
  73. Madrid, R., Janvier, K., Hitchin, D., Day, J., Coleman, S., Noviello, C., Bouchet, J., Benmerah, A., Guatelli, J., and Benichou, S. (2005) J. Biol. Chem. 280, 5032-5044[Abstract/Free Full Text]
  74. Piguet, V., Schwartz, O., Le Gall, S., and Trono, D. (1999) Immunol. Rev. 168, 51-63[CrossRef][Medline] [Order article via Infotrieve]
  75. Arora, V. K., Fredericksen, B. L., and Garcia, J. V. (2002) Microbes Infect. 4, 189-199[CrossRef][Medline] [Order article via Infotrieve]
  76. Op De Beeck, A., Voisset, C., Bartosch, B., Ciczora, Y., Cocquerel, L., Keck, Z., Foung, S., Cosset, F. L., and Dubuisson, J. (2004) J. Virol. 78, 2994-3002[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Virol.Home page
C. R. Langelier, V. Sandrin, D. M. Eckert, D. E. Christensen, V. Chandrasekaran, S. L. Alam, C. Aiken, J. C. Olsen, A. K. Kar, J. G. Sodroski, et al.
Biochemical Characterization of a Recombinant TRIM5{alpha} Protein That Restricts Human Immunodeficiency Virus Type 1 Replication
J. Virol., December 1, 2008; 82(23): 11682 - 11694.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. Pizzato, E. Popova, and H. G. Gottlinger
Nef Can Enhance the Infectivity of Receptor-Pseudotyped Human Immunodeficiency Virus Type 1 Particles
J. Virol., November 1, 2008; 82(21): 10811 - 10819.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
I-C. Huang, W. Li, J. Sui, W. Marasco, H. Choe, and M. Farzan
Influenza A Virus Neuraminidase Limits Viral Superinfection
J. Virol., May 15, 2008; 82(10): 4834 - 4843.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. Khurana, D. N. Krementsov, A. de Parseval, J. H. Elder, M. Foti, and M. Thali
Human Immunodeficiency Virus Type 1 and Influenza Virus Exit via Different Membrane Microdomains
J. Virol., November 15, 2007; 81(22): 12630 - 12640.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
E. Blanchard, S. Belouzard, L. Goueslain, T. Wakita, J. Dubuisson, C. Wychowski, and Y. Rouille
Hepatitis C Virus Entry Depends on Clathrin-Mediated Endocytosis
J. Virol., July 15, 2006; 80(14): 6964 - 6972.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
J. F. Roeth and K. L. Collins
Human Immunodeficiency Virus Type 1 Nef: Adapting to Intracellular Trafficking Pathways
Microbiol. Mol. Biol. Rev., June 1, 2006; 70(2): 548 - 563.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/1/528    most recent
M506070200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sandrin, V.
Right arrow Articles by Cosset, F.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sandrin, V.
Right arrow Articles by Cosset, F.-L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement