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Originally published In Press as doi:10.1074/jbc.M500867200 on February 23, 2005

J. Biol. Chem., Vol. 280, Issue 18, 17969-17977, May 6, 2005
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Functional Relevance of Amino Acid Residues Involved in Interactions with Ordered Nucleic Acid in a Spherical Virus*

Juan Reguera, Esther Grueso, Aura Carreira, Cristina Sánchez-Martínez, José M. Almendral, and Mauricio G. Mateu{ddagger}

From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma de Madrid, Cantoblanco 28049 Madrid, Spain

Received for publication, January 24, 2005 , and in revised form, February 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many fundamental biological processes involve protein-nucleic acid recognition. Double-stranded DNA-protein interactions have been extensively studied, but single-stranded (ss)1 DNA- or RNA-protein interactions are much less characterized. In particular, very little is known about the molecular interactions between the nucleic acid genome of ssDNA or RNA virus and its protein shell, or the functional role of those interactions. Such knowledge may be essential for a better understanding of the life cycle of viruses, and for the design of drugs aimed at disrupting nucleic acid-protein interfaces.

The structure of a helical virus, tobacco mosaic virus, has revealed defined interactions between repeating sets of three nucleotides in its ssRNA genome and each capsid subunit (reviewed in Ref. 1). In the crystal structures of spherical viruses (reviewed in Refs. 2–6) a large part, or all, of the nucleic acid component is invisible, because it is randomly oriented within the viral particles that form the crystal. Fortunately, in a few icosahedral viruses some segments of the nucleic acid molecule (~10–60% of the total) are arranged with the same symmetry as the capsid, and could be visualized. The structural analysis (reviewed in Refs. 4, 7–10) has revealed that the visible capsid-nucleic acid interfaces generally include a limited number of amino acids and some 7–30 nucleotides that in several, but not in all cases, form intramolecular double-helical segments. The tertiary structure acquired by the nucleic acid within the virion may be at least partly independent of the nucleotide sequence (11). Consistent with this observation, a capsid-binding nucleotide motif is not repeated exactly along the sequence, and thus the modeled nucleic acid stretch represents an average of the true individual sequences bound. Accordingly, many of the interactions detected in viral nucleic acid-capsid interfaces are van der Waals and polar interactions with the nonspecific phosphate and pentose moieties, but more specific interactions with the bases are also observed. Ionic interactions between some phosphates and basic side chains protruding from the internal surface of the capsid, or located in disordered N-terminal (Nt) arms of capsid proteins, occur in several ssRNA plant viruses (12–17) and nodaviruses (18–20), and in the ssDNA bacteriophage {Phi}X174 (21–23). Such interactions were not observed in the ssRNA bean pod mottle virus (24) or in ssDNA parvoviruses (25–30), but many nucleic acid-capsid van der Waals contacts and hydrogen bonds were detected in the latter (25–30). A high resolution structural model of the ssRNA satellite tobacco mosaic virus showed that binding of the RNA occurs mainly through an intricate network of direct and water-mediated hydrogen bonds (15). Specific and nonspecific capsid protein-oligonucleotide interactions were also analyzed in detail in artificial complexes formed by a recombinant capsid of the bacteriophage MS2 and identical copies of a unique viral ssRNA fragment (OR) involved in translational repression and initiation of capsid assembly (31, 32). This allowed a structural interpretation of mutational analyses on the formation of a biologically relevant complex between OR and an MS2 capsid protein dimer (32–37).

Several studies have provided evidence for a functional role of the viral nucleic acid and its interactions with viral proteins in the assembly, conformation, stability, and/or disassembly of spherical virions (for some examples see Refs. 4, 5, 9, 38–47). However, and partly due to the limited structural information available, mutational analyses to dissect the individual role(s) of specific capsid side chains that interact with the nucleic acid molecule are scarce (e.g. Refs. 41, 46, 47). Also, no single-residue scanning of capsid side chains involved in major interactions at the viral nucleic acid-capsid interface had, to our knowledge, been described.

The parvoviruses canine parvovirus (CPV) and minute virus of mice (MVM) constitute good models for structure-function studies of residues involved in viral nucleic acid-capsid interactions, because of the structural simplicity of the virion and the relatively well defined viral DNA-capsid interfaces (25–30). The icosahedral T = 1 capsid of MVM (29, 30) or CPV (25–28) is formed by 60 protein subunits that are contributed by three non-identical polypeptides, which are derived from a single gene and show identical fold and core sequence. VP2 is the major capsid component, VP1 includes the VP2 sequence plus a Nt extension, and VP3 results from the cleavage of the Nt of some VP2 subunits (48, 49). The DNA recognition site is essentially equivalent in CPV and MVM (strain i), and the major ordered ssDNA stretch (11 nucleotides) adopts a very similar conformation in both viruses (25, 26, 30). This DNA segment interacts through multiple hydrophobic contacts and/or hydrogen bonds mainly with 7–9 side chains of the capsid of either CPV or MVM (25, 26, 30) (see Table I). Most of these side chains are chemically and sterically identical or similar in both viruses (50) and participate in similar interactions with the DNA. For MVMi, a few shorter DNA stretches, spatially close to the major ordered DNA segment, could also be modeled (29, 30). We have individually truncated to alanine many of those residues of the MVM capsid that were previously (26, 30) found to be more involved in interactions with ordered nucleic acid in the structural model of the virion. The individual effects of the mutations on virus infectivity, viral particle assembly, conformational stability, and DNA packaging have been analyzed.


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  		TABLE I
DNA-protein interactions observed in the structural models of MVMi and CPV

Contact analysis was carried out using the program Whatif (64) and the PDB files 1mvm [PDB] (29, 30) and 4dpv (26, 27), including symmetry-related subunits for completeness. The MVM and CPV residues in each row occupy equivalent positions in the aligned sequence and in the three-dimensional structure. Three DNA stretches of 11, 4 and 1 nucleotides (denoted here as A, B, and C, respectively) were included in the atomic coordinates of MVMi (29, 30), but only the equivalent to stretch A was included in both the unrefined (25) and the refined (26, 27) atomic coordinates of CPV. Residues in parentheses are those not seen to participate in interactions with the visible DNA stretches in either MVM or CPV. Conservation of sequence and conformation is consistent with the possibility that the CPV residues in parentheses could interact with the equivalent to DNA stretches B and C, even though these regions could not be modeled in the crystal structure of CPV.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Plasmids and Mutagenesis—Site-directed mutagenesis of the VP1/VP2 gene of MVM (strain p) was carried out using the QuikChange system (Stratagene), on recombinant plasmid pSVtk-VP1/2, and the mutant sequences were confirmed by automated DNA sequencing. The mutations were introduced by subcloning in the MVMp infectious clone pMM984, originally provided by P. Tattersall (51), and modified to include the VP2 sequence of the MVMp variant used in our laboratories (52). Two HindIII restriction sites, or unique sites XbaI and EcoRI in the VP2 gene were respectively used for mutations located upstream or downstream of the codon corresponding to amino acid residue 402. The mutant infectious clones were subjected to restriction analysis, and the presence of the mutations and the entire sequence inserted were confirmed by DNA sequencing. The concentration of purified DNA was estimated first by agarose gel electrophoresis and quantitated by ultraviolet absorbance at 260 nm.

Electroporation of Mammalian Cells, Immunofluorescence, and Infectivity Assays—For mutational analyses, mammalian NB324K cells at a concentration of 2 x 107 cells/ml were electroporated as described previously (53, 54) with equal, non-saturating amounts (10 µg) of the infectious recombinant plasmid carrying the appropriate mutations, using in each experiment the non-mutated infectious plasmid as a control. Virions were recovered at 72 h from transfected monolayers and titrated in standard plaque-formation assays (55). The results were normalized by quantitation, using SDS-PAGE (10% polyacrylamide) and immunoblotting with anti-MVM antiserum, of the amount of viral capsid proteins present in extracts of the transfected cells (56).

Immunofluorescence assays were as described previously (56), with minor modifications. The primary antibodies used were: (i) a polyclonal antibody that recognizes the MVM capsid proteins (VPs) (52); (ii) a monoclonal antibody (B7) that recognizes a discontinuous epitope specific of the assembled capsid (57); and (iii) a polyclonal antibody elicited against the VP2 Nt that recognizes virions that have encapsidated the genome, and concomitantly externalized the VP2 Nt segment (56).

Purification of Virions and Empty Capsids—Natural infections with MVMp or transfection with its infectious recombinant plasmid yield a large proportion of empty capsids mixed with a minor proportion of infectious virions (49). Partially purified mixtures of empty capsids and infectious virions were obtained essentially as described (56, 58) by centrifugation of the clarified cell extracts in sucrose gradients, followed by extensive dialysis against phosphate-buffered saline (PBS). Empty capsids and infectious virions were purified by centrifugation in cesium chloride (CsCl) density gradients (see below), and dialyzed against PBS. The capsid and virion preparations were found essentially free of contaminants by SDS-PAGE, and the concentration was estimated by densitometry of stained gels and/or UV spectrophotometry (59).

Conformational Stability of MVM Empty Capsids and Virions Followed by Spectrofluorometry—A Varian Cary Eclipse luminescence spectrophotometer equipped with a computer-operated Peltier temperature control unit was used. Empty capsids or purified virions were added to a 2- x 10-mm cell and irradiated with UV light (excitation wavelength, 295 nm). The temperature was continuously increased from 25 °C to 85 °C (or 90 °C) at a constant rate of 1 °C/min, and the intrinsic tryptophan fluorescence at an emission wavelength of 330 nm was determined at 1-min intervals (59, 60). During some experiments small aliquots were taken for the determination of the infectious virus titers (see below). The changes in fluorescence were fitted to sigmoidal, cooperative transitions using the program KaleidaGraph (Abelbeck Software), which allowed a determination of the half-transition temperature (Tm) (59, 60).

Thermal Inactivation of MVM Virions Followed by Infectivity—Two procedures were used: (i) virion preparations were subjected to a thermal gradient as described above (see also 59, 60), and the infectious viruses remaining at different temperatures were determined by titration of aliquots taken from the heated sample. (ii) The kinetics of virus inactivation was determined as follows (61). Virus preparations were diluted in PBS to a concentration of about 1500 plaque-forming units/ml. 100-µl aliquots in thin-walled PCR tubes were incubated at a constant temperature (70 °C) for different amounts of time, and the remaining virus titers were determined in plaque assays. Non-mutated viral particles obtained in parallel transfection experiments were included in each heat-inactivation experiment as a positive control. The use of culture medium (Dulbecco's modified Eagle's medium) supplemented with fetal calf serum instead of PBS did not significantly affect the results.

Analysis of DNA Encapsidation—NB324K cells (1.2 x 107 cells) were transfected by electroporation with 40 µg of the purified MVM plasmids and cultured for 48 h in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The monolayers were washed in PBS and harvested in 20 mM Tris, pH 7.5, 2 mM EDTA, 0.2% SDS (TES buffer) and completely homogenized by freeze-thawing three times, and the cell extracts were used for purification of MVM particles as described (56). Approximately 20 fractions were collected from the CsCl gradients, and the amounts of VPs and of viral DNA were estimated by slot-dot (Hoeffer) analysis. Samples diluted ten times in PBS were bound under vacuum to nitrocellulose filters (Schleicher & Schüll) soaked in the same buffer and left at room temperature until dry. The amount of bound VP (capsid) was estimated immunochemically with a polyclonal anti-MVM capsid serum (52), using an enhanced chemiluminescence method (ECL, Amersham Biosciences). To estimate the amount of viral DNA, the samples bound to membranes were denatured in 0.4 N NaOH, 1.5 M NaCl for 5 min at room temperature, neutralized by incubation with 0.5 M Tris-HCl (pH 7.5), 1.5 M NaCl during 5 min, and bound to the membrane by heating at 80 °C for 2 h. Filters were hybridized under high stringency as described (51) using as a probe a 2.4-kpb EcoRI restriction fragment of the MVM genome (nucleotides 1105–3547 (62)) labeled by random priming with digoxigenin, and incubated with a solution of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate (Roche Applied Science).

Molecular Graphics and Structural Analyses—A Silicon Graphics workstation, the PDB coordinates of the immunosuppressive (i) strain of MVM (1mvm [PDB] (29, 30)) and of CPV (4dpv (27)) and the programs InsightII (Biosym Technologies), RasMol (63), and Whatif (64) were used. The capsid of the MVM prototypic strain (p) used in this study is 98% identical to the immunosuppressive (i) strain (62), with a few non-conserved capsid residues located in surface-exposed loops, far away from the DNA-capsid interface (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Selection for Mutational Analysis of Amino Acid Residues at the Parvovirus Nucleic Acid-Capsid Interface—Based on analyses of the three-dimensional structures of the MVMi and CPV virions (25–30), ten residues of the MVM capsid were chosen for mutagenesis to alanine (Table I and Fig. 1). Each mutation would disrupt any interaction of the truncated side chain (beyond C{beta}) with the DNA while minimizing the possibility of altering the conformation of the polypeptide backbone (65). Some of those side chains are additionally involved in interactions with neighboring amino acid residues, which would also be disrupted by the mutation; this has also been contemplated in our experimental approach (see below).



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  		FIG. 1.
Amino acid residues of the MVM virion chosen for mutational analysis. The amino acid residues are color-coded according to atom (green, C; blue, N; red, O) and labeled. The ordered ssDNA stretches A, B, and C (Table I) in the virion are shown in yellow, cyan, and violet, respectively. The PDB coordinates 1mvm [PDB] (29, 30) were used.

 
The interactions with the major ordered DNA stretch of residues Asn-183, Thr-267, and Asn-491 in the MVMi structural model are similar to those of the equivalent, conserved residues Asn-180, Thr-263, and Asn-492 in the refined CPV model. The sterically similar replacements of Asp-58 and Tyr-270 in MVM by Asn-56 and Phe-266 in CPV do not disrupt the major interactions of these residues with the DNA (Table I). Residues Trp-60 and Asn-491 in MVM appear to be involved in major interactions with two neighboring, shorter DNA stretches that could not be clearly visualized in the CPV structure (30). However, these residues are conserved in CPV (Trp-58 and Asn-492), and their interactions could also be conserved. Finally, residues Lys-278, Lys-471, Asp-273, and Asp-474 in MVM, and their equivalents Arg-274, Lys-472, Asp-269, and Asp-475 in CPV are either identical or similar, and all but Asp-269 of CPV contain the charged groups closest to visible DNA phosphates and could be involved in either attractive or repulsive electrostatic interactions with the DNA molecule. In brief, the ten residues of the MVM capsid chosen for mutation include many of those involved in major interactions with the viral nucleic acid, and most of those residues and their interactions with the DNA are conserved between MVM and CPV.

Several Residues Involved in DNA-Capsid Interactions Are Important for the Infectivity of MVMp—The chosen mutations were individually introduced in an infectious DNA clone of MVMp, susceptible mammalian cells were transfected with quantitated, equivalent amounts of the mutant infectious DNA clones and of the non-mutated control, and the virus yields at 72 h after transfection were determined by titration. The entire experiment was repeated using a new set of infectious DNA preparations that were quantitated and electroporated in a completely independent way. The relative efficiency of the transfection process was determined as described under "Materials and Methods." The normalized relative viral titers were not significantly different from those obtained in the first experiment. The results (Table II) revealed that the infectivity of mutants K278A or N491A was similar to that of the non-mutated control or only slightly reduced, that of mutants D58A, W60A, N183A, T267A, and K471A was clearly reduced by about one order of magnitude, and that of mutants Y270A, D273A, and D474A was drastically reduced by 3–4 orders of magnitude. The experiments described in the following paragraphs were carried out with the aim of identifying the step(s) in the viral life cycle that could be responsible for the reduced infectivity of those mutants.


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  		TABLE II
Effects of the truncation of capsid side chains involved in interactions with the nucleic acid in the MVM virion

 
Expression of VPs, Nuclear Transport, and Capsid Assembly—In situ immunofluorescence assays with cells transfected with equal amounts of plasmids were carried out. VP expression was detected at 48 h after transfection with an antibody that recognizes the VPs (even if non-assembled (56)). All of the mutants and the wild-type expressed similar amounts of capsid proteins (Fig. 2A and Table II). In addition, the presence of intense nuclear fluorescence (Fig. 2A and not shown) revealed that all of these mutant VPs were able to translocate into the nucleus.



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  		FIG. 2.
In situ immunofluorescence of mammalian cells transfected with non-mutated or with some mutant infectious plasmids of MVMp. A and B correspond to the results obtained in two different double-labeling experiments, using specific antibodies to detect expression of viral proteins (VPs), assembled capsids (Capsid), or externalization of the Nt of VP2 (2Nt) (see text); wt and mock indicate transfection with the non-mutated control plasmid or with no viral DNA; all other images correspond to transfection with the indicated mutant plasmid.

 
Capsid assembly within the nucleus was detected with monoclonal antibody B7 that recognizes a capsid-specific, discontinuous epitope located on the virion external surface, at the vertex of the 3-fold spikes (57), and far away from the residues mutated (which are located at the internal capsid surface, and closer to the 2-fold axes). All mutants except Y270A were able to assemble into capsids at levels comparable to the non-mutated control (Fig. 2A). Most of the Y270A-transfected cells that showed positive nuclear reactivity with the anti-VP antibody showed only a very weak reactivity with the anti-capsid antibody, relative to the non-mutated control (Fig. 2A). This result is consistent with previous observations of uncoupling between nuclear transport and capsid assembly (54), and on the nature of the subviral oligomers that translocate across the nuclear membrane.2 Extracts from cells transfected with mutant Y270A, either of two mutants (W60A and D273A) that were positive with the anti-capsid antibody, and the non-mutated control, were subjected to sedimentation in CsCl gradients, and the amount of MVM capsids produced was estimated by quantitation of the hemagglutination (HA) activity present in the density band corresponding to empty capsids. The non-mutated control, W60A, and D273A particles were produced at normal levels (>6400 HA units per 107 transfected cells), whereas Y270A yielded only 100–300 HA units per 107 transfected cells (not shown). This confirmed that mutant Y270A is defective in capsid assembly.

Externalization of the VP2 Nt and Packaging of the Viral DNA—The maturation within the nucleus of MVM virions with an encapsidating genome triggers the externalization of the Nt of VP2 (48), an event that is required for infectivity, as the externalized peptide segment allows the traffic of the particle outward of the nucleus (56). Thus, DNA-containing virions can be specifically detected in cells by in situ immunofluorescence with an antibody specific for epitopes located at the VP2 Nt (56). All mutants except D273A and Y270A reacted strongly with this antibody (Fig. 2B and Table II), indicating that they were able to efficiently externalize the VP2 Nt and to encapsidate the viral genome. As described above, Y270A yielded very low amounts of assembled capsids, and the reactivity with the anti-VP2 Nt antibody was proportionally reduced (Fig. 2B). Remarkably, the D273A mutant, which showed normal reactivity with the anti-VP and anti-capsid antibodies (Fig. 2A), showed an extremely reduced reactivity with the anti-VP2 Nt antibody (Fig. 2B).

It could be argued that mutation D273A may prevent the externalization of the VP2 Nt but not DNA encapsidation. We considered this possibility unlikely, because the two events have been correlated (48, 56), and the mutations are located far away from the pores at the capsid 5-fold axes where the Nt are externalized. However, to analyze this issue further, mutants D273A, Y270A, W60A, and the non-mutated control were subjected to density gradient centrifugation analysis. As shown in Fig. 3, for the non-mutated control and mutant W60A a large amount of viral DNA was detected in the fractions with a density corresponding to virions, whereas viral DNA was detected at low levels in the equivalent Y270A fractions and was barely detectable in the D273A fractions. This result is entirely consistent with that of the immunofluorescence analysis (Fig. 2) and indicates that Y270A maintains a DNA-encapsidation capacity proportional to the low amounts of capsid assembled, whereas D273A does form normal amounts of empty capsids but has a severely impaired ability to encapsidate the viral genome.



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  		FIG. 3.
Analysis of DNA encapsidation. Mammalian cells were transfected with infectious non mutated (wt) or mutant (W60A, Y270A, or D273A) plasmids of MVMp. The assembled empty capsids (buoyant density, 1.32 g/ml) and ssDNA-full virions (buoyant density, 1.39–1.41 g/ml) present in the cellular homogenates were separated according to density by CsCl centrifugation (1.38 g/ml average density). A, samples were obtained from 1.2 x 106 transfected cells; the amount of viral particles in the fractions corresponding to empty capsids (fractions 3–7), and virions (fractions 9–14) were estimated by slot-immunoblot assay. VLPs of MVMp, purified and quantitated as described (56), were used for calibration, and bovine serum albumin was used as a negative control. B, samples were obtained from 1 x 107 transfected cells; the amount of viral DNA in the fractions corresponding to full virions were estimated by DNA hybridization. Different amounts of the MVMp infectious plasmid, quantitated by absorbance at 259 nm and ethidium bromide staining of agarose gels, were used for calibration. Exposure was for 40 h using an intensifying screen.

 
Analysis of the Stability of the Empty Capsid—Most of the side chains involved in interactions with the viral DNA and analyzed in this study either do not interact with neighboring capsid residues, or establish with the latter a few van der Waals contacts only (Table III). However, the side chains of Tyr-270 and Trp-60 are each involved in multiple hydrophobic intrasubunit contacts and a hydrogen bond, and Lys-278 and Asp-474 interact with each other through an intersubunit salt bridge (Table II). Truncation of some of these side chains could potentially affect the stability of the viral particles, not by disruption of DNA-capsid interactions, but because of the disruption of intracapsid interactions. Thus, the contribution of the mutated residues to the stability of the protein shell was estimated using empty capsids, as follows.


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  		TABLE III
Intracapsid interactions in the MVMi virion of the amino acid side chains analyzed in this study

The analysis was carried out using the program Whatif (64) and the PDB file 1mvm [PDB] (29, 30). Intersubunit interactions were analyzed using pairs of symmetry-related subunits. Only the interactions of the side chain (beyond C{beta}) are considered. Cut-off distances and abbreviations are as in Table I. Additional abbreviations are: mc, main chain; sc, side chain; sb, salt bridge.

 
The thermal stability of purified natural empty capsids of MVMp (containing both VP2 and VP1) was determined first, by following the intrinsic fluorescence of the particles in controlled thermal gradients, as previously described for VP2-only empty capsids (virus-like particles or VLPs) of MVMp (59, 60). Superimposed with the linear decrease in fluorescence due to thermal quenching, two transitions were observed. The first transition yielded a Tm = 42 ± 3 °C (Fig. 4A), and the second transition a Tm = 76.0 ± 0.2 °C (Fig. 4B). Comparison with detailed analyses of the structurally very similar viral-like particles (VLPs) of MVMp (59) indicates that these two transitions correspond to a conformational rearrangement of the capsid associated with externalization of the VP2 Nt, and to capsid dissociation, respectively. Partially purified non-mutated empty capsids (containing a very small fraction of virions, which did not significantly contribute to the variation in the fluorescence signal) were then assayed as described above for purified empty capsids. The first transition occurred at a similar temperature, and the second transition, corresponding to dissociation of the capsid, yielded the same Tm, within error (Table II). This result validated the use of partially purified capsids to determine capsid stability in this type of assay. Finally, the thermostability of partially purified mutant empty capsids was compared with that of the non-mutated control, using the same assay. The eight mutant capsids tested showed evidence of a first conformational transition that occurred at a similar temperature in all cases, and dissociated with a Tm indistinguishable (within error) from that of the non-mutated control (Fig. 4C and Table II).



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  		FIG. 4.
Conformational change and dissociation of the MVMp empty capsid probed by intrinsic Trp fluorescence. A and B, relative fluorescence intensity (in arbitrary units, a.u.) emitted at 330 nm as a function of temperature. For reasons of scale and clarity, the complete curve obtained in the same experiment has been split into two parts corresponding to low (A) and high (B) temperature ranges, where the transition corresponding to a conformational change without dissociation, or the transition corresponding to capsid dissociation, respectively, occur (59). C, dissociation of non-mutated empty capsid (circles) and some mutants (triangles, T267A; inverted triangles, N491A; diamonds, D474A) followed by emission fluorescence, as indicated for B. a.u., arbitrary units. For clarity of representation, the curves have been vertically offset. The experimental data were fitted to sigmoidal transitions (continuous lines).

 
Analysis of the Conformational Stability of the Virion—The thermal stability of the non-mutated MVMp virion at neutral pH was analyzed first. The purified virions were exposed to a controlled thermal gradient, and the variation in their intrinsic fluorescence was determined. The virions exhibited a small, but detectable and reproducible transition at a temperature of about 54 °C (Fig. 5A). This transition did not correspond to the conformational transition observed for VLPs (59) or empty capsids at a substantially lower temperature (see above), which is consistent with the fact that in the virions the VP2 Nt is already externalized (48, 56). Instead, the transition detected in the virion may be related to the externalization of the VP1 Nt, which has been shown to occur at a similar temperature (66). The transition corresponding to dissociation of the virion (Fig. 5B) (66) yielded a Tm of 77 °C, which is very similar to that found for VLPs or empty capsids using the same approach (59). Also, no significant difference in stability between DNA-full virions and empty capsids was found when the hemagglutination activity of heated purified particles was compared at neutral pH.3



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  		FIG. 5.
Conformational stability of MVMp virion. A and B, relative fluorescence intensity (in arbitrary units, a.u.) emitted at 330 nm by purified MVMp virions as a function of temperature at neutral pH (pH 7). For reasons of scale and clarity, the complete curve obtained in the same experiment has been split into two parts corresponding to low (A) and high (B) temperature ranges. The experimental data were fitted to sigmoidal transitions (continuous lines). C, relative infectivity of purified MVMp virions as a function of temperature at pH 7. The titer at each temperature was obtained using several dilutions of the sample. No infectious virions were detected at the three highest temperatures tested. The experiment was repeated, and similar results were obtained.

 

Even though the virion particle is disrupted only at high temperatures, its infectivity may be slightly reduced before capsid dissociation occurs (66, 67). Thus, during the thermal gradient experiments using purified non-mutated virions, aliquots were taken at different temperatures, and the remaining infectivity was determined in plaque assays. The results suggest that infectivity may be lost in two steps (Fig. 5C). The first step involved a moderate (~5-fold) reduction in infectivity at about 50–55 °C, a temperature that was approximately coincident with that of the first transition observed by fluorescence (see above). The second step involved a drastic reduction of infectivity (several orders of magnitude) that started at about 70 °C and led to a nearly complete loss of infectivity at about 80 °C, temperatures that corresponded approximately to those of dissociation of the virion, empty capsids, and VLPs (see above and Refs. 59 and 66).

The small virion:empty capsid ratio obtained from infections of cell lines with MVM (49), and the reduced infectivity of most mutants analyzed, made it difficult to obtain the relatively large amounts of each of the purified mutant virions that are needed for the above analysis. However, the conformational stability of mutant infectious virions could be compared by following the reduction of infectivity in thermal inactivation kinetic assays, which required only a small amount of partially purified virions (61). The results of these assays are shown in Fig. 6 for some representative variants and summarized in Table II for all of the mutants analyzed. The stability of mutants D273A, Y270A and, possibly, N491A was not substantially different to that of the non-mutated control. In contrast, the stability of most mutants, namely D58A, W60A, N183A, T267A, K278A, K471A, and D474A was clearly reduced (Table II). To summarize, no mutation tested affected the thermal stability of the empty capsid, but most of them affected the thermal sensitivity of the infectious virion.



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  		FIG. 6.
Thermal inactivation of non-mutated and two representative mutant infectious virions of MVMp as a function of time. Aliquots of the virus preparations were incubated at 70 °C for the periods of time indicated, and the titers determined were normalized using the titer obtained in the absence of heating (t = 0). Black bars, non-mutated virion; gray bars, mutant K471A; white bars, mutant N183A. Titers were determined in triplicate. The standard deviation was in each case about 15% of the average, except for t = 99 min. In the latter case the number of plaque-forming units was very low, leading to somewhat larger errors.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Two aspects related to the experimental approach used in this work may require some discussion. First, the error in atomic positions in the MVM crystal structure (29, 30) in the absence of a complete refinement, and the necessary averaging of the true DNA sequences bound to symmetry-related sites in the capsid, introduce some uncertainty in the amino acid residues and interactions that participate in the DNA-capsid interface as defined in Table I. However, several evidences indicate that major DNA-capsid interactions (hydrogen bonds and multiple hydrophobic contacts), which involve the capsid residues analyzed in this study do occur in the MVM virion: (i) the conformations of the major visible DNA stretch in the MVMi and the CPV models are extremely similar (25, 26, 30); (ii) for many of the visible nucleotides the electron density is distinctive for purine or pyrimidine, and in some cases for individual base type (26, 30); (iii) a complete refinement of the CPV structure did not change in essence the major DNA-capsid interactions observed or the residues involved (25–27); (iv) the capsid residues involved in most interactions with ordered DNA are either conserved or conservatively substituted in both viruses (50); and (v) most of the interactions between these residues and the DNA are similar in both the MVMi and the refined CPV structural models (26, 30).

Second, the use of empty capsids to analyze the contribution to virion stability of interactions between capsid amino acid residues assumes that these interactions are similar in both types of viral particles. The structure of the CPV (68) or the MVM capsid (29, 58) is nearly identical in both the empty particle and in the DNA-full virion. For CPV, a comparison of the refined structures of the virion and the empty capsid showed some local differences in the conformation of several residues in the DNA-binding region (68). However, a detailed contact analysis using the same structural models indicated that the residues equivalent to those of MVM we subjected to mutational analysis are involved in similar interactions in both the virion and the empty capsid: The side chains of Tyr-270 and Trp-60 in MVM, and the equivalent Phe-266 and Trp-58 in CPV, participate in multiple hydrophobic contacts with neighboring amino acid residues in the virion; in the empty capsid of CPV, nearly all of these contacts are preserved (further contacts were observed). Lys-278 and Asp-474 in MVM and the equivalent Arg-274 and Asp-475 in CPV are linked in the virion by an intersubunit salt bridge, and this salt bridge is also preserved in the empty capsid of CPV.

The results obtained, together with the considerations just discussed, allow us to propose defined structural roles for most of the residues that were found to be involved in major interactions with the viral DNA, which can explain the observed functional involvement of these residues in the virus life cycle.

Residues with Moderate Effects on Infectivity—Truncation to Ala of Asp-58, Trp-60, Asn-183, Thr-267, Lys-278, Lys-471, or Asn-491 did not significantly affect capsid protein expression, assembly into capsids inside the nucleus, or externalization of the VP2 Nt. This latter fact indicates that those mutants are also able to encapsidate the DNA, as confirmed by direct encapsidation analysis using mutant W60A. The side chains of Trp-60 and Lys-278, but not the others, not only may interact with the DNA, but are also involved in multiple interactions with other capsid residues as well. However, none of those seven mutations had any significant effect on the stability of the capsid itself. In contrast, all of those mutations (with the possible exception of N491A, which had a lesser inhibitory effect on infectivity) caused a substantial reduction in the thermal sensitivity of the infectious virion. These results indicate that the interactions of the mutated residues with the DNA, and not the interactions with other capsid residues, have a role in preserving the virion conformation needed for the infectivity of the virus particle. The aromatic ring of Trp-60 is oriented perpendicularly to the ring of a neighboring purine base, which allows extensive van der Waals contacts and favorable dipole interactions. Asp-58, and Asn-183 and Asn-491, may be hydrogen-bonded, respectively, to a purine base and to the sugar-phosphate backbone. Thr-267 is involved in several van der Waals contacts. Lys-278 and Lys-471 showed no contacts with the DNA, but they were relatively close to DNA phosphates, and could contribute to medium-range electrostatic interactions.

The results also showed that, even though no neutralization of the charge of most DNA phosphates has been detected, the thermal stability of the MVM virion is not reduced relative to that of the empty capsid at neutral pH. We propose that the manyfold repeated hydrophobic and electrostatic interactions between the viral DNA and Asp-58, Trp-60, Asn-183, Thr-267, Lys-278, and Lys-471, and perhaps other residues involved in capsid protein-viral DNA interactions (Table I) that have not been analyzed in the present study, may help to counteract the potential destabilization caused by repulsion between non-neutralized DNA phosphates in the virion. Truncation of the side chain of any of these residues except Lys-278 led to a reduction of infectivity by about one order of magnitude. The individual contribution to virion stability and infectivity of each of those few residues involved in major interactions between the viral DNA and each capsid subunit is, thus, relatively moderate. However, their combined effect, if additive as expected, could be dramatically important.

Mutation K278A also reduced virion stability, but not infectivity. One possibility to explain this anomalous result (under study) is that the reduction in stability could be compensated by another, favorable effect of the same mutation on another step of the viral cycle.

Residues with Drastic Effects on Infectivity—Truncation of Tyr-270, Asp-273, and Asp-474 did not affect capsid protein expression or nuclear transport. However, contrary to the mutations discussed above, for these three mutants the reduction of infectivity was dramatic, and it does not appear to be caused by a reduction in the thermal stability of the infectious virion.

Mutant Y270A is clearly defective in capsid assembly. Tyr-270 is involved in multiple interactions not only with the DNA, but also with neighboring capsid residues. Thus, the most likely explanation for the inefficient assembly of this mutant, and the dramatic reduction in viral yield, is the removal of some critical intracapsid interactions.

The D474A mutation did not prevent capsid assembly, and capsid stability was not reduced either. The D474A virion did show a reduced stability, but similar or higher reductions in stability were observed for many mutants whose infectivity (viral yield) was reduced by only 10-fold. Thus, the drastic reduction in infectivity of D474A may be related mainly to another, as yet unidentified step in the viral cycle.

Finally, the D273A mutation did not prevent capsid assembly, but it severely impaired the externalization of the Nt of VP2 and the encapsidation of the viral DNA, which explains its drastic effect on the infectivity. Asp-273 is very close to a DNA phosphate, but not to any acidic or basic amino acid residue (the closest basic residue is Lys-494 at a distance of 4.7 Å), and it makes no visible contacts with the DNA or with other amino acid residues. One possibility is that the charge distribution at the capsid internal surface, which may be significantly altered if 60 negative charges are removed by the D273A mutation, may be critical for DNA packaging. In addition, the D273A phenotype provides further support to the previous suggestion that encapsidation of the MVM genome occurs in empty capsids previously assembled inside the cell nucleus (56).

To conclude, the results found with this spherical virus model indicate that several amino acid residues per capsid subunit, which are involved in interactions with the viral nucleic acid molecule, are important for virus infectivity because of their role in preserving the integrity of the infectious virus particle. Because most of these residues are not involved in substantial intracapsid interactions, and none of them contributes to the stability of the capsid itself, their effect may be directly mediated by the multiple non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.


   FOOTNOTES
 
* This work was supported by independent grants from Ministerio de Ciencia y Tecnología and Comunidad Autónoma de Madrid (to the laboratories of M. G. M. (Grants BIO2003-04445 and 07B/0012/2002) and J. M. A. (Grants SAF2001-1325 and 07B/0020/2002)), by EU contract QLK3-CT-2001-01010 (to J. M. A.), and by an institutional grant from Fundación Ramón Areces. 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

{ddagger} To whom correspondence should be addressed: Tel.: 34-91-4978462; Fax: 34-91-4974799; E-mail: mgarcia{at}cbm.uam.es.

1 The abbreviations used are: ss, single-stranded; CPV, canine parvovirus; Nt, N-terminal; MVM, minute virus of mice; MVMi, MVMp, MVM strains i or p, respectively; HA, hemagglutination; PBS, phosphate-buffered saline; VLP, virus-like particle; VP, viral capsid protein; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. Back

2 L. Riolobos, J. Reguera, M. G. Mateu, and J. M. Almendral, manuscript in preparation. Back

3 N. Valle and J. M. Almendral, unpublished results. Back


   ACKNOWLEDGMENTS
 
We acknowledge A. López-Bueno, E. Hernando, and B. Maroto for antibody preparations; P. Tattersall for the DNA clone pMM984; C. R. Parrish for hybridoma B7; and A. L. Llamas-Saiz and N. Hodnett for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stubbs, G. (1989) in Protein-Nucleic Acid Interactions (Saenger, W., and Heinemann, U., eds) pp. 87–109, CRC Press, Boca Raton
  2. Liljas, L. (1986) Prog. Biophys. Mol. Biol. 48, 1–36[CrossRef][Medline] [Order article via Infotrieve]
  3. Rossmann, M. G., and Johnson, J. E. (1989) Annu. Rev. Biochem. 58, 533–573[CrossRef][Medline] [Order article via Infotrieve]
  4. Casjens, S. (1997) in Structural Biology of Viruses (Chiu, W., Burnett, R. M., and Garcea, R. L., eds) pp. 3–37, Oxford University Press, NY
  5. Johnson, J. E., and Speir, J. A. (1999) in Encyclopedia of Virology, 2nd Ed. (Granoff, A., and Webster, R., eds) pp. 1946–1956, Academic Press, London
  6. Chapman, M. S., and Liljas, L. (2003) Adv. Prot. Chem. 64, 125–196[Medline] [Order article via Infotrieve]
  7. Stockley, P. G. (1992) Curr. Opin. Struct. Biol. 2, 143–149
  8. Johnson, J. E., and Rueckert, R. R. (1997) in Structural Biology of Viruses (Chiu, W., Burnett, R. M., and Garcea, R. L., eds) pp. 269–287, Oxford University Press, NY
  9. Jones, S., Daley, D. T. A., Luscombe, N. M., Berman, H. M., and Thornton, J. M. (2001) Nucleic Acids Res. 29, 943–954[Abstract/Free Full Text]
  10. Bink, H. H. J., and Pleij, C. W. A. (2002) Arch. Virol. 147, 2261–2279[Medline] [Order article via Infotrieve]
  11. Tihova, M., Dryden, K. A., Le, T.-V., Harvey, S. C., Johnson, J. E., Yeager, M., and Schneemann, A. (2004) J. Virol. 78, 2897–2905[Abstract/Free Full Text]
  12. Vriend, G., Verduin, B. J. M., and Hemminga, M. A. (1986) J. Mol. Biol. 191, 453–460[CrossRef][Medline] [Order article via Infotrieve]
  13. Sacher, R., and Ahlquist, P. (1989) J. Virol. 63, 4545–4552[Abstract/Free Full Text]
  14. Larson, S. B., Koszelak, S., Day, J., Greenwood, A., Dodds, J. A., and McPherson, A. (1993) Nature 361, 179–182[CrossRef][Medline] [Order article via Infotrieve]
  15. Larson, S. B., Day, J., Greenwood, A., and McPherson, A. (1998) J. Mol. Biol. 277, 37–59[CrossRef][Medline] [Order article via Infotrieve]
  16. Timmins, P. A., Wild, D., and Witz, J. (1994) Structure 2, 1191–1201[Medline] [Order article via Infotrieve]
  17. Speir, J. A., Munshi, S., Wang, G., Baker, T. S., and Johnson, J. E. (1995) Structure 3, 63–78[Medline] [Order article via Infotrieve]
  18. Fisher, A. J., and Johnson, J. E. (1993) Nature 361, 176–179[CrossRef][Medline] [Order article via Infotrieve]
  19. Wery, J.-P., Reddy, V. A., Hosur, M. V., and Johnson, J. E. (1994) J. Mol. Biol. 235, 565–586[CrossRef][Medline] [Order article via Infotrieve]
  20. Tang, L., Johnson, K. N., Ball, L. A., Lin, T., Yeager, M., and Johnson, J. E. (2001) Nat. Struct. Biol. 8, 77–83[CrossRef][Medline] [Order article via Infotrieve]
  21. McKenna, R., Xia, D., Willingmann, P., Ilag, L. L., Krishnaswamy, S., Rossmann, M. G., Olson, N. H., Baker, T. S., and Incardona, N. L. (1992) Nature 355, 137–143[CrossRef][Medline] [Order article via Infotrieve]
  22. McKenna, R., Ilag, L., and Rossmann, M. G. (1994) J. Mol. Biol. 237, 517–543[CrossRef][Medline] [Order article via Infotrieve]
  23. Bernal, R. A., Hafenstein, S., Esmeralda, R., Fane, B. A., and Rossmann, M. G. (2004) J. Mol. Biol. 337, 1109–1122[Medline] [Order article via Infotrieve]
  24. Chen, Z., Stauffacher, C., Li, Y., Schmidt, T., Bomu, W., Kamer, G., Shanks, M., Lomonossoff, G., and Johnson, J. E. (1989) Science 245, 154–159[Abstract/Free Full Text]
  25. Tsao, J., Chapman, M. S., Agbandje, M., Keller, W., Smith, K., Wu, H., Luo, M., Smith, T. J., Rossmann, M. G., Compans, R. W., and Parrish, C. R. (1991) Science 251, 1456–1464[Abstract/Free Full Text]
  26. Chapman, M. S., and Rossmann, M. G. (1995) Structure 3, 151–162[Medline] [Order article via Infotrieve]
  27. Chapman, M. S., and Rossmann, M. G. (1996) Acta Crystallogr. Sect. D 52, 129–142[Medline] [Order article via Infotrieve]
  28. Xie, Q., and Chapman, M. S. (1996) J. Mol. Biol. 264, 497–520[CrossRef][Medline] [Order article via Infotrieve]
  29. Llamas-Saiz, A. L., Agbandje-McKenna, M., Wikoff, W. R., Bratton, J., Tattersall, P., and Rossmann, M. G. (1997) Acta Crystallogr. Sect. D 53, 93–102[CrossRef][Medline] [Order article via Infotrieve]
  30. Agbandje-McKenna, M., Llamas-Saiz, A. L., Wang, F., Tattersall, P., and Rossmann, M. G. (1998) Structure 6, 1369–1381[Medline] [Order article via Infotrieve]
  31. Valegard, K., Murray, J. B., Stockley, P. G., Stonehouse, N. J., and Liljas, L. (1994) Nature 371, 623–626[CrossRef][Medline] [Order article via Infotrieve]
  32. Valegard, K., Murray, J. B., Stonehouse, N. J., van der Worm, S., Stockley, P. G., and Liljas, L. (1997) J. Mol. Biol. 270, 724–738[CrossRef][Medline] [Order article via Infotrieve]
  33. Whiterell, G. W., Gott, J. M., and Uhlenbeck, O. C. (1991) Progr. Nucleic Acids Res. Mol. Biol. 40, 185–220[Medline] [Order article via Infotrieve]
  34. Peabody, D. S. (1993) EMBO J. 12, 595–600[Medline] [Order article via Infotrieve]
  35. Johansson, H. E., Dertinger, D., LeCuyer, K. A., Behlen, L. S., Greef, C. H., and Uhlenbeck, O. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9244–9249[Abstract/Free Full Text]
  36. van der Worm, S. H. E., Stonehouse, N. J., Valegard, K., Murray, J. B., Walton, C., Fridborg, K., Stockley, P. G., and Liljas, L. (1998) Nucleic Acids Res. 26, 1345–1351[Abstract/Free Full Text]
  37. Dertinger, D., Dale, T., and Uhlenbeck, O. C. (2001) J. Mol. Biol. 314, 649–654[Medline] [Order article via Infotrieve]
  38. Hohn, T. (1969) J. Mol. Biol. 43, 191–200[Medline] [Order article via Infotrieve]
  39. Virudachalam, R., Harrington, M. A., and Markley, J. L. (1985) Virology 146, 138–140[Medline] [Order article via Infotrieve]
  40. Li, T., Chen, Z., Johnson, J. E., and Thomas, G. J., Jr. (1992) Biochemistry 31, 6673–6682[CrossRef][Medline] [Order article via Infotrieve]
  41. Ilag, L. L., Tuech, J. K., Beisner, L. A., Sumrada, R. A., and Incardona, N. L. (1993) J. Mol. Biol. 229, 671–684[Medline] [Order article via Infotrieve]
  42. DaPoian, A. T., Johnson, J. E., and Silva, J. L. (1994) Biochemistry 33, 8339–8346[CrossRef][Medline] [Order article via Infotrieve]
  43. Curry, S., Abrams, C., Fry, E., Crowther, J. C., Belsham, G. J., Stuart, D. I., and King, A. M. Q. (1995) J. Virol. 69, 430–438[Abstract]
  44. Dong, X. F., Natarajan, P., Tihova, M., Johnson, J. E., and Schneemann, A. (1998) J. Virol. 72, 6024–6033[Abstract/Free Full Text]
  45. DaPoian, A. T., Johnson, J. E., and Silva, J. L. (2002) J. Biol. Chem. 277, 47596–47602[Abstract/Free Full Text]
  46. Hafenstein, S., and Fane, B. A. (2002) J. Virol. 76, 5350–5356[Abstract/Free Full Text]
  47. Lima, S. M. B., Peabody, D. S., Silva, J. L., and de Oliveira, A. C. (2004) Eur. J. Biochem. 271, 135–145[Medline] [Order article via Infotrieve]
  48. Tattersall, P., Shatkin, A. J., and Ward, D. C. (1977) J. Mol. Biol. 111, 375–394[CrossRef][Medline] [Order article via Infotrieve]
  49. Cotmore, S. F., and Tattersall, P. (1987) Adv. Virus. Res. 33, 91–174[Medline] [Order article via Infotrieve]
  50. Chapman, M. S., and Rosmann, M. G. (1993) Virology 194, 491–508[CrossRef][Medline] [Order article via Infotrieve]
  51. Gardiner, E. M., and Tattersall, P. (1988) J. Virol. 62, 2605–2613[Abstract/Free Full Text]
  52. Ramírez, J. C., Santarén, J. F., and Almendral, J. M. (1995) Virology 206, 57–68[CrossRef][Medline] [Order article via Infotrieve]
  53. Lombardo, E., Ramírez, J. C., Agbandje-McKenna, M., and Almendral, J. M. (2000) J. Virol. 74, 3804–3814[Abstract/Free Full Text]
  54. Lombardo, E., Ramírez, J. C., García, J., and Almendral, J. M. (2002) J. Virol. 76, 7049–7059[Abstract/Free Full Text]
  55. Tattersall, P., and Bratton, J. (1983) J. Virol. 46, 944–955[Abstract/Free Full Text]
  56. Maroto, B., Valle, N., Saffrich, R., and Almendral, J. M. (2004) J. Virol. 78, 10685–10694[Abstract/Free Full Text]
  57. López-Bueno, A., Mateu, M. G., and Almendral, J. M. (2003) J. Virol. 77, 2701–2708[Abstract/Free Full Text]
  58. Hernando, E., Llamas-Saiz, A. L., Foces-Foces, C., McKenna, R., Portman, I., Agbandje-McKenna, M., and Almendral, J. M. (2000) Virology 267, 299–309[CrossRef][Medline] [Order article via Infotrieve]
  59. Carreira, A., Menéndez, M., Reguera, J., Almendral, J. M., and Mateu, M. G. (2004) J. Biol. Chem. 279, 6517–6525[Abstract/Free Full Text]
  60. Reguera, J., Carreira, A., Riolobos, L., Almendral, J. M., and Mateu, M. G. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2724–2729[Abstract/Free Full Text]
  61. Mateo, R., Díaz, A., Baranowski, E., and Mateu, M. G. (2003) J. Biol. Chem. 278, 41019–41027[Abstract/Free Full Text]
  62. Astell, C. R., Gardiner, M. E., and Tattersall, P. (1986) J. Virol. 57, 656–669[Abstract/Free Full Text]
  63. Sayle, R. A., and Milner-White, E. J. (1995) Trends Biochem. Sci. 20, 374–376[CrossRef][Medline] [Order article via Infotrieve]
  64. Vriend, G. (1990) J. Mol. Graph. 8, 52–56[CrossRef][Medline] [Order article via Infotrieve]
  65. Cunningham, B. C., and Wells, J. A. (1989) Science 244, 1081–1085[Abstract/Free Full Text]
  66. Cotmore, S. F., D'Abramo, A. M., Jr., Ticknor, C. M., and Tattersall, P. (1999) Virology 254, 169–181[CrossRef][Medline] [Order article via Infotrieve]
  67. Farr, G. A., and Tattersall, P. (2004) Virology 323, 243–256[CrossRef][Medline] [Order article via Infotrieve]
  68. Wu, H., and Rossmann, M. G. (1993) J. Mol. Biol. 233, 231–244[CrossRef][Medline] [Order article via Infotrieve]

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