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Department of Molecular and Cell Biology, University of California, Berkeley, California, USADepartment of Plant and Microbial Biology, University of California, Berkeley, California, USAHoward Hughes Medical Institute, University of California, Berkeley, California, USA
Few human pathogens have been the focus of as much concentrated worldwide attention as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19. Its emergence into the human population and ensuing pandemic came on the heels of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), two other highly pathogenic coronavirus spillovers, which collectively have reshaped our view of a virus family previously associated primarily with the common cold. It has placed intense pressure on the collective scientific community to develop therapeutics and vaccines, whose engineering relies on a detailed understanding of coronavirus biology. Here, we present the molecular virology of coronavirus infection, including its entry into cells, its remarkably sophisticated gene expression and replication mechanisms, its extensive remodeling of the intracellular environment, and its multifaceted immune evasion strategies. We highlight aspects of the viral life cycle that may be amenable to antiviral targeting as well as key features of its biology that await discovery.
The Coronaviridae family of viruses are enveloped, single-stranded positive-sense RNA viruses grouped into four genera (alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus) that primarily infect birds and mammals, including humans and bats. The seven coronaviruses known to infect humans fall within the alpha- and betacoronavirus genera, whereas gamma- and deltacoronaviruses primarily infect birds. Coronaviruses have been studied for decades using the model betacoronavirus, murine hepatitis virus (MHV), and the human alphacoronavirus HCoV-229E. In humans, the circulating coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 generally cause mild upper respiratory illness and collectively are associated with 10–30% of common cold cases (
). However, within the past two decades, three highly pathogenic coronaviruses have emerged into the human population as the result of spillover events from wildlife that can cause severe respiratory illness: severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2011, and most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019. These outbreaks, together with estimates suggesting that hundreds to thousands of additional coronaviruses may reside in bats alone (
), highlight the potential for future coronavirus zoonotic transmission.
In this article, we provide an overview of the coronavirus life cycle with an eye toward its notable molecular features and potential targets for therapeutic interventions (Fig. 1). Much of the information presented is derived from studies of the betacoronaviruses MHV, SARS-CoV, and MERS-CoV, with a rapidly expanding number of reports on SARS-CoV-2. The first portion of the review focuses on the molecular basis of coronavirus entry and its replication cycle. We highlight several notable properties, such as the sophisticated viral gene expression and replication strategies that enable maintenance of a remarkably large, single-stranded, positive-sense (+) RNA genome and the extensive remodeling of cellular membranes to form specialized viral replication and assembly compartments. The second portion explores the mechanisms by which these viruses manipulate the host cell environment during infection including diverse alterations to host gene expression and immune response pathways. This article is intended as a more in-depth companion piece to our online “Coronavirus 101” lecture (https://youtu.be/8_bOhZd6ieM).
Part I: The viral life cycle
Coronavirus particles consist of a ∼30-kb strand of positive-sense RNA that forms the genome; this genome is coated with nucleocapsid (N) protein and enclosed in a lipid bilayer containing three membrane proteins: spike (S), membrane (M), and envelope (E) (
). For all studied coronaviruses, the M protein is critical for incorporating essential viral components into new virions during morphogenesis, and N protein associates with the viral genome and M to direct genome packaging into new viral particles. The E protein forms an ion channel in the viral membrane and participates in viral assembly. The S protein is required for viral entry, as it binds to the target cell and initiates fusion with the host cell membrane (reviewed in Ref.
). S is homotrimeric, with each subunit consisting of two domains, S1 and S2. S1 contains the receptor-binding domain (RBD) and engages with the host receptor, whereas S2 mediates subsequent membrane fusion to enable the virus to enter the host cytoplasm. Activation of the S protein fusion activity requires prior proteolytic cleavage at two sites. The first cleavage site is at the S1/S2 boundary, leading to structural changes in the S2 domain that place it in a prefusion conformation. This cleavage event also separates S2 from S1, although the two domains remain noncovalently associated. The second cleavage site is at S2′, which drives fusion of the viral and cellular membranes to enable release of the N-coated RNA genome into the cytoplasm.
Whereas coronaviruses use the above general strategy to enter target cells, the receptors and proteases used as well as subcellular sites of S cleavage differ depending on the virus (reviewed in Ref.
). After ACE2 receptor binding, SARS-CoV and SARS-CoV-2 S proteins are subsequently cleaved and activated by the host cell-surface protease TMPRSS2 at the S1/S2 and S2′ sites, leading to membrane fusion (
The extent to which specific coronaviruses fuse at the plasma membrane versus during endocytosis remains incompletely resolved. In the cases of SARS-CoV, MERS-CoV, and MHV, the involvement of endosomal and lysosomal proteases in cleavage of their S proteins suggests that entry can occur during endocytosis. MHV enters predominantly through clathrin-mediated endocytosis and fusion with lysosomal membranes, as lysosomal proteases activate the S protein (
). For SARS-CoV and MERS-CoV, both the endocytic and direct membrane fusion pathways may be used for entry. Studies in which components of endocytosis and endosomal proteases have been blocked demonstrate that SARS-CoV and MERS-CoV can exploit the endocytic pathway to enter target cells (
Glycopeptide antibiotics potently inhibit cathepsin L in the late endosome/lysosome and block the entry of Ebola virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus (SARS-CoV).
). For these viruses, it is likely that the producer and target cell type influence which pathway they use for viral entry. For instance, when MERS-CoV S is precleaved in the producer cell, it gets activated by cell-surface proteases and enters the target cell by direct membrane fusion (
). In contrast, when MERS-CoV S is uncleaved in the producer cell, it enters the target cell through endocytosis and is instead activated by endosomal cathepsins. MERS-CoV with S that has not been precleaved during morphogenesis is incapable of infecting target cell types that have low expression of cathepsins. There are reports demonstrating that inhibition of endosomal cathepsins reduces the efficiency of SARS-CoV-2 entry, suggesting that this virus also exploits endocytosis as another route of entry in addition to direct membrane fusion (
). However, there is a unique salt-bridge interaction present between SARS-CoV-2 S and ACE2, and this may contribute to the enhanced binding affinity. Furthermore, the S1/S2 site in SARS-CoV-2 S contains an insertion of polybasic residues (
). This precleavage event is analogous to the processing of MERS-CoV S and MHV S, both of which also contain a furin cleavage site at S1/S2. A precleavage event at the S1/S2 site implies that SARS-CoV-2 S may only require cleavage at the S2′ site on the target cell surface, which would potentiate the membrane fusion process. Notably, acquisition of polybasic cleavage sites occurs during experimental selection for increased transmissibility and expanded tropism in other viruses, suggesting that it may have played a role in the bat-to-human spillover of SARS-CoV-2 (
). Further investigation into the properties of S protein from SARS-CoV-2 and other closely related viruses may provide insight into the origin of SARS-CoV-2 as well as the mechanism behind its high transmissibility.
Numerous therapeutic strategies are being explored to inhibit SARS-CoV-2 entry, including blocking ACE2 engagement, inactivating host proteases, and inhibiting S2-mediated membrane fusion. Neutralizing antibodies against SARS-CoV S display moderate efficacy in blocking SARS-CoV-2 infection due to significant differences in the epitope region (
). A recent study isolated neutralizing antibodies capable of blocking the interaction between S and ACE2 from convalescent SARS-CoV-2 patients and demonstrated that they effectively reduce viral load in a mouse model, garnering optimism about the possible use of neutralizing antibodies for treatment (
). Not surprisingly, generating protective immunity against the S protein has been the major focus of SARS-CoV-2 vaccine efforts. S protein–directed vaccine platforms under development include production of recombinant S protein, use of nonpathogenic viral vectors to direct expression of S, and nucleic acid–based vaccines in which sequence encoding the S protein is delivered as an mRNA or on a DNA backbone (
). Given the constraints of eukaryotic translation, which generally allow one protein to be translated per mRNA with ribosome scanning beginning near the 5′ end, it is worth pausing to consider how this number of viral proteins can be synthesized from the genome with a single ribosome entry site. Coronaviruses achieve this feat through the use of large, multiprotein fusions (termed polyproteins, described below) that are subsequently processed into individual proteins (
), as well as through synthesis of sub-genome-length mRNAs using an unusual transcription mechanism (discussed in the subsequent section).
All of the viral nonstructural proteins (nsps) are encoded in two open reading frames (ORF1a and -b) that encompass roughly the first two-thirds of the viral genome (Fig. 3). ORF1a/b is translated from the 5′-capped RNA genome by cap-dependent translation to produce a shorter polyprotein (the ∼440–500-kDa pp1a, which includes nsps 1-11) or a longer polyprotein (the ∼740–810-kDa pp1ab, which includes nsp1 to -16), depending on whether the stop codon at the end of ORF1a is recognized or bypassed. Bypassing the ORF1a stop codon occurs through a −1 ribosomal frameshift in the overlapping region between ORF1a and -1b just upstream of the stop codon, enabling production of the larger pp1ab polyprotein. Frameshifting occurs with ∼20–50% efficiency (
The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.
). Whereas nsp1 to -11 from ORF1a are involved in a broad range of functions from blocking the initial immune response to functioning as cofactors for replication and transcription proteins, the core components of the replication and transcription machinery, such as the RNA-dependent RNA polymerase (RdRp), helicase, and other RNA-modifying enzymes, are present in the ORF1b portion of pp1ab. This frameshifting-based translational control strategy helps the virus maintain a stoichiometry of pp1a and pp1ab proteins that is optimal for infectivity and replication (
To liberate the individual nsps, pp1a and pp1ab are proteolytically processed in cis and in trans by two viral proteases encoded by nsp3 and nsp5. Nsp3 contains one or two papain-like proteases (PLpro1 and PLpro2), and nsp5 contains a chymotrypsin-like cysteine protease (3CLpro) (reviewed in Ref.
). The 3CLpro catalyzes the proteolytic cleavage of all nsps downstream of nsp4 and is thus referred to as the main protease. Inhibitors of 3CLpro and PLpro have long been considered as potential drug targets, as their cleavage recognition sequences are distinct from other human proteases and they are essential to viral replication (
). Although PLpro is responsible for fewer cleavage events in pp1a, it additionally functions as a deubiquitinase and deISGylating (removal of conjugated interferon-stimulated gene 15 from cellular proteins) enzyme (
A subset of nsps generated by proteolytic cleavage of the polyproteins come together to form the replication and transcription complexes (RTCs) that copy and transcribe the genome. RTCs reside in convoluted membrane structures (discussed in detail below) derived from rough endoplasmic reticulum (ER) and are anchored in place by viral transmembrane proteins nsp3, nsp4, and nsp6 (
). Similar to other positive-strand RNA viruses, replication of coronaviruses involves synthesis of the complementary full-length negative-strand RNA, which serves as a template for generation of positive-strand progeny genomes (
). These include conserved stem loop structures within ∼500 nucleotides of the 5′ end of the genome, structural elements in the 3′ UTR that are partially conserved across the different coronaviruses, and the 3′ poly(A) tail. Negative-strand synthesis is facilitated by the N protein interacting with both the poly(A) tail and the 5′ end of the genome to bring these termini in proximity (
In addition to genomic replication, the RTCs also carry out synthesis of subgenomic (sg RNA) mRNAs, which encode for the ORFs located in the 3′-proximal one-third of the genome. All sg mRNAs are co-terminal and contain a common 5′ leader sequence that is derived from the 5′ end of the viral genome (
). During negative-strand synthesis, the RdRp complex terminates or pauses at specific sites along the genome called transcription regulatory sequences (TRSs). The TRSs are present downstream of the common leader sequence at the 5′ end of the genome (TRS-L) and 5′ of every viral ORF along the body of the viral genome (TRS-B) except ORF1a and -1b. Complementarity between sequences in TRS-B on the newly synthesized negative sense RNA and TRS-L allows for the transcription complex to switch templates—effectively jumping from a given TRS-B to the TRS-L at the 5′ end of the genome. Transcription then continues, copying the leader sequence to complete the negative-strand sg RNA (
). Secondary structure analysis of the TRS-L region has shown that the context of the sequence and associated structures are important for ensuring that only the TRS-L, and not other TRS-B sequences, acts as the template for strand switching by the RdRp (
). The purpose of the 5′ leader sequence in all sg mRNAs, other than to potentially prime sg mRNA synthesis, is not completely understood. One study with SARS-CoV suggested that the 5′ leader sequence could be important for protection against cleavage by viral nsp1 (
), although the mechanism by which protection is rendered is unclear. The efficiency with which the template switch occurs is an important determinant of the levels of the different sg mRNAs and the ratio of sg mRNAs to genome-length RNA, as failed template switching leads to read-through at TRSs and increases the probability of producing genome-length RNA (reviewed in Ref.
). Most of what is known about this regulation is from studies on arteriviruses, which belong to the same order (Nidovirales) as coronaviruses and synthesize sg mRNAs by a similar mechanism. The levels of several sg mRNAs are correlated with the stability (ΔG) of the duplex between TRS-L and TRS-B (
), and hence duplex stability was thought to be an important regulator of this process. However, a recent sequencing study with an arterivirus showed that some TRS-B sequences with 100% similarity to TRS-L core sequences were not used as switching points for the transcription complex, suggesting that whereas duplex stability is necessary, it is not sufficient to dictate template switching (
Several proteins have also been implicated in regulating the levels of sg mRNAs and the switch between full-length negative-strand synthesis and sg RNA synthesis, although a clear picture of features that favor transcription or replication has not emerged. For example, the viral N protein (
) have been shown to be important for producing full-length negative-strand genomic RNA and long sg RNAs, suggesting a role in read-through of TRSs. However, the N protein also has helicase-like activity (
Composition of the replication/transcription complex
Coronavirus replication, discontinuous transcription, and RNA processing are orchestrated by a remarkably sophisticated replicase complex (Fig. 5). Unlike other RNA viruses, where replication is primarily dependent on the RdRp and a small number of cofactors, coronaviruses appear to use a multiprotein complex, including the RdRp (nsp12), processivity factors (nsp7-8), a helicase (nsp13), single-strand binding protein (nsp9), a proofreading exonuclease (nsp14), other cofactors (e.g. nsp10), and capping enzymes (e.g. nsp16). This is more reminiscent of replisomes from DNA-based organisms and is potentially a consequence of their unusually large genomes (
). Cryo-EM structures of the SARS-CoV and SARS-CoV-2 nsp12-nsp7-nsp8 tripartite complex revealed that nsp8 binds nsp12 as both a heterodimer (nsp7-nsp8) and by itself to stabilize the regions of nsp12 involved in RNA binding (
). However, whereas nsp8 has polyadenylation activity that is stimulated by the presence of a polyU stretch on the template strand, it is unable to incorporate other nucleotides on heteropolymeric templates (
), suggesting that it might not be a primase. Additionally, the cryo-EM structure with nsp7 and nsp12 does not suggest a mechanism for nucleotide incorporation by nsp8. It has been proposed that the presence of polyU sequences at the 5′ end of the negative-strand viral RNA could promote polyadenylation of the viral positive-strand RNAs by nsp8, but this remains to be experimentally validated. The poly(A) tail length also varies during infection (
) with no obvious sequence specificity or function. It binds ssDNA and ssRNA with equal affinity, although ssRNA is the presumed substrate during infection. Structural studies have shown that it dimerizes, and this is important for viral replication but dispensable for RNA binding (
). It is possible that nsp9 binds to single-stranded regions of the viral genome and protects them from nucleases, akin to the role played by ssDNA-binding proteins in DNA replication systems. Indeed, other ss nucleic acid–binding proteins are also known to play roles in recombination and homologous base pairing (
) and several other components of the RTC. The functional role of helicases in replication of RNA viruses is largely unknown, although they are one of the most conserved proteins encoded by coronaviruses (reviewed in Ref.
). Helicases use the energy from nucleotide hydrolysis to translocate on nucleic acids. In addition to its (d)NTPase activity, nsp13 also has a 5′-triphosphophatase activity, suggesting a role for it in RNA capping (
). The helicase domain of MERS and SARS-CoV nsp13 shows remarkable similarity to the cellular Upf1 helicase, a protein involved in the nonsense-mediated decay pathway. Based on this observation, it has been proposed that nsp13 could also play a role in quality control of RNAs (
One of the central outstanding questions about the role of helicases in RNA viruses is whether they function similarly to replicative helicases or if they are involved in unwinding local structures and removing obstacles for the polymerase. Replicative helicases typically work together with the polymerase to unwind the double-stranded nucleic acid ahead of the polymerase. The 5′ → 3′ directionality of the helicase is reminiscent of prokaryotic replisomes, where the helicase and polymerase translocate on different strands and the helicase helps in unwinding the duplex ahead of the polymerase. Thus, during the synthesis of full-length progeny genomes using the negative-strand RNA as a template, nsp13 could be bound to the positive-strand RNA and assist the RdRp as it copies the negative strand (Fig. 5). Cooperativity between the replicative helicase and polymerase is a conserved feature of DNA replisomes. The RdRp stimulates the activity of the helicase (
), but whether the helicase has a reciprocal effect on RdRp activity, similar to DNA replisomes, would be interesting to test. A non-mutually exclusive possibility is that the helicase facilitates RdRp template switching during discontinuous transcription by releasing subgenomic RNAs at TRS sites during negative-strand synthesis, similar to a role played by some other SF1 helicases in recombination (
RNA viruses typically have high mutation rates due to lack of RdRp proofreading activity, which promotes viral genetic diversity and increases their adaptive potential. However, the potential for accumulation of deleterious mutations leading to collapse of the viral population through error catastrophe caps the size of most RNA virus genomes to ∼15 kb (reviewed in Ref.
). The ∼30-kb coronavirus genome far exceeds this threshold, indicating that they must have specialized mechanisms to counteract this mutational burden. In this regard, they are one of the few RNA viruses apart from toroviruses and roniviruses (which are also exceptionally large) that have an exonuclease activity and associated high-fidelity replication (
) showed for the first time the potential for proofreading activity in RNA viruses and explained how coronaviruses maintain their genome integrity. Indeed, the mutation rates of coronaviruses are an order of magnitude lower (10−6 to 10−7) than that of most RNA viruses, and mutating the SARS-CoV or MHV ExoN gene causes the error frequency to jump to that observed in many other RNA viruses (10−3 to 10−5) (
), suggesting that ExoN is important but may not be essential under all conditions. Why MHV and SARS-CoV but not HCoV-229E and TGEV can tolerate ExoN mutants is unclear, although it is possible that ExoN is essential only in alphacoronaviruses (HCoV and TGEV) and not in betacoronaviruses (MHV and SARS-CoV). It is also possible that the active-site mutation in SARS and MHV did not fully deactivate the enzyme or that other proteins in the replicase can compensate for the absence of an active ExoN. For example, nsp10 stimulates the catalytic activity of nsp14-ExoN to remove a mismatched nucleotide at the 3′ end of the RNA by >35-fold (
), providing biochemical evidence for its role in proofreading during transcription/replication. Nsp10 also interacts with nsp16 (a potential RNA-modifying enzyme), and it has been proposed that all of these proteins could come together to form a larger complex during replication similar to DNA replisome complexes. In vitro biochemical studies comparing the activity of ExoN from MHV and SARS-CoV and HCoV-229E together with the accessory proteins could shed mechanistic light on these phenotypic differences between the ExoN mutants.
Replication fidelity is inherently tied to viral fitness and, in most cases, changes to replication fidelity decrease fitness (reviewed in Ref.
). The ExoN mutation did not revert to WT even over 250 viral passages, although it accumulated a variety of mutations that partially compensated for the replication defect and decreased the population sensitivity to mutagens (
). Several components of the replicase complex, including nsp8, nsp9, nsp12, and nsp13, had mutations in the coding region, underscoring the complexity and interdependence of the RTC and how that helps the virus circumvent the consequences of decreased fidelity. A better understanding of the mechanism of replication fidelity will also allow for the exploration of mutants that increase replication fidelity and thereby reduce diversity and potentially fitness of the population, as has been shown for polioviruses (
), may also influence coronavirus diversity. In coronaviruses, recombination occurs as an inherent part of the replication cycle during the synthesis of sg RNAs and is tied to the ability of the RdRp to switch templates from the TRS-B sequence to the TRS-L sequence to copy the leader sequence from the 5′ end of the genome. Such recombination events can also occur between co-infecting coronaviruses with different genotypes (reviewed in Ref.
). Capping typically occurs co-transcriptionally in the nucleus, so RNA viruses that replicate in the cytoplasm encode their own enzymes or incorporate other strategies, such as cap snatching (as in bunyaviruses) (
), to protect the 5′ end of their RNAs. The coronavirus capping mechanism is not completely understood, although it appears to follow the canonical capping pathway. Capping begins with hydrolysis of the γ-phosphate of the 5′ end nucleotide; although not yet directly shown, this is thought to be mediated by the nucleotide triphosphatase activity of nsp13-helicase (
). This is followed by the addition of a guanosine monophosphate to the diphosphate RNA by a guanylyl transferase that has remained elusive in coronaviruses, although the NiRAN domain of nsp12 could be involved in this process (
). Systematic mapping of the interaction between SARS-CoV-2 proteins and the host proteome has revealed interactions between viral proteins and host translation machinery, and an inhibitor of cap-dependent translation initiation reduced viral infectivity in cell culture (
). However, designing nucleoside analogs as inhibitors is particularly challenging for coronaviruses due to the presence of the exonuclease, which can exise incorporated analogs and thus provide resistance. An exception to this has been the adenosine analog remedesivir, which is currently in phase 3 clinical trials for treating coronavirus infections (
). A recent in vitro study with purified RdRp-nsp8 complex from several coronaviruses showed that remedesivir incorporation blocks chain elongation 3 nucleotides downstream of its incorporation site, which potentially protects it from ExoN cleavage (
). Additionally, remedesivir is selectively incorporated by the RdRp over the natural substrate ATP. Better in vitro reconstitution systems incorporating the other components of the RTC (nsp7-, nsp13-, and nsp14-exonuclease) will further help to elucidate the mechanism of inhibition.
It may also be of interest to develop nonnucleoside RdRp inhibitors, as have been developed for other RNA viruses, such as hepatitis C virus (
). Nonnucleoside inhibitors typically function allosterically and hence are potentially immune to the resistance conferred by the exonuclease activity of ExoN. Combining compounds that inhibit ExoN together with nucleoside analogs to inhibit the RdRp or using small molecules that increase the mutation load of the virus by other mechanisms that are not sensitive to the exonuclease are other viable options (
), have also been considered as potential druggable targets.
Coronavirus replication occurs within heavily modified membranes
A defining feature of many positive-strand RNA viruses, including CoVs, is their ability to hijack and reform intracellular membranes to create a cellular niche for the replication of their RNA genome. Ultrastructural characterization of mainly MHV– and SARS-CoV–infected cells has revealed the membranes that anchor RTCs in CoV-infected cells to be quite striking, consisting of double membrane vesicles (DMVs) among other intricate convoluted membrane structures that isolate CoV RNA from the rest of the cellular environment (Fig. 6) (
). Conceptually, RTC formation leads to the concentration of viral replication machinery, spatially separating the sites of viral RNA replication from downstream virion assembly in the endoplasmic reticulum–Golgi intermediate compartment (ERGIC). Additionally, RTCs likely prevent detection of viral dsRNA replication products from innate immune sensors.
The DMVs and convoluted membranes in CoV-infected cells form at the nuclear periphery and are derived from host ER membrane (
). Although biochemical characterization of these proteins is hindered by their hydrophobic nature, protein-protein interaction studies performed in cells suggest that nsp3, nsp4, and nsp6 can oligomerize and form complexes through their luminal loops (
). Because the specific interaction of nsp3 and nsp4 is required for these structures to form, it is hypothesized that nsp3 and nsp4 rearrange membranes and introduce curvature by a “zipper” mechanism, essentially bringing ER membranes together through nsp3/4 interactions (
) (Fig. 6). The nsp3/4 interaction also recruits other proteins, including nsp6, to anchor RTCs. Finally, triple transfection of nsp3, nsp4, and nsp6 together results in the formation of DMVs in uninfected cells (
). Due to the importance of membrane modification during viral replication, CoV transmembrane proteins may be attractive drug targets. In fact, a small molecule screen for antiviral activity yielded a compound that targets the transmembrane protein nsp6 and essentially blocks viral RNA replication and DMV formation (
During CoV infection, the inner membrane of the DMV is sealed while the outer membrane of the DMVs forms a contiguous network with the convoluted membranes and modified ER membranes (Fig. 6). When this network is isolated from cells, it is capable of producing both genomic and subgenomic RNAs in vitro even in the presence of RNases and proteases, but not detergent, thus implicating the membrane network in shielding viral RNA replication (
). The RTC microenvironment also includes numerous host proteins that participate in CoV biology, such as proteins involved in vesicular trafficking and translation initiation factors, the latter of which are suggestive of active translation near sites of viral RNA replication (
). The site of RNA replication inside this membrane network is currently unknown. Whereas viral RTC proteins labeled by immuno-EM primarily localize to convoluted membranes between DMVs, dsRNA (presumed to be of viral origin) labeled by the J2 antibody localizes inside the DMVs (Fig. 6). However, there is no experimental evidence demonstrating whether dsRNAs inside the DMV represent nascent viral transcripts, viral RNA replication byproducts, or even host dsRNAs. Recently, nascent viral RNA was visualized by metabolic labeling and quantitative EM autoradiography, revealing that viral transcription does in fact occur in association with the DMVs rather than convoluted membranes (
). The spatial resolution of this technique, while clearly demonstrating viral transcription within the vicinity of the DMVs, was not sufficient to pinpoint the localization of nascent viral RNA within DMVs and/or in association with DMV membranes. Because no visible pores or openings in the inner membrane of the DMV have been detected with conventional EM techniques, viral RNA synthesis regardless of locale would rely on a yet unidentified transport mechanism capable of moving viral proteins and/or RNA in and out of the DMV inner membrane (
The assembly of an infectious CoV virion requires that its nucleocapsid, consisting of the viral RNA genome coated with N protein, and viral envelope coalesce into the same intracellular space. Viral glycoproteins that are incorporated into the envelope (M, E, and S proteins) are translated in the ER and retained at the site of budding in the ERGIC (Fig. 1). The ERGIC budding site is distinct from the site of viral genome synthesis in the RTC. The nucleocapsid core of the virion traffics from the RTC to ultimately bud into ERGIC membranes, which are decorated with M, E, and S protein and become the lipid envelope of the virion. The most abundant envelope component is the M protein, which plays a central role in viral egress. Outside of the context of infection, M protein expression alone is not sufficient to cause budding of virus-like particles, but co-expression with E (or N in the case of SARS-CoV) can result in virus-like particle formation in the absence of infection (
). The E protein, while not highly abundant in the envelope, is critical for viral envelope curvature and maturation and can form membrane ion channels, although the significance of this latter activity is not yet appreciated (
The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein.
). Although the retention signals are quite divergent among CoVs, all serve to maintain S near the ERGIC-localized M protein, ensuring M-S interaction at the site of virion assembly. Following budding of the nucleocapsid core into the M-, E-, and S-containing ERGIC membranes, the newly enveloped virion then leaves the cell through the exocytic pathway.
Although CoV replication produces an abundance of unique viral RNAs in the cell (positive-strand genomic RNAs, positive-strand sg mRNAs, and negative-strand RNAs), purified CoV virions house mainly full genome-length RNA (
). Conceptually, this specificity is thought to be driven by a packaging signal unique to the genome-length RNA. In MHV, a packaging signal has been mapped to ORF1b within the nsp15 gene (a region absent in sg RNAs) and is predicted to form a bulged stem-loop structure with repeating AGC/GUAAU motifs (
). N protein must have broad RNA-binding activity, as it ultimately coats the length of the viral genome to form the nucleocapsid component of the virion and additionally forms complexes with sg RNAs (
). Thus, an additional role of M in recognizing the packaging signal and selecting full-length genomic RNA is an attractive model for genome packaging specificity, at least in the context of MHV infection (
), leaving us with little understanding of how other CoVs selectively package genome-length RNAs.
Part II: Viral manipulation of the host
Viruses depend on host processes to complete their life cycle. In addition to employing cellular machines like the ribosome to translate their proteins and manipulating cellular membranes during RNA synthesis and viral morphogenesis, several coronavirus proteins modify the cellular environment in ways that may influence viral pathogenesis and replication in vivo. In this section, we discuss the roles of coronavirus proteins in altering the cellular signaling landscape as well as the ability of the virus to modulate host gene expression and its interactions with and counteraction of the host immune response.
Accessory proteins and viral pathogenicity
Coronavirus genomes contain a number of genes concentrated in the 3′ region of the genome that encode for accessory proteins that are largely dispensable for viral replication and growth in vitro (
). Additionally, no significant amino acid sequence similarity is shared between SARS-CoV accessory proteins and other known viral or cellular proteins, providing little insight to predict functional roles (
). Despite being nonessential for viral replication in cultured cells, the accessory proteins presumably modulate virus-host interactions that are important during in vivo infection, including cell proliferation, programmed cell death, pro-inflammatory cytokine production, and IFN signaling (see Table 1) (
27 aa substitutions (6 in RBD, 6 in subdomain, and 4 in known peptide antigen for SARS-CoV); no aa substitutions in CoV-2 polybasic cleavage site compared with CoV consensus sequence; cleavage site is absent in SARS-CoV