Coronavirus multiplication: locations of genes for virion proteins on the avian infectious bronchitis virus genome.

Six overlapping viral RNAs are synthesized in cells infected with the avian coronavirus infectious bronchitis virus (IBV). These RNAs contain a 3'-coterminal nested sequence set and were assumed to be viral mRNAs. The seven major IBV virion proteins are all produced by processing of three polypeptides of ca. 23, 51, and 115 kilodaltons. These are the core polypeptides of the small membrane proteins, the nucleocapsid protein, and the 155-kilodalton precursor to the large membrane proteins GP90 and GP84, respectively. To determine which mRNAs specify these polypeptides, we isolated RNA from infected cells and translated it in a messenger-dependent rabbit reticulocyte lysate. Proteins of 23, 51, and 110 kilodaltons were produced. Two-dimensional tryptic peptide mapping demonstrated that these proteins were closely related to the major virion proteins. Fractionation of the RNA before cell-free translation permitted the correlation of messenger activities for synthesis of the proteins with the presence of specific mRNAs. We found that the smallest RNA, RNA A, directs the synthesis of P51, the nucleocapsid protein. RNA C, which contains the sequences of RNA A, directs the synthesis of the small membrane protein P23. RNA E directs the synthesis of the large virion glycoproteins. These results supported a model in which only the unique 5'-terminal domain of each IBV mRNA is active in translation and enabled us to localize genes for virion proteins on the IBV genome.     

Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins.

The recent finding that the E1 glycoproteins of murine coronaviruses contain only O-linked oligosaccharides suggested that this unusual modification might be a distinguishing feature of coronaviruses and might play an essential role in the life cycle of this family of viruses. To examine these possibilities, we analyzed the oligosaccharide moieties of the membrane proteins of the avian coronavirus infectious bronchitis virus. In addition, we determined the effect of inhibiting the glycosylation of these proteins on viral maturation and infectivity. Infectious bronchitis virus virions contain nine proteins. Four of these proteins, GP36, GP31, GP28, and P23, are closely related structurally and appear to be homologous to the E1 proteins of murine coronaviruses. We found that the oligosaccharides of GP31 and GP28 could be removed with endoglycosidase H and that neither of these glycoproteins was detectable in tunicamycin-treated cells. These two results indicated that GP31 and GP28 contain N-linked oligosaccharides. Therefore, O-linked oligosaccharides are not a universal feature of the small coronavirus membrane glycoproteins. Tunicamycin inhibited glycosylation of all of the viral glycoproteins but did not inhibit production of virions by infectious bronchitis virus-infected cells. The virions released by these cells contained only the three non-glycosylated viral proteins P51, P23, and P14. These particles were not infectious. Therefore, it appears that glycosylated infectious bronchitis virus polypeptides are not required for particle formation. However, the viral glycoproteins are apparently indispensible for viral infectivity.     

Structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus.

We have found six major polypeptides in virions of the avian coronavirus infectious bronchitis virus grown in tissue culture: four glycoproteins, GP84, GP36, GP31, and GP28, and two non-glycosylated proteins, P51 and P23. In addition, we detected three minor species: two glycoproteins, GP90 and GP59, and one non-glycosylated protein, P14. Two-dimensional tryptic peptide mapping showed that GP36, GP31, GP28, and P23 comprise a group of closely related proteins which we have designated the "P23 family," but that the other proteins are distinct. Analysis by partial proteolytic digestion of P23 family, but that the other proteins are distinct. Analysis by partial proteolytic digestion of the P23 family labeled biosynthetically with [35S] methionine, and P23, labeled with [35S] formyl-methionine by in vitro translation of RNA from infected cells, revealed that the proteins of the P23 family differ in their amino-terminal domains. Similar analysis of GP31 and Gp36 labeled with [3H] mannose showed that the partial proteolytic fragments unique to these proteins were glycosylated. This suggests that differences in glycosylation in the amino-terminal domains contributes to the marked polymorphism os the P23 family. The results are discussed with respect to possible models for synthesis of the virion proteins.     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

The Lytic Enzyme of Bacteriophage PRD1 Is Associated with the Viral Membrane

Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity. Protein P15 is a soluble beta-1,4-N-acetylmuramidase that causes phage-induced host cell lysis. We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane. Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane. The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used. The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.     

Genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus

The coronavirus membrane (M) protein is the most abundant virion protein and the key component in viral assembly and morphogenesis. The M protein of mouse hepatitis virus (MHV) is an integral membrane protein with a short ectodomain, three transmembrane segments, and a large carboxy-terminal endodomain facing the interior of the viral envelope. The carboxy terminus of MHV M has previously been shown to be extremely sensitive to mutation, both in a virus-like particle expression system and in the intact virion. We have constructed a mutant, M(Delta)2, containing a two-amino-acid truncation of the M protein that was previously thought to be lethal. This mutant was isolated by means of targeted RNA recombination with a powerful host range-based selection allowed by the interspecies chimeric virus fMHV (MHV containing the ectodomain of the feline infectious peritonitis virus S protein). Analysis of multiple second-site revertants of the M(Delta)2 mutant has revealed changes in regions of both the M protein and the nucleocapsid (N) protein that can compensate for the loss of the last two residues of the M protein. Our data thus provide the first genetic evidence for a structural interaction between the carboxy termini of the M and N proteins of MHV. In addition, this work demonstrates the efficacy of targeted recombination with fMHV for the systematic genetic analysis of coronavirus structural protein interactions.     

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid.

The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus at 37 degrees C resulted in formation of a complex between one of the viral glycoproteins, E1, and the viral nucleocapsid. This was caused by a temperature-dependent conformational change in E1, resulting in aggregation of E1 and interaction with the viral RNA in the nucleocapsid. E1 also bound rRNA. The E1-nucleocapsid complexes can be distinguished on sucrose and Renografin density gradients from native viral nucleocapsids. The separation of the membrane glycoprotein E1 from the peplomeric glycoprotein E2 permitted preparation of antisera against these isolated proteins. A model is proposed for the arrangement of the three major structural proteins in the coronavirus A59 virion in relation to the viral envelope and RNA.     

Structural protein requirements in equine arteritis virus assembly

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP(2b) (previously named G(S)), GP(3), GP(4), and GP(5) (previously named G(L)). Proteins N, M, and GP(5) are major virion components, E occurs in virus particles in intermediate amounts, and GP(4), GP(3), and GP(2b) are minor structural proteins. The M and GP(5) proteins occur in virus particles as disulfide-linked heterodimers while the GP(4), GP(3), and GP(2b) proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP(2b), GP(3), or GP(4) proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP(2b), GP(3), and GP(4) proteins into viral particles. EAV particles lacking GP(2b), GP(3), GP(4), and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP(2b)/GP(3)/GP(4) heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.     

Assembly of human severe acute respiratory syndrome coronavirus-like particles

Viral particles of human severe acute respiratory syndrome coronavirus (SARS CoV) consist of three virion structural proteins, including spike protein, membrane protein, and envelope protein. In this report, virus-like particles were assembled in insect cells by the co-infection with recombinant baculoviruses, which separately express one of these three virion proteins. We found that the membrane and envelope proteins are sufficient for the efficient formation of virus-like particles and could be visualized by electron microscopy. Sucrose gradient purification followed by Western blot analysis and immunogold labeling showed that the spike protein could be incorporated into the virus like particle also. The construction of engineered virus-like particles bearing resemblance to the authentic one is an important step towards the development of an effective vaccine against infection of SARS CoV.     

Pseudotyping Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV): F proteins from group II NPVs are functionally analogous to AcMNPV GP64

GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an AcMNPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from LdMNPV or SeMNPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The AcMNPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.     

The severe acute respiratory syndrome coronavirus 3a is a novel structural protein

The severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein is one of the opening reading frames in the viral genome with no homologue in other known coronaviruses. Expression of the 3a protein has been demonstrated during both in vitro and in vivo infection. Here we present biochemical data to show that 3a is a novel coronavirus structural protein. 3a was detected in virions purified from SARS-CoV infected Vero E6 cells although two truncated products were present predominantly instead of the full-length protein. In Vero E6 cells transiently transfected with a cDNA construct for expressing 3a, a similar cleavage was observed. Furthermore, co-expression of 3a, membrane and envelope proteins using the baculovirus system showed that both full-length and truncated 3a can be assembled into virus-like particles. This is the first report that demonstrated the incorporation of 3a into virion and showed that the SARS-CoV encodes a novel coronavirus structural protein.     

Functional analysis of the transmembrane (TM) domain of the Autographa californica multicapsid nucleopolyhedrovirus GP64 protein: substitution of heterologous TM domains

GP64, the major envelope glycoprotein of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. In the current study, we examined the functional role of the AcMNPV GP64 transmembrane (TM) domain by replacing the 23-amino-acid GP64 TM domain with corresponding TM domain sequences from a range of viral and cellular type I membrane proteins, including Orgyia pseudotsugata MNPV (OpMNPV) GP64 and F, thogotovirus GP75, Lymantria dispar MNPV (LdMNPV) F, human immunodeficiency virus type 1 (HIV-1) GP41, human CD4 and glycophorin A (GpA), and influenza virus hemagglutinin (HA), and with a glycosylphosphatidylinositol (GPI) anchor addition sequence. In transient expression experiments with Sf9 cells, chimeric GP64 proteins containing either a GPI anchor or TM domains from LdMNPV F or HIV-1 GP41 failed to localize to the cell surface and thus appear to be incompatible with either GP64 structure or cell transport. All of the mutant constructs detected at the cell surface mediated hemifusion (outer leaflet merger) upon low-pH treatment, but only those containing TM domains from CD4, GpA, OpMNPV GP64, and thogotovirus GP75 mediated pore formation and complete membrane fusion activity. This supports a model in which partial fusion (hemifusion) proceeds by a mechanism that is independent of the TM domain and the TM domain participates in the enlargement or expansion of fusion pores after hemifusion. GP64 proteins containing heterologous TM domains mediated virion budding with dramatically differing levels of efficiency. In addition, chimeric GP64 proteins containing TM domains from CD4, GpA, HA, and OpMNPV F were incorporated into budded virions but were unable to rescue the infectivity of a gp64 null virus, whereas those with TM domains from OpMNPV GP64 and thogotovirus GP75 rescued infectivity. These results show that in addition to its basic role in membrane anchoring, the GP64 TM domain is critically important for GP64 trafficking, membrane fusion, virion budding, and virus infectivity. These critical functions were replaced only by TM domains from related viral membrane proteins.     

A swine arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny

Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell’s exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly.     

Synthesis of murine mammary tumor viral proteins in vitro

The coding potential of murine mammary tumor viral genomic RNA was investigated by in vitro translation of various size classes of RNAs isolated from the virions. The major products of translation of full-size 35S polyadenylylated virion RNA were gag-related polyproteins of 75,000, 105,000, and 180,000 daltons (P75, P105, and P180, respectively). Studies on the kinetics of translation of these three proteins established that they were synthesized independently and that the smaller proteins were not post-translational cleavage products of the larger proteins. Tryptic peptide mapping showed that almost all of the P75 sequences were contained within P105 and almost all of the P105 sequences were contained within P180. The syntheses of all three proteins were inhibited by m7GTP, indicating that they were translated from capped mRNA's. Although a 24S polyadenylylated RNA had been identified as the intracellular mRNA for env precursor polyprotein, no such protein could be translated from the 24S polyadenylylated RNA isolated from the virions. However, translation of a 14S size class of polyadenylylated virion yielded four prominent proteins of about 36,000, 23,000, 21,000, and 20,000 daltons. These proteins were unrelated to murine mammary tumor viral structural proteins, as suggested from tryptic peptide mapping and immunoprecipitation data. They might be the products of an as-yet-unidentified gene located near the 3' terminus of the murine mammary tumor viral genomic RNA.     

Virus Maturation by Budding

Enveloped viruses mature by budding at cellular membranes. It has been generally thought that this process is driven by interactions between the viral transmembrane proteins and the internal virion components (core, capsid, or nucleocapsid). This model was particularly applicable to alphaviruses, which require both spike proteins and a nucleocapsid for budding. However, genetic studies have clearly shown that the retrovirus core protein, i.e., the Gag protein, is able to form enveloped particles by itself. Also, budding of negative-strand RNA viruses (rhabdoviruses, orthomyxoviruses, and paramyxoviruses) seems to be accomplished mainly by internal components, most probably the matrix protein, since the spike proteins are not absolutely required for budding of these viruses either. In contrast, budding of coronavirus particles can occur in the absence of the nucleocapsid and appears to require two membrane proteins only. Biochemical and structural data suggest that the proteins, which play a key role in budding, drive this process by forming a three-dimensional (cage-like) protein lattice at the surface of or within the membrane. Similarly, recent electron microscopic studies revealed that the alphavirus spike proteins are also engaged in extensive lateral interactions, forming a dense protein shell at the outer surface of the viral envelope. On the basis of these data, we propose that the budding of enveloped viruses in general is governed by lateral interactions between peripheral or integral membrane proteins. This new concept also provides answers to the question of how viral and cellular membrane proteins are sorted during budding. In addition, it has implications for the mechanism by which the virion is uncoated during virus entry.     

Yeast two hybrid analyses reveal novel binary interactions between human cytomegalovirus-encoded virion proteins

Human cytomegalovirus (HCMV) is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH) analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP) experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these "hub" proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.     

Large surface proteins of hepatitis B virus containing the pre-s sequence.

The sequence of hepatitis B virus DNA contains an open reading frame which codes for a not-yet-identified protein of at least 389 amino acids. Only the products starting at the third (GP33/GP36) or the fourth (P24/GP27) initiation signal have been characterized as components of the viral surface antigen. We found a larger protein, P39, and its glycosylated form, GP42, in hepatitis B virus particles and viral surface antigen filaments. Immunological cross-reactions showed that P39/GP42 is partially homologous to P24/GP27 and GP33/GP36. The unique portion of its sequence bound monoclonal antibodies which had been induced by immunization with hepatitis B virus particles. Proteolytic cleavage patterns and subtype-specific size differences suggested that the sequence of P39 starts with the first initiation signal of the open reading frame. Its amino-terminal part (pre-s coded) is exposed at the viral surface and, probably, is highly immunogenic. A model is presented of how the open reading frame for the viral envelope leads to defined amounts of three different proteins.     

HIV-1 assembly: viral glycoproteins segregate quantally to lipid rafts that associate individually with HIV-1 capsids and virions

HIV-1 assembly depends on its structural protein, Gag, which after synthesis on ribosomes, traffics to the late endosome/plasma membrane, associates with HIV Env glycoprotein, and forms infectious virions. While Env and Gag migrate to lipid microdomains, their stoichiometry and specificity of interaction are unknown. Pseudotyped viral particles can be made with one viral core surrounded by heterologous envelope proteins. Taking advantage of this property, we analyzed the association of HIV Env and Ebola glycoprotein (GP), with HIV-1 Gag coexpressed in the same cell. Though both viral glycoproteins were expressed, each associated independently with Gag, giving rise to distinct virion populations, each with a single glycoprotein type. Confocal imaging demonstrated that Env and GP localized to distinct lipid raft microdomains within the same cell where they associated with different virions. Thus, a single Gag particle associates "quantally" with one lipid raft, containing homogeneous trimeric viral envelope proteins, to assemble functional virions.