Identification of a Novel Structural Protein of Arteriviruses

Arteriviruses are positive-stranded RNA viruses with an efficiently organized, polycistronic genome. A short region between the replicase gene and open reading frame (ORF) 2 of the equine arteritis virus (EAV) genome was previously assumed to be untranslated. However, here we report that this segment of the EAV genome contains the 5' part of a novel gene (ORF 2a) which is conserved in all arteriviruses. The 3' part of EAV ORF 2a overlaps with the 5' part of the former ORF 2 (now renamed ORF 2b), which encodes the GS glycoprotein. Both ORF 2a and ORF 2b appear to be expressed from mRNA 2, which thereby constitutes the first proven example of a bicistronic mRNA in arteriviruses. The 67-amino-acid protein encoded by EAV ORF 2a, which we have provisionally named the envelope (E) protein, is very hydrophobic and has a basic C terminus. An E protein-specific antiserum was raised and used to demonstrate the expression of the novel gene in EAV-infected cells. The EAV E protein proved to be very stable, did not form disulfide-linked oligomers, and was not N-glycosylated. Immunofluorescence and immunoelectron microscopy studies showed that the E protein associates with intracellular membranes both in EAV-infected cells and upon independent expression. An analysis of purified EAV particles revealed that the E protein is a structural protein. By using reverse genetics, we demonstrated that both the EAV E and GS proteins are essential for the production of infectious progeny virus.     

Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b,GP3, and GP4) of equine arteritis virus

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP(5) (previously named G(L)), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP(2b) (formerly designated G(S)), GP(3), and GP(4). Analysis of the appearance of the GP(2b), GP(3), and GP(4) proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP(2b) and GP(4) and heterotrimers of GP(2b), GP(3), and GP(4). Shortly after their release from infected cells, virions contained mainly cystine-linked GP(2b)/GP(4) heterodimers, which were subsequently converted into disulfide-bonded GP(2b)/GP(3)/GP(4) trimers through the covalent recruitment of GP(3). This process occurred faster at a higher pH but was arrested at 4 degrees C. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N-ethylmaleimide inhibited the formation of disulfide-bonded GP(2b)/GP(3)/GP(4) trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP(3) with the cystine-linked GP(2b)/GP(4) dimer in freshly released virions, nor did we observe higher-order structures of the GP(2b)/GP(4) or GP(2b)/GP(3)/GP(4) complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP(2b)/GP(3)/GP(4) heterotrimers at 4 degrees C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP(2b)/GP(4) dimer and GP(3) was also inferred from coexpression experiments showing that the presence of GP(3) increased the electrophoretic mobility of the disulfide-bonded GP(2b)/GP(4) dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.     

Intra- and intermolecular disulfide bonds of the GP2b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: EAV virions contain six different envelope proteins. The glycoprotein GP(5) (previously named G(L)) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP(2b) (previously named G(S)), GP(3), and GP(4) are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP(5) and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP(2b), GP(3), and GP(4) proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP(2b) protein in the assembly of the GP(2b)/GP(3)/GP(4) complex. Open reading frames encoding cysteine-to-serine mutants of the GP(2b) protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP(2b) forms an intermolecular cystine bridge with one of the cysteines of the GP(4) protein, while the cysteine residues at positions 48 and 137 of GP(2b) are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP(4) protein is responsible for the covalent association of GP(3) with the disulfide-linked GP(2b)/GP(4) heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.     

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.     

An autophagy-independent role for LC3 in equine arteritis virus replication

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus. Genome replication of EAV has been associated with modified intracellular membranes that are shaped into double-membrane vesicles (DMVs). We showed by immuno-electron microscopy that the DMVs induced in EAV-infected cells contain double-strand (ds)RNA molecules, presumed RNA replication intermediates, and are decorated with the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3). Replication of EAV, however, was not affected in autophagy-deficient cells lacking autophagy-related protein 7 (ATG7). Nevertheless, colocalization of DMVs and LC3 was still observed in these knockout cells, which only contain the nonlipidated form of LC3. Although autophagy is not required, depletion of LC3 markedly reduced the replication of EAV. EAV replication could be fully restored in these cells by expression of a nonlipidated form of LC3. These findings demonstrate an autophagy-independent role for LC3 in EAV replication. Together with the observation that EAV-induced DMVs are also positive for ER degradation-enhancing α-mannosidase-like 1 (EDEM1), our data suggested that this virus, similarly to the distantly-related mouse hepatitis coronavirus, hijacks the ER-derived membranes of EDEMosomes to ensure its efficient replication.     

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.     

体外共表达的猪繁殖与呼吸综合征病毒GP5和M蛋白可形成异源二聚体

为了探讨猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因编码的GP5蛋白和ORF6编码的M蛋白体外共表达特性,分别构建了PRRSV ORF5、ORF6单基因或双基因共表达的真核表达质粒pCI-ORF5、pCI-ORF6和pCI-ORF5/ORF6,转染BHK-21细胞,Western blot检测证实共表达的GP5和M蛋白能够形成异源二聚体。同时,以绿色荧光蛋白(EGFP)和红色荧光蛋白(RFP)为示踪,发现当ORF5-EGFP和ORF6-RFP共表达时,能促进GP5蛋白从内质网向高尔基体转运,提示GP5-M异源二聚体的形成可能与GP5蛋白的翻译后修饰、转运、定位有关。     

表达猪繁殖与呼吸综合征病毒(PRRSV)GP5的重组伪狂犬病毒TK-/gG-/GP5+的构建及其生物学特性初步探讨

以伪狂犬病毒基因缺失标志疫苗株TK/gG-/LacZ 为亲本株,通过同源重组,构建了表达猪繁殖与呼吸综合征病毒(PRRSV)主要免疫原性蛋白GP5的重组伪狂犬病毒TK/gG-/GP5 .经PCR、Southern杂交、Western blot证实构建正确,并能表达GP5.同时,对重组病毒在PK-15细胞中的增殖特性进行了检测,结果与亲本株相比,增殖滴度无显著性差异.将重组病毒免疫BALB/c小鼠,2次免疫后4周,50%(3/6)小鼠产生了低水平的GP5特异性ELISA抗体,但中和抗体均小于1:4.     

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.     

Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity

The two major envelope proteins of arteriviruses, the membrane protein (M) and the major glycoprotein (GP(5)), associate into a disulfide-linked heterodimer that is incorporated into the virion and has been assumed to be a prerequisite for virus assembly. Using an equine arteritis virus (EAV) infectious cDNA clone, we have analyzed the requirement for GP(5)-M heterodimerization and have identified the Cys residues involved in the formation of the GP(5)-M disulfide bond. The single Cys residue (Cys-8) in the M ectodomain was crucial for heterodimerization and virus infectivity. Mutagenesis of any of the five Cys residues in the GP(5) ectodomain or removal of the single GP(5) N-glycosylation site also rendered the full-length clone noninfectious. However, an analysis of revertants yielded an exceptional pseudorevertant in which residues 52 to 79 of the GP(5) ectodomain had been deleted and the original Cys-80-->Ser mutation had been maintained. Consequently, this revertant lacked the GP(5) N-glycosyation site (Asn-56) and retained only a single cysteine residue (Cys-34). By using this GP(5) deletion, we confirmed that Cys-34 of GP(5) and Cys-8 of M are essential for GP(5)-M heterodimerization, a key event in the assembly of the EAV envelope.     

A discrete stage of baculovirus GP64-mediated membrane fusion

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.     

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.     

Persistent equine arteritis virus infection in HeLa cells

The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53→Cys and Val55→Ala), GP2 (Leu15→Ser, Trp31→Arg, Val87→Leu, and Ala112→Thr), GP3 (Ser115→Gly and Leu135→Pro), and GP4 (Tyr4→His and Ile109→Phe) proteins or with a single point mutation in the GP5 protein (Pro98→Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.     

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 ORF7b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is expressed in virus-infected cells and incorporated into SARS-CoV particles

Coronavirus replication is facilitated by a number of highly conserved viral proteins. The viruses also encode accessory genes, which are virus group specific and believed to play roles in virus replication and pathogenesis in vivo. Of the eight putative accessory proteins encoded by the severe acute respiratory distress syndrome associated coronavirus (SARS-CoV), only two-open reading frame 3a (ORF3a) and ORF7a-have been identified in virus-infected cells to date. The ORF7b protein is a putative viral accessory protein encoded on subgenomic (sg) RNA 7. The ORF7b initiation codon overlaps the ORF7a stop codon in a -1 shifted ORF. We demonstrate that the ORF7b protein is expressed in virus-infected cell lysates and from a cDNA encoding the gene 7 coding region, indicating that the sgRNA7 is bicistronic. The translation of ORF7b appears to be mediated by ribosome leaky scanning, and the protein has biochemical properties consistent with that of an integral membrane protein. ORF7b localizes to the Golgi compartment and is incorporated into SARS-CoV particles. We therefore conclude that the ORF7b protein is not only an accessory protein but a structural component of the SARS-CoV virion.