Complete Genome Sequence of Peanut stripe virus Isolated in China

The complete genome sequence of a Laixi isolate of Peanut stripe virus (PStV‐Laixi) from China was determined to be 10, 056 nucleotides in length, excluding the 3′‐terminal poly (A) tail. The viral genome contains a single long open reading frame of 9669 nucleotides encoding a polyprotein of 3222 amino acids. The polyprotein was predicted to be cleaved into ten functional proteins by three viral proteases. An additional protein, termed ‘PIPO’, is also found in the P3 cistron. The complete genome sequence comparison and phylogenetic analysis indicated that PStV‐Laixi was most closely related to three other isolates of PStV (two from USA and one from Taiwan). To our knowledge, this is the first report of the complete sequence of a PStV isolate from China.     

A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing

The single‐stranded, positive‐sense RNA genome of viruses in the genus Potyvirus encodes a large polyprotein that is cleaved to yield 10 mature proteins. The first three cleavage products are P1, HCpro and P3. An additional short open reading frame (ORF), called pipo, overlaps the P3 region of the polyprotein ORF. Four related potyviruses infecting sweet potato (Ipomoea batatas) are predicted to contain a third ORF, called pispo, which overlaps the 3′ third of the P1 region. Recently, pipo has been shown to be expressed via polymerase slippage at a conserved GA₆ sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA₆ sequence, with higher slippage efficiency (～5%) than at the pipo site (～1%). Transient expression of recombinant P1 or the ‘transframe’ product, P1N‐PISPO, in Nicotiana benthamiana suppressed local RNA silencing (RNAi), but only P1N‐PISPO inhibited short‐distance movement of the silencing signal. These results reveal that polymerase slippage in potyviruses is not limited to pipo expression, but can be co‐opted for the evolution and expression of further novel gene products.     

Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites.

Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q3783 S3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q3672 N3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide.     

Open reading frame vpr of simian immunodeficiency virus encodes a virion-associated protein.

The genomes of simian immunodeficiency viruses isolated from rhesus macaques (SIVmac) contain an open reading frame (ORF), vpr, which has a coding potential of 97 to 101 amino acid residues. In this study, a vpr ORF-encoded protein of approximately 11 kDa was identified, and anti-vpr antibodies were detected in rhesus macaques infected by SIVmac. These results provide clear evidence that the vpr ORF is a coding gene of SIVmac. The vpr protein, like the vpx protein which is encoded by another accessory gene of SIVmac, was also found to be associated with viral particles. This observation demonstrates that more than one accessory gene product can be present in the virions of this family of retroviruses and raises the possibility that the vpr protein may have a role in early part of the virus life cycle.     

Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases

Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino‐acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.     

Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains.

The replicase open reading frame lb (ORF1b) protein of equine arteritis virus (EAV) is expressed from the viral genome as an ORF1ab fusion protein (345 kDa) by ribosomal frameshifting. Processing of the ORF1b polyprotein was predicted to be mediated by the nsp4 serine protease, the main EAV protease. Several putative cleavage sites for this protease were detected in the ORF1b polyprotein. On the basis of this tentative processing scheme, peptides were selected to raise rabbit antisera that were used to study the processing of the EAV replicase ORF1b polyprotein (158 kDa). In immunoprecipitation and immunoblotting experiments, processing products of 80, 50, 26, and 12 kDa were detected. Of these, the 80-kDa and the 50-kDa proteins contain the putative viral polymerase and helicase domains, respectively. Together, the four cleavage products probably cover the entire ORF1b-encoded region of the EAV replicase, thereby representing the first complete processing scheme of a coronaviruslike ORF1b polyprotein. Pulse-chase analysis revealed that processing of the ORF1b polyprotein is slow and that several large precursor proteins containing both ORF1a- and ORF1b-encoded regions are generated. The localization of ORF1b-specific proteins in the infected cell was studied by immunofluorescence. A perinuclear staining was observed, which suggests association with a membranous compartment.     

Alternative Translation Initiation of Theiler’s Murine Encephalomyelitis Virus

DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) produce a chronic demyelinating disease in which the virus persists but has a restricted expression. We previously reported that TO subgroup strains, in addition to synthesizing the picornaviral polyprotein, use an alternative initiation codon just downstream from the polyprotein's AUG to translate an 18-kDa protein called L* that is out of frame with the polyprotein (H. H. Chen et al., Nat. Med. 1:927-931, 1995; W. P. Kong and R. P. Roos, J. Virol. 65:3395-3399, 1991). L* is critically important for virus persistence and the induction of the demyelinating disease (Chen et al., 1995; G. D. Ghadge et al. J. Virol. 72:8605-8612, 1998). We have proposed that variations in the amount of translation initiation from the L* AUG versus the polyprotein AUG may occur in different cell types and therefore affect the degree of expression of viral capsid proteins. We now demonstrate that ribosomal translation initiation at the polyprotein's initiation codon affects initiation at the L* AUG, suggesting that ribosomes land at the polyprotein's initiation codon before scanning downstream and initiating at the L* AUG. We also find that the viral 5' untranslated region affects utilization of the L* AUG. Surprisingly, mutant DA cDNAs were found to be infectious despite the presence of mutations of the polyprotein initiation codon or placement of a stop codon upstream of the L* AUG in the polyprotein's reading frame. Sequencing studies showed that these viruses had a second site mutation, converting the reading frame of L* into the polyprotein's reading frame; the results suggest that translation of the polyprotein during infection of these mutant viruses can be initiated at the L* AUG. These data are important in our understanding of translation initiation of TMEV and other RNAs that contain an internal ribosome entry site.     

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.     

The Putative Helicase of the Coronavirus Mouse Hepatitis Virus Is Processed from the Replicase Gene Polyprotein and Localizes in Complexes That Are Active in Viral RNA Synthesis

The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be involved in viral RNA synthesis. Antibodies directed against polypeptides encoded by open reading frame 1b were used to characterize the expression and processing of the MHV helicase and to define the relationship of helicase to the viral nucleocapsid protein (N) and to sites of viral RNA synthesis in MHV-infected cells. The antihelicase antibodies detected a 67-kDa protein in MHV-infected cells that was translated and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known inhibitor of the MHV 3C-like proteinase. When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in patterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infected cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast majority of helicase-containing complexes were active in viral RNA synthesis. Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associated with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and suggests that complexes containing helicase, N, and new viral RNA are the viral replication complexes.     

Subcellular localization and detection of <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> ORF6 protein by immunoelectron microscopy

Members of the genus Tobamovirus represent one of the best-characterized groups of plant positive, single stranded RNA viruses. Previous studies have shown that genomes of some tobamoviruses contain not only genes coding for coat protein, movement protein, and the cistron coding for different domains of RNA-polymerase, but also a gene, named ORF6, coding for a poorly conserved small protein. The amino acid sequences of ORF6 proteins encoded by different tobamoviruses are highly divergent. The potential role of ORF6 proteins in replication of tobamoviruses still needs to be elucidated. In this study, using biochemical and immunological methods, we have shown that ORF6 peptide is accumulated after infection in case of two isolates of Tobacco mosaic virus strain U1 (TMV-U1 common and TMV-U1 isolate A15). Unlike virus particles accumulating in the cytoplasm, the product of the ORF6 gene is found mainly in nuclei, which correlates with previously published data about transient expression of ORF6 isolated from TMV-U1. Moreover, we present new data showing the presence of ORF6 genes in genomes of several tobamoviruses. For example, in the genomes of other members of the tobamovirus subgroup 1, including Rehmannia mosaic virus, Paprika mild mottle virus, Tobacco mild green mosaic virus, Tomato mosaic virus, Tomato mottle mosaic virus, and Nigerian tobacco latent virus, sequence comparisons revealed the existence of a similar open reading frame like ORF6 of TMV.     

Isolation of enzymatically active replication complexes from feline calicivirus-infected cells

A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative "3A-like" protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.     

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.     

Molecular biology of bovine viral diarrhea virus

Bovine viral diarrhea viruses (BVDV) are arguably the most important viral pathogen of ruminants worldwide and can cause severe economic loss. Clinical symptoms of the disease caused by BVDV range from subclinical to severe acute hemorrhagic syndrome, with the severity of disease being strain dependent. These viruses are classified as members of the Pestivirus genus of the Flaviviridae. BVDV are considered primarily a pathogen of cattle but can infect most ruminant species. The virus particle consists of a lipid bilayer membrane surrounding the encapsidated genomic RNA. Inserted in the outer membrane are two virus-encoded glycoproteins that contain the major antigenic determinants of the virus as well as receptor binding and cell fusion functions. A third glycoprotein is weakly associated with the virion, but also possesses unique features that play important roles in suppression of innate immunity. The viral proteins are encoded in a single, large open reading frame. The viral proteins are proteolytically cleaved from the polyprotein by different proteases. The structural proteins are processed by cellular signal peptidases while the processing of the nonstructural proteins is by the viral serine protease. The virus is assembled and matures in the endoplasmic reticulum and golgi bodies of the cell. The virus is released via exocytosis, where viral proteins are not exposed on the surface of the cell.     

The double-stranded RNA genome of yeast virus L-A encodes its own putative RNA polymerase by fusing two open reading frames

The L-A double-stranded RNA virus of Saccharomyces cerevisiae encodes its major coat protein (80 kDa) and a minor single-stranded RNA binding protein (180 kDa) that has immunological cross-reactivity with the major coat protein. The sequence of L-A cDNA clones revealed two open reading frames (ORF), ORF1 and ORF2. These two reading frames overlap by 130 base pairs and ORF2 is in the -1 reading frame with respect to ORF1. Although the major coat protein of the viral particles is encoded by ORF1, the 180-kDa protein is derived from the entire double-stranded RNA genome by fusing ORF1 and ORF2, probably by a -1 translational frameshift. Within the overlapping region is a sequence similar to that producing a -1 frameshift by "simultaneous slippage" in retroviruses. The coding sequence of ORF2 shows a pattern characteristic of viral RNA-dependent RNA polymerases of icosahedral (+)-strand RNA viruses. Thus, the 180-kDa protein is analogous to gag-pol fusion proteins.     

Interaction of the Trans-Frame Potyvirus Protein P3N-PIPO with Host Protein PCaP1 Facilitates Potyvirus Movement

A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses.     

Norovirus regulation of the innate immune response and apoptosis occurs via the product of the alternative open reading frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis.