- Volume 80, Issue 5, 1999

The gene encoding the 36.5 kDa (‘36K’) nonstructural protein located on RNA3 of olive latent virus 2 (OLV-2) was cloned, expressed with the Escherichia coli pGEX-2T system and the purified protein used to raise a polyclonal antiserum. Immunoblot analysis of OLV-2-infected Nicotiana benthamiana plants showed that the 36K protein accumulated in the early stages of infection and was associated with a subcellular fraction enriched in cytoplasmic membranes. In infected cells there were tubular structures, some containing virus-like particles, scattered in the cytoplasm or protruding from or penetrating the cell wall at the plasmodesmata. Immunogold labelling localized the 36K protein in the plasmodesmata of OLV-2-infected cells and showed it to be associated with virus-containing tubules. Leaf trichome cells of N. tabacum plants, transformed with a 36K–green fluorescent protein (GFP) fusion construct, revealed localized fluorescence in the cell walls, possibly due to association of the fusion protein with plasmodesmata. When the same 36K–GFP fusion protein was expressed in N. tabacum protoplasts, long tubular fluorescent structures protruded from the protoplast surface, suggesting that the 36K protein is responsible for tubule induction. The conclusion is drawn that this protein is likely to be the OLV-2 movement protein, mediating cell-to-cell virus movement, and that movement is by a tubule-guided mechanism.

Virions of lettuce infectious yellows virus (LIYV; genus Crinivirus) were purified from LIYV-infected plants and their protein composition was analysed by SDS-PAGE and immunoblotting. Virion preparations contained the major capsid protein (CP), but the minor capsid protein (CPm), p59 and the HSP70 homologue were also identified by immunoblot analysis. Immunogold labelling analysis showed that CP constituted the majority of the LIYV virion capsid, but CPm was also part of the capsid and localized to one end of the virion, similar to the polar morphology seen for viruses in the genus Closterovirus. p59 and the HSP70 homologue were not detected on virions by immunogold labelling, but were always detected in virion preparations by immunoblot analysis. Purified LIYV virions were used for in vitro acquisition analysis with Bemisia tabaci whiteflies and were efficiently transmitted to plants. Infectivity neutralization analyses were done using antisera to the LIYV-encoded CP, CPm, p59 and HSP70 homologue. Only antiserum to the CPm effectively neutralized LIYV transmission by B. tabaci. These data suggest that the LIYV-B. tabaci transmission determinants are associated with purified virions, and that the LIYV virion structural protein CPm is involved in transmission by B. tobaci.

The triple gene block protein 1 (TGBp1) encoded by open reading frame 2 of bamboo mosaic potexvirus (BaMV) was overexpressed in Escherichia coli and purified in order to test its RNA-binding activity. UV crosslinking assays revealed that the RNA-binding activity was present mainly in the soluble fraction of the refolded TGBp1. The binding activity was nonspecific and salt concentration-dependent: activity was present at 0–50 mm NaCl but was almost abolished at 200 mm. The RNA-binding domain was located by deletion mutagenesis to the N-terminal 3–24 amino acids of TGBp1. Sequence alignment analysis of the N-terminal 25 amino acids of the TGBp1 homologues of potexviruses identified three arginine residues. Arg-to-Ala substitution at any one of the three arginines eliminated most of the RNA-binding activity, indicating that they were all critical to the RNA-binding activity of the TGBp1 of BaMV.

Potyviral helper component-proteinase (HC-Pro) is a multifunctional protein involved in aphid transmission, long-distance movement, polyprotein processing, genome amplification and symptom expression. It has been proposed that the active form of HC-Pro is a dimer and that coat protein (CP)-HC-Pro interaction is required for aphid transmission. To test these proposed interactions between CP and HC-Pro of potato A potyvirus (PVA), the yeast two-hybrid system was used. HC-Pro was shown to interact with itself in vivo in yeast cells, as did CP. Taken together with previous observations, we conclude that the functional HC-Pro is a homodimer. Deletion analysis showed that a 24 aa domain in the N-terminal half and the C-terminal proteinase part of HC-Pro were required for the interaction between HC-Pro molecules. No interactions were found between HC-Pro and CP using the genes of aphid-transmissible as well as aphid non-transmissible strains of PVA.

Amino acid differences between helper component-proteinase (HC-Pro) and coat protein (CP) that are putatively associated with biological differences between isolates PVA-B11 and PVA-U of potato A potyvirus (PVA) were studied using an infectious cDNA clone of PVA-B11. Replacement of the entire CP gene of PVA-B11 with the CP gene of PVA-U reduced virus accumulation in tobacco 5-fold, to the level of PVA-U. In contrast, four simultaneous amino acid substitutions made in PVA-B11 HC-Pro (according to PVA-U HC-Pro) increased virus accumulation 2- to 4-fold. A single substitution (S7G) at the CP N terminus reduced virus accumulation 10-fold, but restored aphid-transmissibility of PVA-B11. Simultaneous mutation of HC-Pro and replacement of CP in B11 delayed systemic movement in tobacco and limited cell-to-cell movement in potato. These results imply coordinated functions of HC-Pro and CP in accumulation and movement of PVA, because the phenotypic effects caused by simultaneous mutation of the two genes were different from the expected ‘sum’ of phenotypic changes observed following mutation of only one gene at a time.

The complete nucleotide sequence of a virus infecting winter wheat in Shandong province, China has been determined. This was previously thought to be soil-borne wheat mosaic virus but, while the two viruses are related, they are only 75% (RNA1) and 63% (RNA2) identical at the nucleotide level, while the amino acid sequences share from 62% (19 kDa RNA2 product) to 84% (RNA1 replicase) identity. The analysis shows that the Chinese virus should be considered a new member of the genus Furovirus and has been named Chinese wheat mosaic virus (CWMV). A Cys-Gly…Cys-Gly-X-X-His amino acid pattern was identified in the cysteine-rich protein of CWMV and those of several other plant virus genera, which seems likely to have some functional significance.

Bovine viral diarrhoea virus (BVDV) belongs to the genus Pestivirus of the family Flaviviridae. Both a noncytopathic (ncp) and an antigenically related cytopathic (cp) BVDV can be isolated from persistently infected animals suffering from mucosal disease. In every case studied so far, the genomic changes leading to the cp biotype correlate with the production of the NS3 nonstructural protein, which, in the ncp biotype, is present in its uncleaved form, NS23. This report shows that, in contrast to ncp BVDV, the cp biotype induces apoptosis in cultured embryonic bovine turbinate cells. Early in the process of apoptosis, cells show a rise in the intracellular level of reactive oxygen species, which is indicative of oxidative stress. This precedes two hallmarks of apoptosis, caspase activation as shown by cleavage of the caspase substrate poly(ADP-ribose) polymerase, and DNA fragmentation. Cells were protected from apoptosis only by certain antioxidants (butylated hydroxyanisole and ebselen), whereas others (N-acetylcysteine, pyrrolidine dithiocarbamate, lipoic acid, dihydrolipoic acid and tiron) turned out to be ineffective. Antioxidants that protected cells from apoptosis prevented oxidative stress but failed to block virus growth. These observations suggest that oxidative stress, which occurs early in the interaction between cp BVDV and its host cell, may be a crucial event in the sequence leading to apoptotic cell death. Hence, apoptosis is not required for the multiplication of the cp biotype of BVDV.

The glycoproteins Erns of classical swine fever virus (CSFV) and Erns and E2 of bovine viral diarrhoea virus (BVDV) are shown to be located at the surface of infected cells by the use of indirect immunofluorescence and by cytofluorometric analysis. The positive immunostaining of the cell surface was further analysed by immunogold electron microscopy and it could be shown that only extracellular virions were labelled. Gold granules were not seen at the cellular plasma membrane. In contrast to BVDV E2, the CSFV E2 of virions sticking to the plasma membrane was not accessible to the respective monoclonal antibodies. However, CSFV particles isolated from culture supernatant were able to bind both monoclonal anti-Erns and anti-E2 antibodies. For CSFV and BVDV, binding of anti-Erns antibodies to the virions was more pronounced than that of anti-E2. This finding was unexpected since E2 is considered to be the immunodominant glycoprotein.

The crystal structure coordinates of the hepatitis C virus NS3 protease (HCVpro) were used to develop an homology model of the dengue 2 virus NS3 protease (DEN2pro). The amino acid sequence of DEN2pro accommodates the same alpha-helices, beta-sheets and protein-binding domains as its HCVpro counterpart, but the model predicts a number of significant differences for DEN2pro and its interactions with substrates and cofactor. Whereas HCVpro contains a Zn2+-binding site, there is no equivalent metal-binding motif in DEN2pro. It is possible that the structural role played by the zinc ion may be provided by a salt bridge between Glu93 and Lys145. The two-component viral protease comprises NS3 and a virus-encoded cofactor, NS4A for HCV and NS2B for DEN2. Previous studies have identified a central 40 amino acid cofactor domain of the dengue virus NS2B that is required for protease activity. Modelling of the putative interactions between DEN2pro and its cofactor suggests that a 12 amino acid hydrophobic region within this sequence (70GSSPILSITISE81) may associate directly with NS3. Modelling also suggests that the substrate binds in an extended conformation to the solvent-exposed surface of the protease, with a P1-binding site that is significantly different from its HCV counterpart. The model described in this study not only reveals unique features of the flavivirus protease but also provides a structural basis for both cofactor and substrate binding that should prove useful in the early design and development of inhibitors.

The phosphoprotein NS5A of hepatitis C virus has recently been suggested to control PKR protein kinase for resistance to interferon. To investigate other functions of NS5A, studies were initiated on the regulation of transcription of important cellular genes and of cell growth by this protein. The results suggested that NS5A protein represses transcription of the cell cycle regulatory gene p21WAF1, while it activates the human proliferating cell nuclear antigen gene in murine fibroblasts and human hepatoma cells. Furthermore, introduction of NS5A into murine fibroblasts (NIH3T3) promoted anchorage-independent growth and tumour formation in nude mice. Thus, NS5A appears to exhibit a role in cell growth regulation.

Hepatitis E virus (HEV) is a non-enveloped, positive-strand RNA virus, with the genome encoding three open reading frames (ORFs) of which ORF 2 directs translation of the capsid protein, PORF2. Following pulse-labelling and cell fractionation of PORF2 expressed in mammalian cells using the Semliki Forest virus replicon, the capsid protein was detected as three major species of 78 (PORF2), 82 and 86 kDa, with P82 and P86 being N-glycosylated (gPORF2 and ggPORF2, respectively). Although gPORF2 and ggPORF2 species represented 79% of total PORF2 after 20 min metabolic labelling and were largely membrane-associated, the glycosylated PORF2 species were much less stable than non-glycosylated PORF2, which was present in the cytosol and represented the major product accumulated in the cell. In the absence of detectable surface expression or export of PORF2, this suggests that glycosylated ORF 2 proteins may not be intermediates in HEV capsid assembly.

Recombinant Semliki Forest virus (rSFV) vaccines encoding louping ill virus (LIV) genes prME and NS1 were examined. Cells transfected with rSFV-prME RNA showed correct processing of the precursor prME and the release into the medium of M and E proteins in particulate form, whilst rSFV-NS1-transfected cells secreted glycosylated, heat-labile NS1 dimers. Mice immunized with rSFV particles produced antibodies against prME and NS1 that were mainly of the IgG2a subtype, indicating that a T-helper 1 immune response was induced. Immunization with prME- or NS1-encoding particles induced T-cell proliferation. Mice vaccinated intraperitoneally (i.p.) with rSFV-prME and/or rSFV-NS1 were significantly protected from lethal i.p. challenge by two strains of LIV, the virulent LI/31 strain, from which the commercial LIV vaccine is derived, and the less-virulent LI/I antibody-escape variant. Intranasal (i.n.) vaccination was protective for rSFV-prME only against LI/31 challenge and not against challenge with LI/I. Immunization with rSFV-NS1 was protective against i.p. and i.n. challenge with both virus strains when given i.p., but was not protective when given i.n. For unvaccinated mice infected with LIV, all animals showing clinical signs had severe degenerative and inflammatory lesions in the central nervous system. None of the rSFV-vaccinated mice that survived challenge showed central nervous system pathology, with the exception of mild leptomeningitis in a minority of LI/31-infected mice. This suggests that protection following immunization with rSFV must occur at early stages of LIV infection.

RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66% similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side.

The G protein sequences of fourteen animal rhabdoviruses, representing all four recognized genera (Vesiculovirus, Lyssavirus, Ephemerovirus and Novirhabdovirus) and the ungrouped sigma virus, were aligned using CLUSTAL W and adjusted to account for obvious sequence similarities not detected by the algorithm. Analysis of the alignment indicated remarkable preservation of G protein structural features including cysteine residues, antigenic sites and significant elements of secondary structure (α-helices, β-strands and loops). Twelve highly conserved cysteine residues were assigned numbers (CI to CXII) according to their location in the alignment. Other cysteine residues were assigned numbers (C0 to CXIIe) according to their position relative to the conserved cysteines. The pattern of conservation of cysteine residues and the structural characteristics of identified discontinuous antigenic sites were used to deduce a model for G protein structure. Six absolutely conserved cysteines are predicted to associate in three disulphide bridges (CI-CXII; CVIII-CXI; CIX-CX) that form the core of the G protein structure and define the common discontinuous antigenic site. The associations of six other highly conserved cysteines (CII-CIV; CIII-CV; CVI-CVII) are predicted by the absence of a specific pair in all viruses within a genus. Of the other cysteines, one pair occurs only in ephemeroviruses and novirhabdoviruses (C0-CXIIa); two pairs occur only in ephemeroviruses (CIb-CVIIIa; CXIIb-CXIIe); and two pairs occur only in lyssaviruses (CIa-CVIIIb; CXIIc-CXIId). The structures predicted by the model account for the preservation of conformational antigenic sites, accommodate genus-specific variations, and are generally consistent with previous observations of G protein structure.

To study the molecular basis of virulence of viral haemorrhagic septicaemia virus (VHSV), we used a cross-reactive neutralizing MAb to select MAb-resistant (MAR) mutants with reduced pathogenicity for fish. From sequence determination of the G gene of MAR mutants, attenuated laboratory variant and avirulent field strains, we identified two distant regions of the glycoprotein associated with virulence: region I (aa 135-161), homologous to the putative fusion peptide of both rabies virus (RV) and vesicular stomatitis virus (VSV), and region II (surrounding aa 431-433), homologous to RV and VSV domains controlling the conformational changes necessary for the fusion process to take place. Simultaneous mutations in both regions resulted in the most attenuated phenotype and we obtained genetic evidence that regions I and II may be structurally linked. As the MAR mutants had mutations in or near domains involved in fusion, the fusion properties of VHSV and its variants were analysed. This work allowed us to postulate that the fusion domain of VHSV is probably constituted of two distinct regions of the protein connected through a disulfide bridge between cysteines 110 and 152. Finally, we obtained evidence suggesting that the pH threshold for fusion is a determinant for virulence: restriction of fusion to a more acidic pH was associated with attenuation for the variant tr25 which had a shift of the threshold for maximal fusion from pH 6.30 (for the parental strain) to pH 6.00; conversely, two field strains which had maximal fusion at pH 6.60 were the most virulent.

A highly pathogenic simian/human immunodeficiency virus (SHIV), designated C2/1, was obtained by serum passages in cynomolgus monkeys of p-SHIV, an SHIV strain that contains the env gene of pathogenic human immunodeficiency virus type 1 89.6. CD4+ lymphocyte depletion was induced within 1 week of the SHIV-C2/1 infection in peripheral blood as well as in various lymphoid organs in all the animals tested, with symptoms of diarrhoea and no increase in body weight, followed by intense viraemia. Serum antibody against Env protein was detected from 4 weeks after the virus infection, while the anti-Gag antibody response was absent in the SHIV-C2/1-infected animals. In contrast, both anti-Gag and anti-Env antibody responses were present in animals infected with p-SHIV or the non-pathogenic SHIV-MN. Sequencing of the env gene of isolates of SHIV-C strains showed conserved amino acid changes in the Env C2 and V3 regions that included changes to negatively charged amino acids, in the cytoplasmic region of gp41 that included a 42 amino acid deletion, and in the Nef protein. The pathogenic SHIV-C2/1-monkey model suggests that virus-specific pathogenicity in SHIV infection may be associated with the absence of anti-Gag antibody responses in animals and may be caused by genetic changes during serum passage in vivo.

Primary isolates of human and simian immunodeficiency viruses (HIV and SIV) use the chemokine receptor CCR5, in association with CD4, as coreceptor. During AIDS progression, HIV-1 and HIV-2 often adapt to use additional cofactors, particularly CXCR4. In contrast, SIV isolates do not use CXCR4, but other coreceptors such as BOB/GPR15 and Bonzo/STRL33. Only limited information is currently available on usage of BOB/GPR15 and Bonzo/STRL33 by HIV-1. Therefore, we investigated a panel of gp160 clones from 15 primary isolates, representing 5 different subtypes, for utilization of these cofactors. The majority of HIV-1 envelopes mediated entry into BOB/GPR15-expressing cells, albeit often with low efficiency. Usage of Bonzo/STRL33 was less common and usually inefficient. To investigate if HIV-1 entry via these orphan receptors is sufficient to allow virus replication, 15 uncloned primary HIV-1 isolates and 7 molecular clones were used to infect target cells expressing CD4 and Bonzo/STRL33 or BOB/GPR15. Three primary isolates and two molecular clones replicated efficiently in cells expressing BOB/GPR15. Two of these isolates were X4-tropic, two were R5X4-tropic and one was R5-tropic. In contrast, none of the HIV-1 variants showed significant levels of replication in Bonzo/STRL33-expressing cells. Our data show that some HIV-1 isolates of different genetic subtype and of different biological phenotype use BOB/GPR15 for productive infection and suggest that this cofactor may play a role in HIV-1 pathogenesis and transmission.

Chronic hepatitis B treatment has been significantly improved by interferon (IFN) treatment. However, some studies have suggested that hepatitis B virus (HBV) might have a direct effect on the resistance to IFN. Defective particles, generated by spliced HBV RNA and associated with chronic hepatitis B, have been previously characterized; expression of these particles leads to cytoplasmic accumulation of the capsid protein. The aim of this study was to investigate the role of these defective genomes in IFN resistance. The global antiviral activity of IFN was studied by virus yield reduction assays, the expression of three IFN-induced antiviral proteins was analysed by Western blotting and confocal microscopy, and the regulation of MxA gene expression was studied by Northern blotting and the luciferase assay, in Huh7 cells transfected with a complete or the defective HBV genome. Results showed that the expression of the defective genome reduces the antiviral activity of IFN and that this modulation involves a selective inhibition of MxA protein induction by the HBV capsid protein. Our results also show the trans-suppressive effect of the HBV capsid on the MxA promoter, which might participate in this phenomenon. In conclusion, this study shows a direct interplay between the IFN-sensitive pathway and the capsid protein and might implicate this defective HBV genome in virus persistence.

To determine the potential of replication-competent (E3-deleted) bovine adenovirus-3 (BAV-3) as a delivery system for vaccine antigens in calves, we evaluated the ability of recombinant BAV-3 expressing different forms of of bovine herpesvirus-1 (BHV-1) glycoprotein gD to protect against BHV-1 infection in calves that had pre-existing BAV-3 specific antibodies. Three- to four-month-old calves, vaccinated intranasally with recombinant BAV-3 expressing full-length gD (BAV3.E3gD) or a truncated version of gD (gDt) (BAV3.E3gDt), or with E3-deleted BAV-3 (BAV3.E3d; control), were challenged with BHV-1 strain 108. Vaccination with BAV3.E3gD or BAV3.E3gDt induced gD-specific antibody responses in serum and nasal secretions, and primed calves for gD-specific lymphoproliferative responses. In addition, all calves developed complement-independent neutralizing antibodies against BHV-1. Protection against viral challenge was observed in calves vaccinated with recombinant BAV3.E3gD or BAV3.E3gDt as shown by a significant reduction in body temperature and clinical disease, and a partial reduction in the amount and duration of virus excretion in nasal secretions. These results indicate that replication-competent BAV-3-based vectors can induce protective immune responses in calves (the natural host) that have pre-existing BAV-3-specific antibodies.