- Volume 80, Issue 6, 1999

The grapevine fanleaf virus (GFLV) RNA2-encoded polyprotein P2 is proteolytically cleaved by the RNA1-encoded proteinase to yield protein 2A, 2B(MP) movement protein and 2C(CP) coat protein. To further investigate the role of the 2B(MP) and 2C(CP) proteins in virus movement, RNA2 was engineered by alternatively replacing the GFLV 2B(MP) and 2C(CP) genes with their counterparts from the closely related Arabis mosaic virus (ArMV). Transcripts of all chimeric RNA2s were able to replicate in Chenopodium quinoa protoplasts and form tubules in tobacco BY-2 protoplasts in the presence of the infectious transcript of GFLV RNA1. Virus particles were produced when the GFLV 2C(CP) gene was replaced with its ArMV counterpart, but systemic virus spread did not occur in C. quinoa plants. In addition, chimeric RNA2 containing the complete ArMV 2B(MP) gene was neither encapsidated nor infectious on plants, probably because polyprotein P2 was incompletely processed. However, chimeric RNA2 encoding ArMV 2B(MP), in which the nine C-terminal residues were those of GFLV 2B(MP), formed virus particles and were infectious in the presence of GFLV but not ArMV 2C(CP). These results suggest that the nine C-terminal residues of 2B(MP) must be of the same virus origin as the proteinase for efficient proteolytic processing of polyprotein P2 and from the same virus origin as the 2C(CP) for systemic virus spread.

In order to elucidate the function of the C-terminal region of cowpea chlorotic mottle bromovirus (CCMV) movement protein (MP) in cell-to-cell movement, a set of deletions ranging from 10 to 80 amino acids (deltaMP10, deltaMP20, deltaMP33, deltaMP43, deltaMP60 and deltaMP80) was engineered into the MP gene encoded by the biologically active clone C3/deltaCP-EGFP, a variant of CCMV RNA3 that contained wild-type (wt) MP and the enhanced green fluorescent protein (EGFP) gene in place of the coat protein (CP). The effect of each MP deletion on cell-to-cell movement was examined in three susceptible host plants: Chenopodium quinoa, Nicotiana benthamiana and cowpea (Vigno sinensis cv. Black Eye). The results indicate that, except for mutant deltaMP43, infections resulting from the deletion mutants remained subliminal. Interestingly, infections resulting from inoculating mutant deltaMP43, which lacked the 43 most C-terminal amino acids, spread rapidly between cells and the number of infected cells expressing EGFP approached that of control inoculations made with C3/deltaCP-EGFP. To verify whether the presence of wt CP altered the movement behaviour of these mutants, each MP deletion was also incorporated into the genetic background of wt CCMV RNA3 (pCC3) and inoculated independently to all three hosts. The results suggest that the overall movement process exhibited by each MP mutant is influenced profoundly by the presence of CP and the particular host plant tested.

The brown alga Ectocarpus siliculosus frequently carries an endogenous virus, E. siliculosus virus (EsV-1), the genome of which is a circular, double-stranded DNA molecule of about 320 kbp. After infection, which occurs in the unicellular spores or gametes, the virus is present latently in all somatic cells of the host. Virus multiplication is restricted to cells of the reproductive organs. It has been an open question whether the latent viral DNA occurs as a free episome or becomes integrated into the host genome. PCR studies showed that viral DNA co-migrates with high molecular mass DNA in pulsed-field gel electrophoresis, which confirms that latent viral DNA is integrated into the host genome.

We have investigated whether poly(C)-binding protein (PCBP)-1 and PCBP-2 interact with the hepatitis C virus (HCV) 5′ untranslated region. Our results demonstrate that glutathione S-transferase (GST)-PCBP-1 and GST-PCBP-2 fusion proteins bind specifically to the HCV 5′ untranslated region. An antiserum raised against PCBP-2 induced a supershift after incubation with RNA-protein complexes formed between proteins in a HeLa cell cytoplasmic extract and the HCV 5′ untranslated region, indicating that this interaction occurs intracellularly. The complete internal ribosome entry site was necessary for efficient binding, suggesting that maintenance of the secondary structure was necessary for recognition of the binding site by the PCBPs.

Hepatitis C virus (HCV) has high genomic variability and, since its discovery, at least six different types and an increasing number of sub-types have been reported. The HCV genotype may influence virus replication, the natural course of disease and the response to therapy. HCV has been described as a dynamic population of heterogeneous, closely related variants, designated quasispecies. In order to study the degree of genetic variability of strains isolated in Montevideo, Uruguay, sequence data obtained from the 5′ non-coding region of type 1-infected patients were compared with published sequences from 53 different strains of all types isolated all over the world. The phylogenetic analysis revealed that type 1 strains isolated in Montevideo represent a different genetic lineage from major sub-types 1a and 1b strains and indicates an increasing diversification of HCV viruses.

Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP0-P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.

The capacity of DNA vaccines to prime CD8+ T cells makes them excellent candidates for vaccines that are designed to emphasize cellular immunity. However, the long-term stability of CD8+ T cell memory induced by DNA vaccination is poorly characterized. Here, the quality of CD8+ T cell recall responses in mice was investigated more than 1 year after DNA vaccination with the Sendai virus nucleoprotein gene. Cytotoxic T lymphocyte (CTL) activity specific for both dominant and subdominant epitopes could be recalled readily 1 year after vaccination and the frequencies of CTL precursors specific for both of these epitopes were relatively high. These CTL responded strongly to subsequent Sendai virus infection in terms of their ability to migrate to the lung and to differentiate into effector cells. In addition, the recall response to virus infection, as determined by CTL activity in the lungs and IFN-gamma responses in the spleen, was both faster and greater in magnitude than that in control-immunized mice. Significantly, virus titres were reduced at least 100-fold in the lungs of mice that were immunized more than 1 year before infection, as compared with control mice. These data demonstrate that CD8+ T cell memory elicited by DNA vaccination is functionally relevant and persists for at least 1 year.

Respiratory syncytial virus (RSV)-specific cytotoxic T lymphocytes (CTL) or neutralizing antibodies can protect against RSV infection when induced separately by immunization with synthetic peptides. In the work described here, RSV-specific neutralizing antibodies and CTLs were induced after immunization with a cocktail of peptides consisting of a B-cell mimotope (S1S-MAP), a T-helper epitope (SH:45-60) and a CTL epitope linked to a fusion (F) peptide (F/M2:81-95) that were comparable to those induced by the peptides alone. Following challenge, a 190-fold reduction in RSV titre was observed in the lungs of peptide cocktail-immunized mice. The combination of RSV-specific humoral and cellular immunity induced by the peptide cocktail was thus more effective at clearing RSV than peptide-induced humoral or cellular immunity alone.

G2 rotavirus was prevalent in a 1993 epidemic of acute gastroenteritis in Taiwan. In this study, the genetic relationship among G2 rotavirus strains was analysed. The VP7 genes were amplified and sequenced. Except for one strain isolated in 1981, the nucleotide sequences of the VP7 genes of most of the G2 rotaviruses were very similar (identity > 97%) and were closely related to that of a Japanese G2 reference strain, S2. The genetic relatedness of G2 rotaviruses was analysed further by RNA-RNA hybridization. The genomes of the major G2 strains of 1993 did not hybridize well with those of the G2 strains of previous seasons in RNA segments 1, 6 and 7. Partial nucleotide sequences of the VP1 gene were analysed and appeared to be similar among the major G2 strains from the same epidemic (identity > 98%), whereas the identity of the VP1 genes of the major G2 strains of the 1993 epidemic to those of previous seasons was only about 84%. Since the numbers of mutations accumulated in the VP1 and VP7 genes over a period of 10 years were comparable, the significant change in the VP1 genes of the major strains of the 1993 epidemic suggests that these G2 rotaviruses had evolved by genetic reassortment.

The effects of passive antibodies on protection and active immune responses to human rotavirus were studied in gnotobiotic pigs. Pigs were injected at birth with saline or sow serum of high (immunized) or low (control) antibody titre and subsets of pigs were fed colostrum and milk from immunized or control sows. Pigs were inoculated at 3-5 days of age and challenged at 21 days post-inoculation (p.i.) with virulent Wa human rotavirus. Pigs receiving immune serum with or without immune colostrum/milk were partially protected against diarrhoea and virus shedding after inoculation, but had significantly lower IgA antibody titres in serum and small intestinal contents at 21 days p.i. and lower protection rates after challenge compared with pigs given control or no maternal antibodies. IgG antibody titres were consistently higher in small than in large intestinal contents. Pigs given control serum with control colostrum/milk had lower rates of virus shedding after inoculation than those given control serum alone. In summary, high titres of circulating maternal antibodies with or without local (milk) antibodies provided passive protection after inoculation but suppressed active mucosal antibody responses. These findings may have implications for the use of live, oral rotavirus vaccines in breast-fed infants.

Human T-cell leukaemia/lymphoma virus type 1 (HTLV-1) is a pathogenic retrovirus responsible for a number of inflammatory pathologies and adult T-cell leukaemia. Although T-cell tropic in vivo, HTLV-1 can infect a wide variety of cell types in vitro. Cell-to-cell spread of HTLV-1 may require specific binding of envelope to its cellular receptor, with other cell-surface molecules facilitating fusion. Here it is shown that intercellular adhesion molecule-1 or -3 (ICAM-1, ICAM-3) or vascular cell adhesion molecule-1 (VCAM-1) are required for syncytium formation of K562 with HTLV-1-infected MT2 cells but not C91-PL cells. The effect of ICAMs and VCAM-1 on MT2-induced fusion can be blocked by antibodies that bind beta-integrins. These fusion co-factor molecules are effective only when present in combination with HTLV-1 receptor-bearing cells and are not sufficient to mediate syncytium formation alone. The results suggest that engagement of HTLV-1-infected cells with susceptible target cells requires the simultaneous binding of viral envelope glycoprotein to the cellular receptor and co-factor molecules to beta-integrins. The tissue-specific expression of adhesion molecules might therefore influence HTLV-1 virus tropism and pathogenic changes associated with syncytium formation.

Ovine lentiviruses and caprine arthritis-encephalitis virus (CAEV) are prototypic lentiviruses that replicate predominantly in macrophages of infected animals. In situ hybridization of pathologically affected tissues from diseased animals has shown that viral RNA exists in permissive macrophages as well as in non-macrophage cell types that do not support productive virus replication. These findings raise questions about the cellular tropism of these viruses in vivo and how this may relate to their pathogenesis and the establishment of persistent infections. In this study, the susceptibility of macrophages and fibro-epithelial cells derived from goat synovial membrane (GSM) to infection by 14 North American ovine lentivirus strains was examined. All 14 strains were macrophage-tropic, as indicated by expression of viral proteins and by fusion and development of syncytial cytopathic effects following co-culture of infected macrophages with GSM cells. In contrast, neither viral DNA nor viral proteins was detected in GSM cells inoculated with cell-free virus from nine of the 14 strains. Specific virus proteins were immunoprecipitated from restrictive GSM cells following culture with infected macrophages and serial passage of GSM cells to remove the macrophages. The lack of infection of GSM cells by cell-free virus from some ovine lentivirus field strains was circumvented by cell-associated virus infection from infected macrophages to GSM cells following cell-to-cell contact. This strategy could be one of the mechanisms involved in the escape from immune surveillance and establishment of persistent infection in infected animals.

Foamy viruses (FVs) make use of a replication strategy which is unique among retroviruses and shows analogies to hepadnaviruses. The presence of an integrase (IN) and obligate provirus integration distinguish retroviruses from hepadnaviruses. To clarify whether a functional IN is required for FV replication, a mutant in the highly conserved DD35E motif of the active centre was analysed. This mutant was found to be able to express Gag and Pol protein precursors and cleavage products and to generate and deliver cDNA. However, this mutant was replication-deficient. The junctions of individual foamy proviruses with cellular DNA were sequenced. The findings suggest that FV integration is asymmetrical, because the proviruses started with what is believed to be the U3 end of the free linear DNA to generate the conventional TG dinucleotide, while apparently two nucleotides from the U5 end were cleaved to create the complementary CA dinucleotide. Alignment of known FV genome sequences indicated that this mechanism of integration is not restricted to the two FV isolates from which integrates were studied, but appears to be a common feature of this retrovirus subfamily. In conclusion, with respect to the necessity of a functionally active IN for virus replication FVs behave like other retroviruses; their mechanism of integration, however, is probably unique.

The two major characteristics of pathogenesis in African swine fever virus (ASFV) infections of domestic pigs are massive B-cell apoptosis and haemorrhage. The effects of ASFV on porcine B cells have therefore been systematically examined in vivo, by using virus-infected pigs and SCID-Beige mice reconstituted with porcine bone marrow, and in vitro, by using porcine B-cell lines and B cells from normal and ASFV-infected pigs. Secretion of porcine Ig was stimulated by ASFV both in vivo and in bone marrow cultures in vitro, with the virulent Malawi isolate of ASFV being the most effective. Stimulation of Ig secretion in vitro depended on the presence of ASFV-infected macrophages and did not occur with supernatants from ASFV-infected macrophages. Although the virus alone did not stimulate proliferation of purified B cells in vitro, it was co-stimulatory with CD154 (CD40 ligand). The B cells recovered from ASFV-infected porcine lymphoid tissue were of activated surface marker phenotypes and, interestingly, expressed diminished levels of the B-cell co-stimulatory surface molecule CD21. In addition, they were highly sensitive to IL-4 and CD154. These results may be integrated into a model of pathogenesis in which those B cells activated indirectly as a result of virulent ASFV infection of macrophages are not rescued from apoptosis through interaction with CD154, due to the drastic depletion of T cells that occurs early in infection. The consequently diminished specific anti-ASFV antibody response would favour survival of the virus, with the non-specific hypergammaglobulinaemia being perhaps another example of pathogen-mediated immune deviation.

A vaccinia virus-bacteriophage T7 RNA polymerase hybrid transient expression vector has been developed for complementation analysis of late gene functions in vaccinia virus. The conditionally defective virus ts21 was modified to express the bacteriophage T7 RNA polymerase. The derived virus, vtsT7, was conditionally defective in viral late gene expression but produced high levels of a target protein under the control of a T7 promoter at non-permissive temperatures. The level of beta-galactosidase expression under the control of a T7 promoter was slightly lower in vtsT7 infections than those with the prototypical T7 RNA polymerase vector vTF7.3. However, the levels of expression for the human immunodeficiency virus envelope gene, a protein which undergoes post-translational modification, was slightly higher in vtsT7 infections, suggesting that some proteins may be expressed better in the absence of vaccinia virus late gene expression. Infections using vtsT7 at a low m.o.i. at 39 degrees C resulted in the accumulation of high molecular mass, non-linear replicative intermediates of vaccinia virus DNA replication and high levels of expression of a transfected gene proximal to a T7 promoter. The virus vtsT7 provides a means for the analysis of potential trans-acting factors participating in vaccinia virus late processes such as resolution of DNA replicative intermediates.

Studies were performed to determine if the unique N-terminal domain of the R1 subunit from herpes simplex virus (HSV) type 1 ribonucleotide reductase is a substrate for casein kinase 2 (CK2). Transphosphorylation assays demonstrated that R1 was highly phosphorylated by this enzyme with multiple phosphorylation sites mapped to the N terminus between residues 1 and 245. Immunoprecipitation pull-down assays using R1-specific antisera failed to demonstrate a stable interaction between R1 and CK2 but residual amounts of CK2 present after immunoprecipitation efficiently transphosphorylated R1. Activity assays with a peptide substrate identified CK2 in R1 immunoprecipitated from infected-cell extracts but did not detect activity in R1 proteins immunoprecipitated from bacterial extracts. However, Western blotting identified potential E. coli homologues of the CK2 alpha and beta subunits. These results support conclusions that the N-terminal domain of HSV R1 is not a protein kinase and that all previous results can be explained by contaminating kinases, principally CK2.

In cattle, bovine herpesvirus-1 (BHV-1) can cause a mild genital disease known as infectious pustular vulvovaginitis (IPV) and a more severe respiratory disease known as infectious bovine rhinotracheitis (IBR). On the basis of epidemiological data, it has been proposed that these diseases are caused by strains with different genotypes (IBR by BHV-1.1 and IPV by BHV-1.2 strains). By using a panel of 237 BHV-1 isolates, a monoclonal antibody (MAb 71) was found that failed to react with all 54 putative IPV strains in the panel, and another MAb (77) was found that did not react with 16 of these 54 IPV strains. Because MAbs 71 and 77 also failed to react with a BHV-1.1 glycoprotein C (gC)-deletion mutant, it was hypothesized that both MAbs recognize BHV-1.1 gC. By marker-rescue experiments and by expressing fragments of the BHV-1.1 gC gene in recombinant baculoviruses, it was shown that both MAbs indeed recognize BHV-1.1 gC. MAb 71 recognizes the N-terminal half and MAb 77 recognizes the C-terminal half of BHV-1.1 gC. In a PEPSCAN analysis with 12-mer oligopeptides, MAb 71 reacted with overlapping peptides containing gC amino acid residues 75-80 and MAb 77 did not react in this analysis. The differences in gC found in this study may contribute to the biological differences between BHV-1.1 and BHV-1.2.

Human herpesvirus-6 (HHV-6), like other betaherpesviruses, shows cell fusion with wild-type strains, and this cellular spread is mediated by the glycoprotein gH/gL complex. Anti-fusion monoclonal antibodies (MAbs) specific for HHV-6 glycoprotein gH inhibit infection and prevent cellular spread by syncytia formation. Reactivity of these MAbs with gH deletion mutants suggests a conserved C-terminal fusion-associated domain. A conserved motif here has an N-glycosylation site and characteristics of a beta turn. Motif deletion abrogated MAb recognition while co-expression with glycoprotein gL restored this conformational epitope, indicating the importance of folding and not glycosylation at this site. Our previous studies showed gL binding to gH at an N-terminal domain specific for betaherpesviruses. To further examine the function of this N-terminal domain, a betaherpesvirus-specific motif was deleted. This mutant gH still bound gL, and was recognized by the anti-fusion MAbs; however, recognition was now primarily in the immature form and reduced during processing to the mature form. A model is discussed whereby gL binding gH at the N-terminal domain acts to draw together the C-terminal extracellular domain and this interaction affects a functional conformation during glycoprotein maturation.

Human cytomegalovirus (HCMV) strains can be classified into different glycoprotein B (gB) genotypes. In a previous study, frequent intragenic variation of the gB gene was shown. The aim of this study was to analyse whether gB variation was due to homologous recombination. The gB gene of DNA extracts derived from the peripheral blood leukocytes of 14 immunosuppressed patients was amplified by PCR and cloned. Three variable sites of gB were analysed by restriction fragment analysis and DNA sequencing and compared with published prototypic strains. In three patients doubly infected with two distinct HCMV gB strains, prototypic (60-85%) and non-prototypic recombinant strains (5-40%) were detected. To demonstrate that homologous recombination is driving HCMV gB variability, cells were coinfected with plaque-purified prototypic gB strains and recombinant gB genes were selectively amplified by PCR. gB recombinants were detected after 15 days of coculture and cross-over sites were determined by sequencing. These data indicate that homologous recombination contributes to the variability of the gB gene in vitro and in vivo.