- Volume 80, Issue 11, 1999

In order to investigate the molecular mechanism of the reported negative effect of the Nef protein of human immunodeficiency virus type 1 (HIV-1) on the cellular transcription factors NF-κB and AP-1, human T cell lines (both populations and subclones) expressing the nef gene from HIV-1 clone pNL432 were constructed. Functional expression of the nef gene was confirmed by downregulation of CD4 and MHC class I proteins on the cell surface as measured by fluorescence-activated cell sorter analysis. However, contrary to previous reports, no significant difference was found in the induced level of NF-κB and AP-1 activity between nef + and nef − cell lines upon stimulation by phorbol 12-myristate 13-acetate and phytohaemagglutinin, as measured by transient transfection and electromobility shift assays. These data indicate that the Nef protein does not have a negative effect on the induction of NF-κB and AP-1.

Co-encapsidation of retroviral RNAs into virus particles allows for the generation of recombinant proviruses through events of template switching during reverse transcription. By use of a forced recombination system based on recombinational rescue of replication- defective primer binding site-impaired Akv–MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5′ untranslated region harbouring the supposed RNA dimer-forming cis -elements and (ii) that copackaged retroviral RNAs can recombine despite pronounced sequence dissimilarity at the cross-over site(s) and within parts of the genome involved in RNA dimerization, encapsidation and strand transferring during reverse transcription. We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species.

We analysed the role of neuraminidase (NA) on haemadsorption by the haemagglutinin (HA) protein of influenza B virus. The influenza B virus mutant ts-7 has a temperature-sensitive mutation in the NA protein. At high temperature, cells infected with this virus did not exhibit haemadsorption activity, but the addition of bacterial neuraminidase (bNA) restored haemadsorption activity. COS cells transfected with HA cDNAs of B/Kanagawa/73 or B/Lee/40 virus showed no evidence of haemadsorption. However, with the addition of bNA or co- transfection with NA cDNA of the B/Lee/40 virus, haemadsorption was observed. Experiments with point-mutated HA cDNAs of B/Lee/40 virus showed that two N-acetyl glycosylation sites at amino acid residues 160 and 217 were responsible for the inability of the HA protein to adsorb to erythrocytes. These results indicated that haemadsorption by the HA protein of influenza B virus required the involvement of NA. Because the NA inhibitor Zanamivir was reported not to penetrate cells, we investigated the action of this inhibitor and found that Zanamivir inhibited haemadsorption on MDCK cells infected with B/Kanagawa/73 or B/Lee/40 virus. After removing Zanamivir by washing, the addition of bNA restored the haemadsorption activity on the infected cells. Scanning electron microscopy indicated that at 0·4 μM Zanamivir, HA protein did not adsorb to erythrocytes but retained the ability to aggregate virions. However, at 4 μM Zanamivir, distinct virion formation could not be observed.

Substitution of Val113 in Sendai virus (SeV) M protein generates non-functional polypeptides, characterized by their exclusion from virus particles and by their ability to interfere with virus particle production. These phenotypic traits correlate with a single-band PAGE migration profile, in contrast to wild-type M (Mwt ), which separates into two species, one of which is a phosphorylated form. The single-band migration is likely to result from a conformational change, as evidenced by the lack of maturation of a native epitope and by a particular tryptic digestion profile, and not from the phosphorylation of all M molecules, an assumption consistent with the PAGE migration feature. One of the M mutants (HA–M30 , an M protein carrying Thr112Met and Val113 Glu substitutions tagged with an influenza virus haemagglutinin epitope) was characterized further in the context of SeV infection, i.e. under conditions of co-expression with Mwt. HA–M 30 is shown (i) to bind mainly to membrane fractions, (ii) not to co-precipitate Mwt, as HA–Mwt does, (iii) to interfere with the binding of nucleocapsids to membranes and (iv) to accumulate in perinuclear regions, in contrast to HA-Mwt , which is also found at the cell periphery. Such mutants constitute potential tools for the identification of critical steps in paramyxovirus assembly and budding.

Recombinant lentogenic Newcastle disease virus (NDV) of the vaccine strain Clone-30 was reproducibly generated after simultaneous expression of antigenome-sense NDV RNA and NDV nucleoprotein, phosphoprotein and RNA-dependent RNA polymerase from plasmids transfected into cells stably expressing T7 RNA polymerase. For this purpose, the genome of Clone-30, comprising 15186 nt, was cloned and sequenced prior to assembly into a full-length cDNA clone under control of a T7 RNA polymerase promoter. Recombinant virus was amplified by inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. Two marker restriction sites comprising a total of five nucleotide changes artificially introduced into noncoding regions were present in the progeny virus. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth characteristics in cell culture and in embryonated eggs. Moreover, an intracerebral pathogenicity index of 0·29 was obtained for both viruses as determined by intracerebral inoculation of day-old SPF chickens, proving that the recombinant NDV is a faithful copy of the parental vaccine strain of NDV.

By using mice with a targetted disruption in the gene encoding inducible nitric-oxide synthase (iNOS), we have studied the role of nitric oxide (NO) in lymphocytic choriomeningitis virus (LCMV)-induced, T cell-mediated protective immunity and immunopathology. The afferent phase of the T cell-mediated immune response was found to be unaltered in iNOS-deficient mice compared with wild-type C57BL/6 mice, and LCMV- induced general immunosuppression was equally pronounced in both strains. In vivo analysis revealed identical kinetics of virus clearance, as well as unaltered clinical severity of systemic LCMV infection in both strains. Concerning the outcome of intracerebral infection, no significant differences were found between iNOS-deficient and wild-type mice in the number or composition of mononuclear cells found in the cerebrospinal fluid on day 6 post-infection. Likewise, NO did not influence the up-regulation of proinflammatory cytokine/chemokine genes significantly, nor did it influence the development of fatal meningitis. However, a reduced virus-specific delayed-type hypersensitivity reaction was observed in iNOS-deficient mice compared with both IFN-γ-deficient and wild-type mice. This might suggest a role of NO in regulating vascular reactivity in the context of T cell-mediated inflammation. In conclusion, these findings indicate a minimal role for iNOS/NO in the host response to LCMV. Except for a reduced local oedema in the knockout mice, iNOS/NO seems to be redundant in controlling both the afferent and efferent phases of the T cell-mediated immune response to LCMV infection.

Primary human hepatocytes were used to develop a culture model for in vitro propagation of hepatitis C virus (HCV). Production of positive- strand full-length viral RNA in cells and culture supernatants was monitored by PCR methods targeting three regions of the viral genome: the 5′ NCR, the 3′ X-tail and the envelope glycoprotein E2. De novo synthesis of negative-strand RNA was also demonstrated. Evidence for a gradual increase in viral components over a 3 month period was obtained by two quantitative assays: one for evaluation of genomic titre (quantitative PCR) and one for detection of the core antigen. Production of infectious viral particles was indicated by passage of infection to naive hepatocyte cultures. Reproducibility of the experiments was assessed using cultures from three liver donors and eleven sera. Neither the genotype, nor the genomic titre, nor the anti-HCV antibody content, were reliable predictive factors of serum infectivity, while the liver donor appeared to play a role in the establishment of HCV replication. Quasispecies present in hepatocyte cultures established from three different liver donors were analysed by sequencing hypervariable region 1 of the E2 protein. In all three cases, the complexity of viral quasispecies decreased after in vitro infection, but the major sequences recovered were different. These data strongly suggest that human primary hepatocytes are a valuable model for study of persistent and complete HCV replication in vitro and for identification of the factors (viral and/or cellular) associated with successful infection.

Striped jack nervous necrosis virus (SJNNV), the causative agent of viral nervous necrosis in marine fish, is a member of the family Nodaviridae whose genome consists of two positive-sense RNA molecules encapsidated in a single virion. In this study, the nucleotide sequence of SJNNV RNA1 was determined. The SJNNV RNA1 was 3081 bases long and contained a single ORF encoding 983 aa of approximately 110 kDa. The sequence identities between RNA1 of SJNNV and RNA1 of insect nodaviruses were 28% at the nucleotide and amino acid levels, although the conserved motifs for the RNA-dependent RNA polymerase were located at almost the same positions in the amino acid sequences. The present study, together with our previous work on SJNNV RNA2, suggests that a new genus, Piscinodavirus, should be created in the family Nodaviridae.

Striped jack nervous necrosis virus (SJNNV), a fish nodavirus, is the causative agent of viral nervous necrosis in marine fishes. The fish nodaviruses are divided into four different genotypes based on the nucleotide sequence of the coat protein gene. In the present study, partial coat protein genes of fish nodaviruses were expressed. This allowed the serological relationship among the different virus genotypes to be analysed and neutralizing epitopes on the coat protein to be mapped. Western blot analysis revealed that SJNNV and other fish nodavirus genotypes shared a significant number of common antigenic determinants, although SJNNV was serologically distinguishable. The results suggested that the SJNNV determinant for neutralizing MAbs was a linear epitope, which consisted of a repeated amino acid sequence within the coat protein. One of the neutralizing epitopes of SJNNV was deduced to be PAN at aa 254–256 in the coat protein.

Epidemiological studies on the VP7 serotype prevalence of human rotaviruses in South Africa and the United Kingdom identified several strains which could not be serotyped as G1–G4 by monoclonal antibodies. Further analysis of these strains with a G8-specific monoclonal antibody and with probes for human rotaviruses confirmed them as G8 rotaviruses. These G8 strains exhibited a high degree of sequence identity when compared with each other and with other rotavirus G8 strains. Five South African strains were further characterized as VP6 subgroup I, but with a long RNA electropherotype, which is similar to the G8 strains previously isolated in Finland. In the UK strains, one was VP6 subgroup II with a long RNA electropherotype (similar to the Italian G8 strain). The other two were subgroup I with a short RNA electropherotype. None of these strains exhibited the super-short RNA electropherotype described in the prototype G8 strains recovered from Indonesia (69M).

Expression cassettes containing the codons for the pestivirus E rns signal peptide (Sig) followed by a chemically synthesized ORF that encoded the bovine viral diarrhoea virus (BVDV) strain C86 glycoprotein E2, a class I membrane glycoprotein, were constructed with and without a chimeric intron sequence immediately upstream of the translation start codon, and incorporated into the genome of bovine herpesvirus-1 (BHV-1). The resulting recombinants, BHV- 1/SigE2syn and BHV-1/SigE2syn-intron, expressed comparable quantities of glycoprotein E2, and Northern blot hybridizations indicated that the presence of the intron did not increase significantly the steady-state levels of transcripts encompassing the SigE2syn ORF. In BHV-1/SigE2syn- infected cells, the 54 kDa E2 glycoprotein formed a dimer with an apparent molecular mass of 94 kDa, which was further modified to a 101 kDa form found in the envelope of recombinant virus particles. Penetration kinetics and single-step growth curves indicated that the incorporation of the BVDV E2 glycoprotein in the BHV-1 envelope, which apparently did not require BHV-1-specific signals, interfered with entry into target cells and egress of progeny virions. These results demonstrate that a pestivirus glycoprotein can be expressed efficiently by BHV-1 and incorporated into the viral envelope. BHV-1 thus represents a promising tool for the development of efficacious live and inactivated BHV-1-based vector vaccines.

Bovine herpesvirus type 5 (BHV-5) is the causative agent of a fatal meningo-encephalitis in calves and is closely related to BHV-1 which causes infectious bovine rhinotracheitis. The gene encoding BHV-5 glycoprotein gH was sequenced. A high degree of conservation was found between BHV-1 and BHV-5 deduced gH amino acid sequences (86·4%), which is also observed for all alphaherpesvirus gH sequences. Transcriptional analysis revealed a 3·1 kb mRNA as the specific gH transcript which was detected 2 h post-infection (p.i.). Twelve out of twenty-one MAbs directed against BHV-1 gH immunoprecipitated a 108–110 kDa glycoprotein, which was then designated BHV-5 gH. Synthesis and intracellular processing of BHV- 5 gH was analysed in infected MDBK cells using gH cross-reacting MAbs. Glycoprotein gH was expressed as a beta-gamma protein, detected by radioimmunoprecipitation as early as 3 h p.i. Glycosylation studies indicated that BHV-5 gH contains N-linked carbohydrates which are essential for the recognition of the protein by the MAbs. This suggests that N-linked glycans are involved in protein folding or are targets for the gH cross-reacting MAbs. Plaque- reduction neutralization assays showed that at least one BHV-1 gH antigenic domain is lacking in BHV-5 which may possibly relate to in vivo differences in virus tropism.

The membrane-impermeable thiol blocker 5′5-dithiobis 2- nitrobenzoic acid (DTNB) blocked infectivity of human cytomegalovirus (CMV) although the virus still bound to cells. DTNB-treated CMV regained 65% of its infectivity after incubation with the disulfide bond-reducing agent dithiothreitol. These observations suggest that free thiol groups on CMV are required for infectivity and may participate in disulfide bond formation during virus entry.

Following extensive propagation in fibroblasts, human cytomegalovirus (HCMV) loses tropism for a number of otherwise natural host cells, in particular, endothelial cells. In this study, the hypothesis was tested that loss of endothelial tropism is associated with the appearance of genomic variants. Initial quantitative focus expansion assays on endothelial monolayers demonstrated that, while the laboratory strains AD169 and Towne failed to form detectable foci, 29 out of 30 recent clinical HCMV isolates had the potential to expand in endothelial cell culture. By long-term adaptation in fibroblast cultures, nonendotheliotropic strains could be selected from clinical HCMV isolates, while long-term endothelial-adapted strains of the same isolates retained both fibroblast tropism and endothelial tropism. Such differentially adapted isolate pairs always displayed genomic differences in restriction fragment length analyses. Coinfection of endothelial cells by two nonendotheliotropic HCMV strains yielded an endotheliotropic recombinant HCMV variant combining portions of the genomes of both parental viruses. When DNA purified from various isolates was transfected into fibroblasts, progeny virus retained the specific tropism of parental virus from which the DNA was isolated. These findings demonstrate that endothelial tropism is an inherent property of most clinical HCMV isolates and is determined by the viral genome. Although the specific determinants of HCMV cell tropism are still unknown, this study provides the first evidence for a genetic contribution.

Epstein–Barr virus (EBV) encodes putative helicase–primase proteins BBLF4, BSLF1 and BBLF2/3, which are essential for the lytic phase of viral DNA replication. The BSLF1, BBLF4 and BBLF2/3 proteins were expressed in B95-8 cells after induction of a virus productive cycle, possessing apparent molecular masses of 89 kDa, 90 kDa and 80 kDa, respectively. The anti-BSLF1 or anti-BBLF2/3 protein-specific antibody, which recognizes its target protein in both Western blotting and immunoprecipitation analyses, immunoprecipitated all of the BSLF1, BBLF4 and BBLF2/3 proteins from the extract of the cells with a virus productive cycle, indicating that these viral proteins are assembled together in vivo . To characterize their protein–protein interactions in detail, recombinant baculoviruses capable of expressing each of these viral gene products in insect cells were constructed. The assembly of the three virus replication proteins was reproduced in insect cells co- infected with the three recombinant baculoviruses, indicating that complex formation does not require other EBV replication proteins. Furthermore, experiments performed by using the extracts from insect cells co-infected with each pair of the recombinant viruses demonstrated that the BSLF1 protein could interact separately with both the BBLF4 and BBLF2/3 proteins and that the BBLF2/3 protein also interacted with the BBLF4 protein. These observations strongly suggest that within the BBLF4–BSLF1–BBLF2/3 complex each component interacts directly with the other two, resulting in helicase–primase enzyme activity.

Subnuclear distribution of the human herpesvirus-8 (HHV-8)- encoded nuclear protein LNA-1 was analysed at high resolution in body cavity (BC) lymphoma-derived cell lines, in cell hybrids between BC cells and various human and mouse cells and in freshly infected K562 and ECV cell lines. Three-dimensional reconstruction of nuclei from optical sections and quantitative analysis of the distribution of LNA-1 fluorescence in relation to chromatin showed that LNA-1 associates preferentially with the border of heterochromatin in the interphase nuclei. This was further confirmed in the following systems: in endo- and exonuclease-digested nuclei, in human–mouse (BC-1–Sp2- 0) hybrids and on chromatin spreads. LNA-1 was found to bind to mitotic chromosomes at random. Epstein–Barr virus (EBV), but not HHV-8, was rapidly lost from mouse–human hybrid cells in parallel with the loss of human chromosomes. HHV-8 could persist on the residual mouse background for more than 8 months. In early human–mouse hybrids that contain a single fused nucleus, LNA-1 preferentially associates with human chromatin. After the gradual loss of the human chromosomes, LNA-1 becomes associated with the murine pericentromeric heterochromatin. In human–human hybrids derived from the fusion of the HHV-8-carrying BCBL-1 cells and the EBV-immortalized lymphoblastoid cell line IB4, LNA-1 did not co-localize with EBNA-1, EBNA-2, EBNA-5 or EBNA-6. LNA-1 was not associated with PML containing ND10 bodies either. DNase but not RNase or detergent treatment of isolated nuclei destroys LNA-1 bodies. In advanced apoptotic cells LNA- 1 bodies remain intact but are not included in the apoptotic bodies themselves.

It has been shown recently that the residual virulence of vaccinia virus (VV) is an important factor that influences the outcome of immunization with VV recombinants. This study focused on the correlation of the residual virulence of several VV recombinants with antibody responses against the strongly immunogenic extrinsic glycoprotein E of varicella-zoster virus and the weakly immunogenic extrinsic protein preS2–S of hepatitis B virus and against VV proteins, with mice used as a model organism. Furthermore, the effects of mixing different recombinants on the antibody response were studied. The results obtained indicated that: (i) the antibody response depended on the residual virulence of the recombinants, more so in the case of the weakly immunogenic protein; (ii) the residual virulence, the growth rate of the VV recombinants in extraneural tissues and the immunogenicity were associated features; (iii) immunization with mixtures of two differently virulent recombinants or with unequal amounts of two similarly virulent recombinants sometimes led to the suppression of antibody response. The appearance of this suppression was dependent on three factors: the residual virulence of the recombinants, the immunogenicity of the extrinsic proteins and the ratio of the recombinants in the mixtures. Thus, the data obtained demonstrate that there are various limitations to the use of replicating VV recombinants for immunization purposes.