- Volume 76, Issue 8, 1995

Several studies have demonstrated a functional role for the V1-V2 region of the human immunodeficiency virus type 1 (HIV-1) envelope surface glycoprotein gp120 in the membrane fusion processes underlying viral entry and syncytium induction. In a study with chimeric primary envelope genes, we have previously demonstrated that the exchange of V2 regions was sufficient to transfer syncytium-inducing capacity to a non-syncytium-inducing envelope protein. The exchanged V2 regions, comprising a number of variable amino acids, conferred changes to both the predicted secondary structure and to the net positive charge of the V2 loops. In a syncytium-forming assay based on transient envelope protein expression in CD4+ SupT1 cells, we have extended this observation by mutating the variable positions of the V2 region to determine the relative contribution of individual amino acids to syncytium formation. It can be shown that simultaneous mutation of multiple amino acids is needed to interfere with the V2 region-determined syncytium-inducing phenotype. Single amino acid changes either influencing charge or predicted secondary structure of the V2 loop proved to be insufficient to abolish V2 region-controlled syncytium formation. This robust V2 organization may allow the virus to accumulate mutations, while retaining its biological phenotype.

The p121 protein, a small hydrophobic protein encoded by the human T cell leukaemia/lymphotropic virus type I pX region, contains a proline-rich region located between two putative transmembrane (TM) domains. The p121 protein is associated with cellular endomembranes, and physically binds to the 16 kDa subunit of the vacuolar H+-ATPase proton pump. To investigate the nature of the 16 kDa and p121 interaction and to determine the oncogenic domain of p121, we constructed p121 mutant proteins in which various portions of the TM domains were deleted, as well as a p121 mutant containing a single amino acid substitution. These mutants were tested for binding to the 16 kDa subunit of the vacuolar H+-ATPase in HeLa/Tat cells and for the capability to potentiate transformation by bovine papillomavirus type 1 E5 oncoprotein in mouse C127 cells. The results indicated that both TM domains of the p121 protein were dispensable for its interaction with the 16 kDa protein, whereas partial or complete deletion of the proline-rich region resulted in decreased or no binding of the p121 protein to the 16 kDa subunit. Immunofluorescence analysis of HeLa/Tat cells transfected with the p121 mutants showed that deletion of the proline-rich region did not alter the subcellular localization of these mutant p121 proteins, suggesting direct involvement of the proline-rich domain in binding rather than the failure of these p121 mutants to reach the appropriate cellular compartment. Mapping of 16 kDa subunit mutants in binding with the p121 protein suggested that molecular determinants located between the second and third TM domain of the 16 kDa protein might be involved in this interaction. Finally, most of the p121 mutants lost the ability to potentiate transformation of C127 cells indicating that binding of p121 to the 16 kDa subunit does not directly correlate with oncogenicity.

The retrovirus protease (PR), an aspartic PR, is composed of two identical subunits, each containing a conserved tripeptide sequence present at the active site of the enzyme. Asp-Ser-Gly is found in avian sarcoma leukaemia viruses (ASLV) and Asp-Thr-Gly in mammalian oncoretroviruses. We have mutated the conserved sequence at the active site of ASLV PR by converting the Ser and Gly residues to Thr and Ala, respectively. Replacement of Gly with Ala yielded an ASLV PR devoid of proteolytic activity. The Ser to Thr conversion did not alter the substrate specificity of the enzyme. Both wild-type and mutated PRs correctly cleaved viral precursors expressed in bacterial cells, as well as synthetic peptides homologous to ASLV and human immunodeficiency virus type 1 cleavage sites. Bacterially produced ASLV PR with Thr instead of Ser had increased enzymatic activity, as shown by hydrolysis of synthetic peptides. However, this mutation reduced the production of reverse transcriptase-containing particles and infectious virus following transfection of permissive cells with virus DNA.

A recombinant of herpes simplex virus (HSV) was constructed in which the HSV thymidine kinase (TK) gene was deleted and the varicella-zoster virus (VZV) TK gene was introduced into the US5 region under the control of the human cytomegalovirus IE promoter. Infection with the recombinant (R18) led to the induction of TK although the kinetics of synthesis resembled those of a ‘late’ gene product. The recombinant was virulent in the zosteriform mouse model with the pattern of pathogenesis similar to that of wild-type HSV-1. The sensitivity of the recombinant to several nucleoside analogues was assessed and in most cases (BVaraU, ACV and GCV) it resembled VZV rather than HSV. The enhanced sensitivity of the recombinant to BVaraU compared with wild-type HSV resulted in a far greater response to treatment with BVaraU as assessed in the mouse model.

Cytosine DNA methyltransferases (MTases) first recognize specific nucleotide sequences and then transfer a methyl group from S-adenosylmethionine to cytosine. This division of function is reflected in five highly conserved motifs shared by cytosine MTases. The region containing the first four motifs is responsible for the catalytic function whereas the region containing the fifth motif V provides specificity of binding to DNA. In at least one case, two separate proteins, one containing the first four motifs and the second containing the last motif combine to provide full functional activity. In the frog virus 3 (FV3) genome we have identified an open reading frame (ORF) whose deduced amino acid (aa) sequence contains motifs characteristic of prokaryotic as well as eukaryotic MTases. The ORF consists of 642 bp which codes for a protein of 214 aa with a predicted molecular mass of 24.8 kDa. This ORF contains the first four highly conserved motifs of cytosine MTases but the fifth motif, responsible for DNA binding specificity, is missing. Presumably, FV3 MTase is composed of two subunits. Northern blot analysis showed that the putative MTase ORF is transcribed into two transcripts belonging to the delayed-early class of FV3 messages. These two transcripts appear to be initiated at two different start sites but terminate in the same 3′ region of the gene. The transcription start sites are not preceded by any known promoter sequences, but two regions of hyphenated dyad symmetry are present at the 3′ end of the message. A protein with a molecular mass of ∼ 28 kDa was synthesized by a rabbit reticulocyte lysate programmed with capped runoff transcripts from the cloned gene, suggesting that the ORF can be transcribed into a message coding for a viral protein. Overall, our results suggest that we have identified a gene for a subunit of MTase in the FV3 genome.

The early genes E6 and E7 from human papillomaviruses (HPVs) play a key role in the development of cervical cancer. Modulation of E6 and E7 gene expression may alter tumour progression; therefore, modifiers of viral transcription such as hormones or growth factors are potential risk factors in cancer development. We have analysed the effects of epidermal growth factor (EGF) on E6/E7 mRNA from human papillomavirus type 16 (HPV-16) by Northern blot in two cell lines, SiHa cervical carcinoma cells, and HPK IA, an HPV-16-immortalized keratinocyte cell line. E6/E7 mRNA is EGF-inducible in SiHa cells, with the earliest response after 2 h. In contrast, in HPK IA cells no increase in E6/E7 RNA is observed, suggesting a differential EGF response of viral transcription in tumour cells compared with keratinocytes. We demonstrate that the cell type-specific HPV-16 enhancer is a target of EGF-induced signals, as its activity is amplified by EGF in SiHa cell transfections. However, when transfected into HPK IA keratinocytes, the viral enhancer shows no EGF response. The enhancer contains two binding sites for the transcription factor AP-1, a potential mediator of the EGF signalling cascade. Enhancer subfragments with single AP-1 binding sites are also EGF-responsive in SiHa cells. Mutating either AP-1 site in the complete enhancer decreases the EGF response, whereas a double mutation causes a complete loss of EGF regulation, suggesting that the EGF induction of HPV-16 early transcription requires AP-1 activation. We conclude that alterations of EGF responsiveness that increase viral oncogene expression may contribute to cervical cancer progression.

A variety of recombinant proteins derived from protein pVI of human adenovirus type 2 (Ad2) were analysed for their ability to bind Ad2 hexon in vitro. As pVI is also required for activation of the adenovirus-coded protease, the same pVI derivatives were assessed for their ability to activate recombinant adenovirus-coded 23K protease. Two regions, between amino acid residues 48–74 and 233–239 of pVI, were required for the interaction with hexon. These regions are highly conserved amongst mastadenovirus pVI proteins. Both these regions are capable on their own of binding hexon weakly but must be provided in cis for strong hexon binding. In addition, we found evidence to indicate that conformation as well as sequence was important for good hexon binding in our assays. Authentic processing of the appropriate recombinant pVI derivatives, by the recombinant protease, was obtained without the addition of other cofactors. These findings are discussed in relation to the role of pVI in triggering the adenovirus maturation pathway.

Human adenovirus (Ad) vectors are being used increasingly for a variety of applications in vaccination and gene therapy. The ability of vectors to enter cells and the efficiency of promoters expressing the therapeutic gene or vaccine antigen are critical to the outcome of such experiments. To identify promoters which might be suitable for use under a variety of conditions we have investigated the expression of a rotavirus antigen, VP7sc, employing several commonly used promoters carried in E1-substituted Ad vectors both in cell types which support virus replication and in cells which do not. Although not all gene constructions were identical, wide variations in promoter function were evident even in human 293 cells which support virus replication. The simian virus type 40 (SV40) early and β-actin promoters expressed poorly; the SV40 late promoter was somewhat better. The human IE94 cytomegalovirus (CMV) promoter and a modified Ad major late promoter were best, functioning equally well but with different kinetics. In other human cell lines the CMV promoter was more versatile, generally providing sustained expression at a significant level, in one case for at least 6 days. In addition, as mouse, rabbit and pig models of rotavirus infection are under investigation and VP7sc is a vaccine antigen, we also investigated the ability of the recombinant adenoviruses to infect cells from these and other sources. VP7sc expression was detected in several heterologous cell types, illustrating the ubiquity of the human Ad receptor and the versatility of human Ad as vectors when suitable promoters are used.

The major neutralization antigen VP7 of rhesus rotavirus (RRV) was expressed in a baculovirus recombinant system. The expressed VP7 showed the same molecular mass as native VP7, and was recognized by hyperimmune sera as well as neutralizing and non-neutralizing monoclonal antibodies (MAbs) raised against RRV. Intraperitoneal administration of the expressed VP7 in mice elicited the production of serum antibodies which were able to immunoprecipitate VP7 from RRV-infected cell lysates and to neutralize the virus in vitro. Sera from immunized mice competed for binding to RRV in an ELISA with both neutralizing and non-neutralizing MAbs specific for VP7. Using a passive protection model of rotavirus disease, vaccination of mice with the recombinant VP7 induced partial protection from infection. These results suggest that the baculovirus-expressed VP7 may be useful in priming a protective immune response to rotavirus infection.

Complementary DNAs encoding ORFs 2 to 7 of equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (GL) was identified as the most reactive to EAV-positive equine sera and an immuno-dominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a GL-specific synthetic peptide (residues 75 through 97) induced EAV-neutralizing antibody in vaccinated horses. The defined antigenic region of GL is likely to be exposed on the surface of the native EAV virion and consequently may be useful in the development of diagnostic tests and vaccines.

The Mengo virus specific cytotoxic T lymphocyte (CTL) response was investigated after intraperitoneal infection of mice with the attenuated Mengo virus strain vMC24. A high level of CTL activity was detected in spleen cell cultures obtained from infected C3H/HeJ (H-2k) or C57BL/6 (H-2b) mice after a secondary in vitro stimulation with Mengo virus-infected cells. The CTL activity, which was MHC class I-restricted, was shown to be mediated by CD8+ T cells. Recombinant vaccinia viruses that expressed capsid proteins VP0, VP1 or VP3 were produced and used to identify the protein(s) recognized by the Mengo virus-specific CTLs. In both C3H/HeJ and C57BL/6 mice, analysis of CTL activity against target cells expressing each capsid protein showed that VP0 was the only capsid protein recognized by the CD8+ CTLs. The CTL epitope(s) could be further located in the C-terminal half of VP0, i.e. in capsid protein VP2. Moreover, using unlabelled target cells expressing VP0 as cold competitors, we were able to almost completely inhibit recognition and lysis of Mengo virus-infected cells by specific CD8+ CTLs. Thus, the CTL response directed against VP2 was immunodominant in both C3H/HeJ- and C57BL/6-infected mice.

In May 1993, a severe epidemic of respiratory disease began in horses in Inner Mongolia and spread throughout horses in China. The disease affected mules and donkeys as well as horses but did not spread to other species, including humans. The severity of the disease raised the question of whether the outbreak might have been caused by the new avian-like influenza viruses detected in horses in China in 1989 or by current variants of A/equine/Miami/1/63 (H3N8) (equine-2) or by a reassortant between these viruses. Antigenic and sequence analysis established that all gene segments of the influenza virus causing the epidemic were of recent equine-2 origin and that the virus was not a reassortant. Serological analysis of post-infection horse sera provided evidence for the continued circulation of the A/Equine/Jilin/1/89 (Eq/Jilin) (H3N8) avian-like viruses in horses in Heilongjiang province with original antigenic sin-like responses. It is noteworthy that prior infection with the avian-like Eq/Jilin strain did not afford cross-protection against a current equine-2 strain. Serological evidence for the continued circulation of the avian-like H3N8 influenza virus in horses indicates that this virus has probably established itself in horses in Asia.

Aujeszky′s disease virus (ADV) is a well known neurotropic virus in pigs. In the present study the mechanism of spread of ADV along the maxillary nerve and the role of the viral envelope glycoproteins gC, gE and gI in this process was examined in pigs. The Ka parental strain of ADV and its gC-, gE- and gI-deleted mutants were inoculated intranasally in pigs, after which virus dissemination in the maxillary nerve and the trigeminal ganglion was monitored at time intervals by means of virus isolation. The parental strain was isolated from both the nasal mucosa and the trigeminal ganglion at 21 h post-inoculation (p.i.), whereas the middle part of the connecting maxillary nerve was positive only after 48 h p.i. It appears, therefore, that ADV travels from the nasal mucosa via the nerve towards the ganglion in a non-infectious form, and then replicates in the neuronal somas, after which infectious virus is transported towards the nasal mucosa. Although all mutants were present at 48 h p.i. in the nasal mucosa and the trigeminal ganglion, the appearance of infectious virus in the maxillary nerve was clearly delayed with the gE− and gI− mutants. It is suggested that glycoproteins gE and gI are involved in the axonal transport of infectious ADV away from neuronal cell bodies, also called anterograde transport.

A diagnostic PCR assay was designed based on conserved regions of previously sequenced densovirus genomic DNA isolated from mosquitoes. Application of this assay to different insect cell lines resulted in a number of cases of consistent positive amplification of the predicted size fragment. Positive PCR results were subsequently confirmed to correlate with densovirus infection by both electron microscopy and indirect fluorescent antibody test. In each case the nucleotide sequence of the amplified PCR fragments showed high identity to previously reported densoviruses isolated from mosquitoes. Phylogenetic analysis based on these sequences showed that two of these isolates were examples of new densoviruses. These viruses could infect and replicate in mosquitoes when administered orally or parenterally and these infections were largely avirulent. In one virus/mosquito combination vertical transmission to progeny was observed. The frequency with which these viruses were detected would suggest that they may be quite common in insect cell lines.

The ultrastructure of human astrovirus serotype 2 (H-Ast2) grown in cell culture was analysed by electron microscopy of thin sections and negatively stained preparations. Infected LLCMK2 cells, as visualized in thin sections, contained cytoplasmic aggregates of dense or hollow-cored particles that aggregated in quasi-crystalline arrays and were specifically labelled using a rabbit polyclonal anti-Ast2 antiserum. H-Ast2 particles from the supernatant of infected LLCMK2 cells in thin sections after flat-embedding were similar in size to intracellular virions. In negatively stained preparations, these virus particles had an external diameter of 41 nm and exhibited a well defined layer of surface spikes. Pentagonal and hexagonal contours were occasionally visible, and probably correspond to the projections of icosahedral structures. Star-like morphologies and particles with surface triangular hollows were seen in dark areas of the preparations only after a short treatment of the viruses at pH 10. Incubation of the viruses at pH 10.5 induced a rapid disassembly of the virus particles. The finding that the particles with icosahedral geometry and surface spikes are fully infective allows an alternative morphological model to the traditional one for astroviruses to be proposed.

Determinants of attenuation in the Sabin type 1 strain of poliovirus are located in the 5′ noncoding region, the capsid coding region and the viral RNA-dependent RNA polymerase (3D pol ) coding region. These mutations also contribute to a temperature sensitive phenotype of virus replication. We have cloned and expressed the Sabin 1 virus 3D pol protein which contains three amino acid differences from the wild-type (Mahoney) sequence, as well as a wild-type polymerase containing only a single Sabin amino acid substitution at nt 6203. These enzymes have been examined and compared for temperature sensitive polymerase activity. Wild-type and mutated polymerases demonstrated identical specific activities at 30, 35 and 39 °C. All three showed the same kinetics of heat inactivation after pre-incubation at elevated temperatures. Thus the contribution of Sabin 3D pol sequences to the inability of the virus to grow at elevated temperatures must lie in a function or activity of the enzyme other than RNA polymerization. A likely reaction is the initiation step of RNA chain synthesis.

Membrane cofactor protein (CD46) has been firmly established as the major high affinity receptor for measles virus (MV). In addition, another protein, moesin, has been shown to be linked with the susceptibility of human cells to MV infection. Murine cells are largely resistant to MV infection, although a number of cell types can be productively infected. As murine cells do not express CD46 an additional mechanism for the uptake of MV is likely. Murine cells possess a functional homologue of CD46 (Crry/p65) in addition to murine moesin, which has nucleotide and amino acid homology to human moesin. We report that anti-moesin monoclonal antibodies 119 and 38/87 reduce the number of infectious centres attributed to MV in murine cell lines NS20Y and L929, whereas polyclonal antisera specific for Crry/p65 and CD46 had no effect on MV infection of these cells. We suggest that moesin may be important in the non-CD46-mediated uptake of MV strain Edmonston by susceptible murine cell lines.

The genome of infectious pancreatic necrosis virus (IPNV) is composed of two segments of dsRNA. The larger segment contains a small ORF partly overlapping the 5′ end of the polyprotein reading frame. Yet very little is known about this possible new gene, which presumably codes for a 17 kDa polypeptide (VP5). The region of the viral genome which encompasses the small ORF was reverse-transcribed and amplified by PCR before cloning and sequencing. Analysis of the sequences obtained from five different virus strains revealed that the small ORF is not found on one of them, and that it is truncated on two others. Moreover, the deduced amino acid sequences did not appear to be well conserved. Despite the large variations between IPNV strains at the genomic level, all predicted VP5 are arginine-rich basic polypeptides. To verify whether the small ORF is translated into protein in fish cells, the 17 kDa polypeptide of the VR-299 strain was expressed as a fusion protein in a prokaryotic expression vector and used to produce a specific antiserum. This antiserum reacted with concentrated virus in an immunodot assay, indicating that VP5 is synthesized in infected cells, but probably only in small quantities. When tested with 12 other IPNV strains, results were less conclusive than those obtained with strain VR-299. Nevertheless, three of the 12 viruses gave a clearly negative signal in the immunodot assay, suggesting that possibly more than one viral strain lacks the small ORF.