- Volume 74, Issue 2, 1993

A cDNA copy of the murine coronavirus [otherwise known as murine hepatitis virus (MHV)] surface (S) glycoprotein gene was isolated and expressed in DBT cells by using a recombinant vaccinia virus system. The expressed S protein induced extensive syncytium formation at neutral pH. Oligonucleotide mutagenesis was used to engineer an S protein gene in which codons for the proteolytic cleavage site, Arg-Arg-Ala-Arg-Arg, were replaced with an equal number of codons for amino acids with aliphatic or aliphatic hydroxyl side-chains. The mutated S protein was stably expressed in DBT cells and, in contrast to the wild-type protein, was not proteolytically cleaved. Nevertheless, the non-cleaved protein induced extensive syncytium formation. These results clearly indicate that the non-cleaved form of the MHV S protein is able to mediate cell membrane fusion. Thus proteolytic cleavage is not an absolute requirement for fusion activity.

A total of 38 neutralization escape mutant viruses have been selected from a cloned stock of human rhinovirus serotype 2 (HRV-2), using either of two monoclonal antibodies (MAbs) which recognize overlapping epitopes as judged by competition binding. The mutant viruses were analysed for their sensitivity to a panel of antiviral MAbs by antibody binding and virus neutralization assays. The position and nature of the selected mutations was determined by sequencing of the virus RNAs, and the location of the substituted amino acids on the three-dimensional structure of the virus predicted from the co-ordinates determined for the closely related HRV-1A. Escape from neutralization could be attributed to single amino acid substitutions in all but one case, which had a deletion of four amino acids. In all cases in which the same mutation was found more than once, these mutations were transitions. The ratio of transition to transversion mutations was about 5:1 overall or about 1·7:1 if only unique substitutions are considered. Each antibody selected for a discrete cluster of mutations and the area of these clusters was considerably less than that determined to be in contact with antibodies from X-ray crystallographic analyses of antibody/protein complexes. One mutation did not occur within the cluster of others selected with the same antibody. This substitution occurred at the base of a small loop and may cause conformational changes at the virus surface.

A lentivirus has been isolated from a Finnish ewe with ovine progressive pneumonia in a closed upstate New York flock. We demonstrated that the virus, designated ovine lentivirus strain CU1 (OLV-CU1), is biologically, biochemically and molecularly related to, but distinct from, previously described sheep and goat lentiviruses. Nine of 32 ewes (from the affected flock) with precipitating antibodies for ovine lentivirus also produced antibodies that were able to neutralize the infectivity of OLV-CU1. The virus replicated in cultured sheep fibroblasts and caused the formation of large multi-nucleated cells. OLV-CU1-specific RNA transcripts found in infected cells and virion antigenic proteins were similar to those of other small ruminant lentiviruses. However, the virus was distinguished from other isolates at the DNA level by nucleic acid hybridization, restriction endonuclease mapping and partial sequencing of the virus genome.

The entire envelope gene of human T cell leukaemia virus type I (HTLV-I) has been successfully expressed in a baculovirus non-fusion vector system. The HTLV-I envelope protein accumulated within the insect cells as inclusion bodies which allowed efficient recovery of the recombinant protein. In an attempt to study the role of the HTLV-I envelope glycoprotein as an immunogenic target, mice were immunized with the envelope protein inclusion bodies (env-I.B.) in the presence or absence of an adjuvant. Antibodies of broad specificity were produced against the HTLV-I envelope protein in the presence or absence of an adjuvant as detected by Western blotting, radioimmunoprecipitation and peptide ELISA. Neutralizing antibody was detected when env-I.B. immunizations were carried out in the presence of high doses of a new adjuvant composed of a mycobacterial cell wall extract. In a combined immunization regimen, env-I.B. were found to enhance and broaden the antibody response to the HTLV-I envelope glycoprotein, following priming with various recombinant vaccinia virus (RVV) constructs expressing either the entire native HTLV-I envelope (gp46 and gp21) or just the surface envelope protein (gp46). Increased titres of neutralizing antibodies were observed following priming with the RVV expressing gp46 only. Results indicate that immunization regimes that involve priming with RVV expressing HTLV-I envelope followed by boosting with recombinant baculoviral HTLV-I envelope might be useful in eliciting protective immune responses in vivo.

Sequential isolates of human immunodeficiency virus type 1 (HIV-1) were obtained from patients with AIDS on short and long term treatment with zidovudine (3′-azido-3′-deoxythymidine; AZT). The isolates were tested for resistance to zidovudine by monitoring the inhibition of syncytium formation, HIV-1-specific immunofluoresence and p24 production in C8166 cells. The reverse transcriptase (RT) genes of zidovudine-sensitive (< 1 μm) and -resistant (10 to 15 μm) strains were amplified using the polymerase chain reaction and the products were sequenced directly. The predicted amino acid sequences of the RTs of zidovudine-sensitive and-resistant isolates showed 95 to 97% identity to the corresponding sequence of HIV-1SF2 which was used as a reference. Amino acid changes at positions 41, 67, 70, 215 and 219 which are known to be associated with zidovudine resistance were present in some, but not all isolates exhibiting zidovudine resistance in vitro. This indicates that mutations in the RT of HIV-1, other than those already identified, may be involved in conferring resistance to zidovudine.

The isolation and characterization of two avian polyomaviruses, from chicken (BFDV-2) and a parrot (BFDV-3), is reported. Both isolates are closely related to the non-mammalian polyomavirus budgerigar fledgling disease virus (BFDV) isolated from budgerigars (now called BFDV-1), and all three viral genomes are shown to have the same basic size of 4981 bp. A 151 bp insertion was, however, observed in the non-coding region of BFDV-2 which represented an exact duplication of the left half of the non-coding region, including the putative early promoter and amino terminus of the large T antigen. With a further 15 base pairs exchanged elsewhere throughout the three genomes, these viruses have distinct degrees of tropism for various avian species. The production of antibodies directed against a β-galactosidase-large T antigen fusion protein of BFDV-1 is described. These antibodies detected the large T antigen, with an M r of approximately 80K, and the small t antigen, with an M r of approximately 24K, in cells infected with BFDV isolates. Whereas these antibodies bind with low affinity to the large T antigen of simian virus 40 (SV40), SV40- or mouse polyomavirus-specific antibodies will not bind to the BFDV large T antigen. Antibodies directed against BFDV structural polypeptides exhibit broad, reciprocal cross-reactivities with all three structural proteins of mammalian polyomaviruses. The significance of polyomavirus infections in various avian species is discussed. Based on unique structural and biological properties we propose that these viruses should be placed in a distinct subgenus (Avipolyomavirus) within the polyomaviruses.

To study the immune response to human papillomavirus type 16, a mouse model was developed using a mouse keratinocyte cell line expressing the E7 protein. This line was grafted onto syngeneic mice to form a differentiated epithelium, thus closely mimicking the natural infection. A delayed-type hypersensitivity response could be demonstrated after intradermal challenge with a vaccinia virus recombinant expressing the E7 protein. This response appeared to be specific for the E7 polypeptide and was mediated by CD4+ cells.

The product encoded by the latent membrane protein (LMP) gene of Epstein—Barr virus (EBV) has been implicated as a transforming protein by a number of studies. We have examined the effects of LMP expression in FDCP-mix cells, a growth factor-dependent multipotential murine ‘stem cell’ line. Our studies show that LMP reduces the generation of clonogenic cells and leads to the production of cells expressing a marker (lysozyme M) characteristic of mature monocytes and macrophages. Furthermore, cells expressing LMP are compromised in their ability to produce mature neutrophils. These data suggest that expression of LMP in primitive cells can modulate their self-renewal and differentiation potential and provide evidence in support of the suggestion that EBV may be involved in some of the maturation defects of haemopoiesis.

The structure and heparin binding properties of a family of human cytomegalovirus (HCMV) disulphide-linked glycoprotein complexes designated gC-II were analysed. gC-II complexes contain two groups of glycoproteins designated Group 1 and Group 2. These glycoproteins were separated from each other by short exposure of virions to a reducing agent. This showed that the disulphide bonds between these glycoproteins were on the external surface of the virion. Although these glycoproteins were no longer associated they were not released from the virion, suggesting that they were transmembrane glycoproteins. Approximately 75 to 90% of the gC-II complexes and 18% of the complexes containing the HCMV gB glycoprotein obtained from the virion envelope bound immobilized heparin. When virions were incubated with [3H]heparin, gC-II complexes bound more heparin than gB complexes, by approximately threefold. These data showed that gC-II complexes had a greater heparin-binding capacity. After treatment of virions with a reducing agent the affinity of gC-II glycoproteins for heparin was greatly reduced whereas the affinity of gB glycoproteins was only slightly reduced. Thus, higher order structure was important for heparin binding by gC-II complexes but not by those of gB. Relative to gC-II Group 1 glycoproteins, a greater portion of gC-II Group 2 glycoproteins still bound to heparin after reduction, suggesting that Group 2 glycoproteins may be the important heparin binding component of the gC-II complexes. Both gB and gC-II complexes were eluted from immobilized heparin with soluble heparin or 0·65 m-NaCl suggesting that both formed ionic bonds with heparin. Chondroitin sulphate was not effective at eluting HCMV envelope glycoproteins from immobilized heparin. Thus, the structure of the glucosaminoglycan backbone is important to the binding of HCMV glycoproteins to heparin.

Polymorphonuclear leukocytes (PMNL) have been shown to harbour human cytomegalovirus (HCMV) in viraemic patients, but to date PMNL of asymptomatic healthy subjects have not been examined directly to determine whether this is a normal site of HCMV persistence. Using the polymerase chain reaction (PCR), paired DNA samples prepared from adherent peripheral blood mononuclear cells (PBMC), which are known to be a site of persistence of HCMV, and PMNL of 10 healthy adults were analysed. All of seven individuals who were HCMV seropositive, and one of three who were seronegative gave a reproducible signal for HCMV DNA in their adherent PBMC, whereas none of the paired PMNL DNA samples gave a positive result. The remaining two seronegative subjects showed no HCMV DNA in either the PBMC or PMNL samples. In every case where PCR for HCMV was negative, PCR amplification of a control human gene was used to show there was no inability to amplify the DNA. We conclude that within the leukocyte population of normal asymptomatic HCMV carriers, PMNL do not appear to harbour persistent HCMV whereas adherent PBMC in the same subjects are a site of persistence.

We studied the effect of two cytokines, basic fibroblastic growth factor (bFGF) and transforming growth factor beta (TGF-β) on human cytomegalovirus (HCMV) replication in cultured human lung fibroblasts. We show that TGF-β increases HCMV production, probably by a transcriptional mechanism, and that bFGF represses HCMV replication in a dose-dependent manner. These actions were antagonistic and the mechanisms involved were independent of the effects of these factors on cell DNA synthesis and proliferation.

Protection against equine herpesvirus type 1 (EHV-1) infection in a mouse model has been studied by means of delayed-type hypersensitivity (DTH) and adoptive transfer of immune spleen cells. Mice were found to develop a positive DTH response to EHV-1 antigen which was sustained for several months after primary inoculation. The response was found to cross-react with EHV-4-derived antigen. Immune cells (from mice primed with live or heat-inactivated EHV-1) conferred an enhanced DTH response on recipients; however, only the immune cells that were previously primed with live EHV-1 gave protection against re-infection. The degree of protection was also dependent on the number of spleen cells transferred. Immune cells from mice primed with heat-inactivated EHV-1 appeared to enhance virus replication following subsequent inoculation. The serum antibody response to EHV-1 appeared to be slightly suppressed in recipients of spleen cells from mice primed with either live or heat-inactivated virus. These results support the important role for cell-mediated responses in protective immunity to EHV-1 and provide clues to the nature of protection and immunopathology in the natural host.

Gene expression of the cancer-associated human papillomavirus (HPV) type 18 is modulated by cis-regulatory elements located within the viral upstream regulatory region (URR). All cellular factors identified so far involved in viral gene control bind to the 3′ portion of the HPV-18 URR. In contrast, very little is known about regulatory elements within the 5′ portion of the URR. We therefore analysed this region of unknown function to delineate potential cis-regulatory elements contained therein. By utilizing transient expression assays, an 84 bp fragment could be identified in the 5′ portion of the URR that exhibits orientation-independent cis-stimulatory activity in HeLa cervical carcinoma cells and primary human fibroblasts. Gel retardation assays and competition experiments indicated specific binding of cellular proteins to the 84 bp fragment. By functional dissection, a regulatory element with intrinsic cis-stimulatory activity could be mapped within the 84 bp fragment. Binding studies indicate that this cis-stimulatory element contains a sequence-aberrant Sp1 recognition site. Transient luciferase assays performed with mutated templates demonstrate that this Sp1 binding site behaves as a functional Sp1 element in vivo and is a main determinant of the cis-stimulatory activity exerted by the 84 bp fragment. These data show that the 5′ portion of the HPV-18 URR contains cis-activating elements and indicate an important functional role for the cellular transcription factor Sp1 in their regulation.

The gene encoding the envelope glycoprotein of Omsk haemorrhagic fever (OHF) virus was cloned and sequenced. A freeze-dried preparation of infected suckling mouse brain suspension was used as the source material for viral RNA. The derived cDNA was amplified using the polymerase chain reaction and the cloned DNA sequenced by dideoxynucleotide sequencing. Alignment of the OHF virus sequence with those of other known tick-borne flaviviruses showed that they shared N-glycosylation sites, cysteine residues, the fusion peptide and a hexapeptide (EHLPTA) that identifies tick-borne flaviviruses. OHF virus was distinguishable from the other viruses but shared closest amino acid identity (93·0%) with the tick-borne encephalitis viruses. A sequence of three amino acids (AQN; amino acids 232 to 234), which was previously shown to be specific for the tick-borne encephalitis viruses, was altered to MVG in OHF virus. This is predicted to have a higher hydrophobicity than the AQN sequence and may therfore have significant implications for the phenotypic characteristics of OHF virus. The results demonstrate close phylogenetic relationships between these viruses but also show their distinct evolutionary development. Sequence changes within the envelope glycoprotein of OHF virus have been identified that may be responsible for the distinct tropism of this flavivirus. These results support and enlarge upon previous data obtained from serological analysis.

Two Aedes albopictus (mosquito) subclones, C7–10 and C6/36, were examined by immunofluorescence and immunoelectron microscopy for the distribution of Sindbis virus structural and non-structural proteins. Both the viral glycoproteins, E1 and E2, and the non-structural proteins, nsP1 and nsP2, were found within vesicles and electron-dense, amorphous matrices associated with Sindbis virus infection. The labelling patterns indicated that both replication of viral RNA and production of virus particles were localized within the same structures in the infected cell. The data support previous reports that alphavirus infection is contained within specific structures in the cytoplasm and provide additional evidence that the C6/36 and C7–10 subclones may represent different tissue types in the adult insect.

We have cloned several cDNAs derived from the P gene of rinderpest virus. One of these, derived from a bicistronic N-P mRNA, has been sequenced in its entirety. Sequencing of a section of the others, and comparison with the genome sequence, showed that P gene transcripts, as for other morbilliviruses, were variable; non-templated Gs could be added at a site resembling the normal stop transcription site. Primer extension analysis showed that about half the transcripts were edited. Sequences of the P, C and V proteins encoded by the normal and edited transcripts were compared with those of other morbilliviruses and with those of the more distantly related paramyxoviruses.

Cloned bovine lymphoblastoid cell lines, transformed by the protozoan parasite Theileria parva were infected with cell culture-attenuated rinderpest virus vaccine. The virus grew readily in lymphoid B cells, CD4+ and CD8+ alpha/beta T cells and gamma/delta T cells producing new infectivity, viral antigens, c.p.e. and total cell death. There did not appear to be a predilection for any particular phenotype of lymphoblast. The results imply that if the vaccine causes immunosuppression, it could do so through a variety of mechanisms.

The virulence of avian influenza A viruses depends on the cleavability of the haemagglutinin (HA) by an intracellular protease at multiple basic amino acids. Although previous studies have demonstrated the importance of these amino acids for processing by the cellular protease, with emphasis on conserved residues near the cleavage site, the minimal requirements for cleavage remain unknown. By expressing site-specific mutants of the HA of a virulent avian influenza virus, A/turkey/Ireland/1378/85 (H5N8), in the simian virus 40 system and testing for their cleavability by an endogenous protease in CV-1 cells, and their fusion activity in a polykaryon formation assay, we were able to show that glycine at the amino terminus of HA2 is not essential for cleavage and that maximal cleavage requires at least five basic residues at the cleavage site, when carbohydrate is nearby. Moreover, we confirmed, that a conserved proline upstream of the cleavage site is not essential for HA cleavage or fusion activity, and that lysine replacement of the carboxyl-terminal arginine of HA1 abolishes cleavability. These findings should help identify the proteases responsible for intracellular cleavage of the HA of virulent avian influenza viruses.