- Volume 75, Issue 8, 1994

A lymphocyte proliferation assay was used to examine the helper T cell response to rotavirus in mice following parenteral immunization with the UK strain of bovine rotavirus. Mixed populations of lymphocytes prepared from spleen or peripheral lymph nodes were tested for proliferation in the presence of UK strain rotaviruses, prepared as cell culture lysates, ultracentrifuged (pelleted) lysates, sucrose-purified virus and caesium chloride-purified virus. Live rotavirus induced non-specific stimulation of lymphocytes, which was not observed in response to inactivated virus. Putative helper T cells of the L3T4+ phenotype were prepared as an enriched population from UK strain-immunized mice or grown in vitro as a polyclonal T cell line. The response of L3T4+-enriched cells from mice immunized with inactivated virus was dependent on antigen-presenting cells (APCs). Cells obtained following immunization with live virus did not require further addition of APCs. The response of the L3T4+ T cell line was wholly dependent on APCs. UK strain-specific L3T4+ cells responded to whole UK rotavirus and to isolated VP6 of both UK and C486 rotavirus strains. The results indicate that virus-specific L3T4+ T cells are induced following rotavirus immunization and can respond to epitopes on VP6. UK strain-primed L3T4+ cells also responded to an avian rotavirus strain, Ch2, which shares only minimal serological cross-reactivity with the UK strain. T cell recognition of rotavirus may thus be broadly cross-reactive.

Rotavirus strain A253, isolated from the faeces of a diarrhoeic piglet in Venezuela, was classified as serotype G11 by cross-neutralization studies and by comparison of the deduced amino acid sequence of the VP7 surface protein. The epitopes involved in neutralization of the two G11 porcine rotavirus strains A253 and YM were analysed using neutralization-resistant mutants selected with seven neutralizing monoclonal antibodies (MAbs), monotype-specific (M-) MAbs and serotype-specific (S-) MAbs, produced against VP7 of strain A253. Crossneutralization tests and sequence analysis of the escape mutants selected from strains A253 and YM indicated the presence of two antigenic sites, one common to both M-MAbs and S-MAbs in region A (positions 87, 91 and 96) and the other defined by one S-MAb in region C (position 223). All A253 variants selected with M-MAbs and two S-MAbs, although having different amino acid substitutions, had a change at amino acid position 87, whereas YM variants involved residues 91 and 96, part of the same antigenic site. Compared to strain A253, the YM strain presents an amino acid substitution at position 87 and was not recognized by M-MAbs. These results suggest that in the VP7 of G11 serotype specificity, the amino acid at position 87 is an important component of a neutralization site associated with region A and the intraserotypic variation between strains A253 and YM may account for the selection of mutations at different positions by a single MAb.

We have shown recently that the human poliovirus receptors (hPVRs) expressed on the surface of cultured cells are 80K glycoproteins, whereas the previously reported 67K forms are partially glycosylated intermediate glycoforms. Both the membrane-bound 80K and 67K forms of hPVR are glycosylated derivatives of the two isoforms hPVRα and hPVRδ, where the latter two can be resolved only by SDS-PAGE upon enzymatic deglycosylation. Here we report the N-terminal sequence analysis of the mature 80K as well as the intermediate 67K glycoforms of hPVR which has allowed us to identify the signal peptidase cleavage site of the unprocessed hPVR. The signal sequence that directs translocation of hPVR across the membrane of the endoplasmic reticulum on its route to the glyco-processing pathway has thus been defined. We compare this signal sequence with those of the putative monkey poliovirus receptor and the mouse poliovirus receptor homologue.

Sequence comparison of the RNA-dependent RNA polymerases of small round-structured viruses (SRSVs) from 10 recent U.K, outbreaks of gastroenteritis revealed significant genetic variation. Computer analyses indicated that these viruses can be divided into two discrete groups. SRSV group I contains the previously characterized antigenic type 1 Norwalk and type 3 Southampton viruses. The amino acid sequences of the RNA polymerase, capsid and ORF3 of these two viruses are relatively similar (about 92%, 69% and 72% amino acid identity, respectively). A representative member of group II SRSVs, Bristol virus, was subjected to a detailed genetic analysis. Bristol virus is a recent antigenic type 2 isolate from a U.K. hospital outbreak of gastroenteritis. Using a single clinical sample the 3′-terminal 3881 nucleotide cDNA sequence [excluding the poly(A) tail] of this virus was determined. Analysis of the sequence revealed significant differences from those of group I viruses with the RNA polymerase region, capsid and ORF3 showing only about 62%, 43% and 30% amino acid identity respectively with the equivalent proteins of the Norwalk and Southampton viruses. These data suggest that the morphologically identical SRSVs belong to at least two genetically distinct groups.

The nucleotide sequence of the bovine ephemeral fever virus (BEFV) genome has been determined from the 3′ terminus to the end of the nucleoprotein (N) gene. The 3′ leader sequence comprises 50 nucleotides and shares a common terminal three nucleotides (3′-UGC-) and a downstream U-rich domain with vesicular stomatitis virus (VSV) and rabies virus. The N gene comprises 1328 nucleotides from the transcription initiation consensus sequence (AACAGG) to the conserved transcription termination-poly(A) sequence [CATG(A)7] and encodes a polypeptide of 431 amino acids with an estimated M r of 49159 and a pI of 5·4. The deduced amino acid sequence of the BEFV N protein is similar to those of other mammalian rhabdoviruses and is more closely related in sequence to vesiculoviruses (VSV Indiana and New Jersey, Piry, Chandipura) than to lyssaviruses (rabies and Mokola). An almost full-length clone, 1301 bp in length, of the BEFV N gene and clones derived from 5′-terminal (559 bp) and 3′-terminal (742 bp) fragments were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. A panel of 12 BEFV N protein-specific monoclonal antibodies was shown to react in immunoblots with fusion proteins containing the almost full-length N protein and the C-terminal fragment, but not the N-terminal fragment. Two of these antibodies also reacted with baculovirus-expressed rabies virus N protein. Polyclonal mouse ascitic fluids derived from BEFV, rabies virus and several other related viruses were also shown to cross-react in immunoblots with purified preparations of rabies virus and BEFV N proteins.

The gibbon ape leukaemia virus (GaLV) family of type C retroviruses consists of five closely related viral isolates, GaLV SF, GaLV SEATO, GaLV Br, GaLV H and simian sarcoma-associated virus. The cDNA encoding the human receptor for GaLV SEATO had previously been isolated. We now demonstrate that all of the above GaLVs can use the human form of the GaLV receptor to infect cells. All murine cells analysed to date have been found to be resistant to infection by GaLVs owing to the absence of a functional GaLV receptor. We have now identified a murine cell line which is unique in its susceptibility to GaLV infection. This cell line was established from a Japanese feral mouse, Mus musculus molossinus. We cloned and sequenced the cDNA for the receptor expressed in these cells and compared it to the cDNA for the GaLV receptor expressed in resistant murine cells such as NIH 3T3 (derived from M. m. musculus) and MDTF (derived from M. dunni tail fibroblasts). The crucial region for GaLV infection (the fourth extracellular domain) from the functional M. m. molossinus GaLV receptor is quite divergent from the same region of the M. m. musculus and M. dunni proteins, but similar to that of the functional human GaLV receptor. These results confirm the importance of the amino acids of this region in GaLV receptor function.

Human T cell leukaemia virus type I (HTLV-I) is a direct activator of human resting T lymphocytes. The present study was undertaken to delineate further the role of viral particles and to define the involvement of envelope glycoproteins in the induction of T cell mitogenic stimulation. Virus-producing cells treated with paraformaldehyde (PFA) were found to be unable to induce the formation of syncytia, but still able to trigger the proliferation of resting T cells. Likewise, PFA-treated virus particles were still mitogenic. These results suggest that the mitogenic event is triggered before the fusion of the envelope with the cell membrane. Furthermore, HTLV-I envelope-expressing cells obtained after infection of C8166/45 cells (HTLV-I- transformed, but defective in virion production) with an HTLV-I envelope recombinant vaccinia virus were unable to activate normal T cells. Human immunodeficiency virus type 1 particles produced by C8166/45 cells were also devoid of mitogenic ability. However, when HTLV-I viral preparations were purified by chromatography, only the virion-containing fractions were found to be mitogenic for human resting T lymphocytes. This mitogenic activity was partially abolished by preincubating the purified virus with a monoclonal antibody directed to the surface envelope glycoprotein. Finally, treatment of HTLV-I-transformed cells by tunicamycin, an inhibitor of N-linked glyco-sylation, led to the production of virus particles with a decreased mitogenic activity. Collectively, these observations suggest that the HTLV-I mitogenic activity is triggered by the contact of HTLV-I virions with T cells.

H3B cells, a laboratory clone of H9 cells persistently infected with the HTLV-IIIB strain of human immunodeficiency virus (HIV), contained significant levels of cell-associated reverse transcriptase (RT) activity measured by in vitro assays using either exogenous or endogenous templates. The cell-associated RT activity detected using exogenous template was almost wholly in a soluble (non-sedimentable) form whereas endogenous activity sedimented as a particulate structure associated with viral RNA. Despite this, H3B cells did not contain episomal HIV DNA detectable by Southern blot, indicating that in vivo reverse transcription was not occurring to any significant extent in these cells. However, when susceptible HUT 78 cells were infected by co-cultivation with H3B cells, dramatic synthesis of episomal HIV DNA occurred. Concurrently with this de novo initiation of reverse transcription, however, we found no detectable change in intracellular levels or cleavage profiles of immunoprecipitable RT polypeptides. Finally, actinomycin D pre-treatment of H3B cells to prevent de novo transcription from donor cell proviral DNA after co-cultivation did not affect the initiation of in vivo reverse transcription following cell-to-cell HIV infection. These results demonstrated that cells persistently infected with HIV contained significant fully cleaved cell-associated RT in a form that was active in vitro but not in vivo and that following cell-to-cell transmission of HIV infection to susceptible cells, de novo reverse transcription was initiated without detectable evidence of further synthesis or proteolytic processing of HIV RT. The nature of this initiation process requires further study.

Neurological dysfunction in AIDS occurs in the absence of productive infection of neurons, and may involve modulation of neuronal cell function by viral or cellular products released from surrounding infected cells. The human immunodeficiency virus type 1 (HIV-1) transactivator protein Tat may be one such factor, as it can act as a neurotoxin, induces marked morphological changes in neurons and astrocytes in primary embryonic rodent brain cultures, and is released by certain HIV-1-infected cells. In addition, Tat can alter expression of cellular genes in several non-neuronal cell types. To explore the possibility that Tat may also mediate neuronal dysfunction in AIDS through non-lethal effects on neurons, we determined the trans-activating ability of Tat in human neuronal cells. We generated human neuronal cell lines stably expressing several HIV-1 tat genes, and also tested human neuronal cells exposed to extracellular recombinant Tat protein. Both endogenously expressed Tat as well as exogenous recombinant Tat protein up-regulated HIV-1 long terminal region (LTR)-driven gene expression by several hundred-fold. Only brief exposure to recombinant Tat was necessary and no toxic effects were seen at levels sufficient for trans-activation. Furthermore, Tat significantly enhanced virus expression in neuronal cells transfected with molecular clones of HIV-1. These results show that Tat is trans-activationally active in human neuronal cells, and can be taken up from the extracellular compartment by these cells in a biologically active form. Neurons represent an important potential target for Tat-mediated cellular dysfunction.

The biological properties of homogeneous populations of feline immunodeficiency viruses derived from infectious molecular clones of the TM1, TM2 and Petaluma strains were compared. Differences in in- fectivity for Crandell feline kidney (CRFK) cells, and in syncytium formation and replication kinetics in a feline T lymphoblastoid cell line (MYA-1 cells) were observed. To investigate the basis of these differences between the TM2 and Petaluma strains, we first compared the basal promoter activity of the long terminal repeat which is a highly divergent region, but no significant difference in activities was found in CRFK cells. We then constructed two recombinant chimeric clones which carry gag, pol, vif and ORF A from the heterologous virus. From analyses using the chimeric clones, it was revealed that efficient virus growth in CRFK cells and MYA-1 cells was regulated by the gag, pol, vif and ORF A regions, whereas viral determinants of infectivity for CRFK cells, and syncytium formation and cytopathogenicity in MYA-1 cells, were located in the env region.

The mechanism for down-regulation of E1a expression by products encoded in the E3 transcription unit of human adenovirus types 2 and 5, that occurs in infected L929 cells, has been investigated further. We show that the phenomenon occurs in different mouse cells and also in some human cells suggesting that the observations have relevance to natural human infections. We also provide evidence that probably all viral proteins are down-regulated by E3 products, although to different extents, but that host proteins are unaffected. Whereas E1a protein levels and synthesis are reduced in the presence of E3 products, E1a protein half-life and polysomal Ela RNA levels and size distribution are not. These data suggest that E3 products down-regulate E1a protein levels by interfering with the translation of E1a- specific mRNA. Studies were additionally carried out with mutant adenoviruses containing different defects in the E3 transcription unit. Based on these studies it seems likely that the E3 14·5K and 10·4K proteins are crucially involved in Ela down-regulation. Our data are discussed in terms of strategies for immune evasion by group C human adenoviruses.

We have previously shown that the E5a gene of human papilloma virus type 11 (HPV-11/HPV-6c) is a transforming oncogene. In order to dissect the biological consequences of E5a gene expression we utilized the lac operator/repressor system to manipulate E5a gene expression. Cells were cotransfected with the lac repressor gene and the E5a gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence. The expression of E5a gene could therefore be repressed by binding of lac repressor to the lac operator sequence in proximity to this SV40 regulatory region. The transfected cells were cultured in the presence of the inducer IPTG and under G418 selection. IPTG derepressed E5a gene expression by binding to the repressor and reducing its affinity for the lac operator sequence. In these studies, we found that E5a-transformed cells still maintained the transformed phenotype as judged by growth density, cell morphology and anchorage-independent growth when E5a gene expression was repressed. We also found that c-jun expression was induced 3 h after E5a expression was induced by IPTG and c-jun expression was not shut down after repression of E5a expression. This is the first demonstration that the E5a gene of HPV-11 initiates transformation of NIH 3T3 cells but is dispensable for maintenance of the transformed phenotype.

Enhancin, an infectivity-enhancing protein from Trichoplusia ni granulosis virus (TnGV) was tested for its ability to increase Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) infection in the larvae of four lepidopteran insects. Enhancin increased the mortality of AcMNPV infection in all the four insect species tested. Peritrophic membrane (PM) assays showed altered protein profiles in PMs treated with enhancin in all the four species. This supports the hypothesis that enhancin affects virus infection by altering the structural integrity of the PMs. The binding of enhancin to the midgut brush border membranes (BBMs) was determined and specific binding sites were found on the BBM of Pseudaletia unipuncta. No specific binding sites were found on the BBMs of T. ni, Helicoverpa zea or Spodoptera exigua. Therefore, specific binding of enhancin to the midgut cell membrane may not be necessary for the enhancement of baculovirus infection in insects.

The C-polyhedrin genes of two different geographic isolates of a type 5 cytoplasmic polyhedrosis virus (CPV) were cloned. A CPV infecting Orgyia pseudotsugata (OpCPV), isolated in the Pacific Northwest of the U.S.A., and a CPV infecting Heliothis armigera, isolated in South Africa, were studied. Both genes were found to be 883 nucleotides in length and encoded a predicted protein of 246 residues (M r of 28890). Comparison of the nucleotide sequences of these two viruses with another type 5 geographic isolate, infecting Euxoa scandens (EsCPV; isolated in Eastern Canada), showed that there were only 17 nucleotide differences among the three genes. The only nucleotide variation that had an effect on the encoded protein was a deletion of nucleotide 774 in the gene of EsCPV. The deletion introduces a frameshift mutation resulting in the alteration of the carboxyl-terminal amino acid sequence. Sequence alignment of the OpCPV C-polyhedrin showed little homology to a type 1 CPV (infecting Bombyx mori) or with analogous proteins (N-polyhedrins) from two baculoviruses infecting O. pseudotsugata. Interestingly, most of the conserved residues between the N- and C-polyhedrins were either basic or aromatic amino acids.

A PCR protocol was established that not only allows the detection of, but also the differentiation of species of the genus Orthopoxvirus. This assay was accomplished by the selection of oligonucleotides located within the gene that encodes the A-type inclusion protein of cowpox virus. The primer pair flanked a region exhibiting distinct and specific DNA deletions in the corresponding sequences of vaccinia, mousepox, monkeypox and camelpox virus. For this reason, PCR resulted in DNA fragments of different sizes. The presented PCR protocol, combined with BglII restriction digests, allowed the unequivocal assignment of 42 orthopoxvirus (OPY) strains and isolates to the correct OPY species. The resulting classification corresponded exactly with known biological data for the OPV strains investigated. Furthermore, 13 out of 22 cowpox virus isolates could be subtyped by the presence or absence of a small BglII fragment. DNA sequencing showed that the lack of this BglII fragment was caused by a deletion of 72 nucleotides.

Injection of swine peripheral blood mononuclear cells into mice with severe combined immunodeficiency (SCID), resulted in the stable long-term establishment of a functional swine immune system (SCID-sw). Swine immunoglobulins were present in the serum of SCID-sw mice and swine cells were detected in the blood as well as in lymph nodes and spleen using monoclonal antibodies raised against cell subpopulations. Swine lymphocytes from reconstituted SCID mice responded in vitro to specific antigens or mitogens. When SCID-sw mice were challenged with African swine fever (ASF) virus, ASF virus-infected cells were detected in blood and spleen, and antiviral antibodies and virus-specific T cells were generated.

During an active human cytomegalovirus (HCMV) infection, leukocytes harbouring the HCMV lower matrix protein pp65 (ppUL83) are present in the peripheral blood and can be detected with the HCMV antigenaemia assay. In the present study, it was investigated whether the presence of pp65 in these cells was due to transcription of the virus genome or might be the result of uptake of this viral protein. Peripheral blood leukocytes of transplant recipients and AIDS patients with an active HCMV infection were investigated for the presence of HCMV immediate early (IE) antigen and pp65 using well characterized monoclonal antibodies, and for the presence of the corresponding mRNAs using non-radioactive in situ hybridization. Both mononuclear and polymorphonuclear cells were found to contain IE antigen and pp65. However, only mRNAs encoding IE antigen were found in these cells, whereas mRNAs encoding pp65 were not detected. In contrast, both IE antigen and pp65, as well as their corresponding mRNAs, were detected in the circulating late-stage HCMV-infected endothelial cells that were also present in the leukocyte fractions. These findings demonstrate that a restricted viral gene expression (transcription of IE genes) does occur in mononuclear and polymorphonuclear leukocytes. However, the abundant presence of the early antigen pp65 without detectable presence of the corresponding mRNA in these cells strongly indicates uptake of this protein by the phagocytic leukocytes, rather than de novo synthesis.

Epstein-Barr virus (EBV) replicates in a latent or a lytic way in the infected organism, depending on the type and level of differentiation of the host cell. The switch between latency and lytic replication was previously shown, for Burkitt’s lymphoma cell lines, to depend on the viral BZLF1 gene product. Protein-DNA assays were used to identify the cis-acting elements that represent the link between regulating signal transduction pathways and the viral cascade of gene expression. Specific binding of proteins to several sites of the BZLF1 promoter during latency was shown. Induction of the lytic cycle by stimulation with 12-O-tetradecanoyl- phorbol 13-acetate abolished the binding of these proteins to the distal promoter (positions −227 to −551), suggesting a functional role for the down- regulation of promoter activity during latency. Computer analysis identified a multiply repeated sequence motif, HI, in this region and exonuclease III footprints confirmed that these sites act as specific protein recognition sites. Using a set of reporter plasmids we were able to demonstrate a negative regulatory effect of the HI motif in some B lymphoid cell lines, in contrast to epithelial HeLa cells. The HI silencer elements are different from other silencer elements described so far in respect of their sequence and protein-binding pattern during the activation of BZLF1.