- Volume 73, Issue 8, 1992

The order of appearance of human immunodeficiency virus type 1 (HIV-1) nucleic acids was examined in monocyte-derived macrophages following a high multiplicity infection with macrophage-tropic virus. Using the polymerase chain reaction, viral DNA was first detected 2 h after infection and continued to accumulate over the next 24 h. Transcripts representing tat, rev and nef splicing were detected by 24 h, and transcripts representing env splicing were detected by 48 h after infection. Coincident with the appearance of env transcripts, new synthesis of cellular and extracellular p24 antigen began, multinucleated giant cells formed and progeny infectious virus emerged. This analytical system provides a foundation for further studies on the effects of antiviral agents and cellular factors on the replication cycle of HIV-1 in non-transformed, primary monocyte-derived macrophages.

The external glycoproteins of human immunodeficiency virus type 1 (HIV-1) (gp120) and HIV-2 (gp105) are responsible for binding the cellular receptor CD4. The proteins are functionally identical although their affinity for CD4 varies, with gp120 binding 10- to 20-fold more efficiently than gp105. To investigate the structural requirements for CD4 binding in each molecule we have constructed a number of hybrid glycoproteins in which sequences are exchanged between the two molecules via conserved residues and subsequently tested for their ability to bind to CD4. We found that two constructs in which the V1/V2 or V3 loops of gp105 are exchanged for those of gp120 continue to bind to CD4. Surprisingly, however, all other domain exchange mutants failed to bind to CD4 suggesting that long-range interactions within the molecule are sequence-specific. Mixing mutant molecules in vitro did not rescue CD4 binding. However, coexpression of a number of mutant glycoprotein pairs within the same cell produced complementation of CD4 binding ability; complementing molecules were shown to be heteromeric in structure. Alignment of the molecules within each complementation group allowed the interactive sequences necessary for receptor binding to be determined. These sequences constitute a novel target for the disruption of gp120 function.

Bovine leukaemia virus (BLV) resides in infected lymphocytes in a latent, repressed state but becomes expressed a few hours after the cells are cultured in vitro. We have identified several conditions and factors affecting the expression of BLV in short-term cultures of naturally infected lymphoid cells. The presence of foetal calf serum in the culture medium greatly stimulates virus expression. This stimulation is not due to cellular proliferation. Transcription of BLV RNA and synthesis of p25 in the cultures of peripheral blood lymphocytes are preceded by a lag period of several hours. Synthesis of BLV p25 in these cultures takes place almost immediately after viral RNA synthesis. Extending previous results, we demonstrate that the plasma and lymphatic fluid of cattle contain factors that suppress and stimulate BLV expression. As a result of systematic examination of several parameters, we have developed reproducible assays for the detection of these factors. It is very likely that their relative concentration in the host is an important determinant of susceptibility and resistance to the development of lymphosarcoma and persistent lymphocytosis in BLV-infected cattle.

The Fv-4 resistance (Fv-4r ) gene is a truncated endogenous murine leukaemia virus (MuLV) containing a 3′ portion of pol, the entire env gene and the 3′ long terminal repeat. Env expression renders mice resistant to infection by ecotropic MuLVs, probably via receptor interference. Previous studies have suggested that the flanking cellular sequences are also important for Fv-4 env gene expression. To establish how the truncated retrovirus was generated and the nature of the cellular sequences involved, the Fv-4 susceptible (Fv-4s ) allele DNA was cloned, and its restriction map and nucleotide sequence were compared with those of the Fv-4r allele. A likely mechanism for generation of the truncated endogenous MuLV is suggested by the results; integration of a prototype MuLV provirus at a site within the Fv-4s allele about 6 to 8 kb downstream of a non-retroviral promoter region, followed by deletion of the 5′ half of the provirus, with an accompanying loss of only 7 or 10 bp of cellular flanking sequences. The deletion may have led to the expression of the Fv-4r env gene under control of the non-retroviral promoter.

The cDNA sequence of genomic segment 5 of bovine rotavirus (RF strain) has been inserted into baculovirus transfer vectors, downstream of the polyhedrin promoter. Recombinant baculoviruses containing gene 5 were selected and the protein was expressed to high yields in Spodoptera frugiperda cells. The recombinant protein was inoculated into rabbits and mice to produce specific hyperimmune antisera. The polyclonal antiserum reacted with a protein in rotavirus-infected MA104 cells and with a protein translated in vitro. This serum was also used to confirm that the gene 5 protein is not a structural protein. Recently, the gene 5 product has been predicted to be a zinc finger protein and reported to contain a highly conserved arrangement of cysteine residues; here, we demonstrate that the recombinant gene 5 protein binds zinc and is an RNA-binding protein as are several other zinc finger proteins.

The primary amino acid sequence of the VP4 proteins of human rotavirus strain AU-1 and feline rotavirus strain FRV-1 was deduced from nucleotide sequence analysis of full-length genome segment 4 cDNAs produced by a combined reverse transcription-polymerase chain reaction. The VP4 genes were 2359 nucleotides in length and contained one long open reading frame capable of encoding a protein of 775 amino acids. Strain AU-1 and FRV-1 VP4s were 98.8% similar at both the nucleotide sequence and amino acid level. Given that most of the genome segments of strains AU-1 and FRV-1 formed hybrids under stringent hybridization conditions, the relationship between their VP4 gene sequences is best explained by feline rotavirus being transmitted to human hosts as whole virions relatively recently. Of added interest is that AU-1 and FRV-1 VP4 both exhibit high degrees of similarity (96.0% nucleotide identity and 97.2 to 97.5% amino acid identity) with serotype G1 human rotavirus strain K8 VP4, which is distinct from any other sequenced VP4 allele. This suggests that strain K8 VP4 was derived by natural gene reassortment from a feline rotavirus or a strain AU-1-like human rotavirus.

Neutralizing epitopes present on the five serotypes of bluetongue virus (BTV) which have been isolated in the United States were investigated with a panel of monoclonal antibodies (MAbs). Neutralizing MAbs were raised against the U.S. prototype viruses of BTV serotypes 2, 10, 11, 13 and 17, and were reacted with each virus in both neutralization and immune precipitation assays. All MAbs neutralized and precipitated VP2 of the virus against which they were raised. Five MAbs raised against BTV-10 also precipitated VP2 of the prototype strain of BTV-17, and four of these MAbs neutralized BTV-17. To characterize further the neutralizing epitopes of BTV, the MAbs raised against BTV-10 and BTV-17 were reacted by immune precipitation and neutralization assays with four field strains each of BTV-17 and BTV-10 isolated from ruminants in the U.S. All MAbs raised against BTV-10 both precipitated VP2 and neutralized the four field isolates of BTV-10, whereas none of the MAbs raised against BTV-17 reacted with these viruses. By contrast, all seven MAbs raised against BTV-17 and four of the seven MAbs raised against BTV-10 precipitated VP2 of the four BTV-17 field isolates. Another MAb raised against BTV-10 precipitated VP2 of three of the four field isolates of BTV-17. Whereas neutralization of the BTV-17 field isolates by several MAbs was inconsistent, all 10 isolates of BTV-10 and BTV-17 were neutralized by three MAbs raised against BTV-10. Results of this and other studies indicate that multiple neutralizing epitopes exist on each serotype of BTV. Some of these epitopes are conserved whereas others apparently vary in their significance to the neutralization of individual field isolates of BTV-17 and perhaps other BTV serotypes. These findings have implications for the future development of efficacious subunit vaccines to prevent BTV infection of ruminants.

Cultures of adherent and non-adherent bovine peripheral blood mononuclear (PBM) cells were inoculated with bluetongue virus (BTV) serotype 10. Some cultures of non-adherent cells were stimulated with interleukin 2 (IL-2) and concanavalin A for 24 h prior to virus inoculation. Cells were harvested at various intervals up to 72 h after inoculation. A panel of leukocyte differentiation antigen-specific monoclonal antibodies (MAbs), specific for bovine CD2, CD4 or CD8, monocytes and granulocytes, B cells, γδ T cells or the IL-2 receptor (IL-2r), was directly conjugated to fluorescein isothiocyanate, and a MAb specific for the BTV major core protein VP7 was directly conjugated to phycoerythrin. Cells were labelled with conjugated MAbs in single- and double-label immunofluorescence studies to identify specifically the BTV-infected cells in inoculated cultures. The viability of cells was determined by propidium iodide exclusion, and all analyses were done using flow cytometry. Productive infection of cultures of PBM cells was confirmed by virus titration. The data revealed a clear difference between subsets of bovine PBM cells in susceptibility to infection with BTV in vitro. Monocytes were readily infected with BTV, as were stimulated CD4+ cells, and infection was cytopathic to monocytes and stimulated lymphocytes. The proportion of infected cells decreased after 24 h and virus titres dropped markedly by 72 h in all cultures. CD4+ cells in cultures of unstimulated non-adherent cells inoculated with BTV showed increased expression of IL-2r. The possible relevance of these findings to the pathogenesis of BTV infection of cattle is discussed.

The reovirus receptor on mammalian cells has not been fully characterized and controversy exists over the nature of this receptor. We report here that the expression of this receptor is dependent on the differentiation status of a human promyelocytic leukaemia cell line (HL60). Phorbol treatment of HL60 cells for 24 h, at a concentration range of 160 nm down to 1 nm, led to differentiation of these cells towards monocytes and a loss of approximately 80% of their ability to bind reovirus in a fluorescence assay. These cells also lost their susceptibility to T1 and T3 reovirus infection. DMSO treatment for 24 h at a concentration of 1.25% (v/v) led to differentiation towards granulocytes. This was accompanied by an increase of approximately 15% in binding of reovirus to these cells. After being infected by T1 or T3 reovirus, the granulocytes produced higher titres of progeny virus than did untreated HL60 cells. Similar differences were noted when virus binding to HL60 cells was assayed using radiolabelled reovirus. These effects were not detected when murine L fibroblasts were treated with DMSO or phorbol. ATCC-derived murine R1.1 cells did not bind reovirus. Competition data indicated that there may be two reovirus receptors on HL60 cells, and that T1 can bind to only one receptor whereas T3 can bind to both receptors. Our data also suggested that the β-adrenergic receptor was unlikely to act as the reovirus receptor on HL60 cells.

Using immunoelectron microscopy, the distribution of influenza A virus neuraminidase (NA) glycoproteins was examined, after performing immunoreactions to virions on the grid. With polyclonal antibody, the immunolabels of the glycoproteins were found to be homogeneously distributed, whereas with monoclonal antibody they were found to be distributed in clusters. After destruction of haemagglutinin (HA) but not of NA activity with a high concentration of trypsin, the remaining visible spikes were evenly distributed. This finding was consistent with the absence of immunolabelling with anti-HA antibody, and the homogeneous pattern of immunolabels with anti-NA polyclonal antibody, but not with the clustered labelling with the anti-NA monoclonal antibody. Thus, the immunolabelling image with anti-NA polyclonal antibody was considered to reflect the true one.

The amino acid sequence of the poliovirus 2C protein contains two highly conserved stretches, GSPGTGKS136 and MDD177, which correspond to the consensus ‘A’ and ‘B’ motifs (GXXXXGKS/T and DD/E, respectively) found in nucleoside triphosphate-binding proteins. To assess the functional importance of these amino acid sequences, we changed conserved and non-conserved amino acids. The replacement of the non-conserved Thr133 residue with Ser or Ala did not markedly change the virus phenotype. Similarly, replacement of the non-conserved Pro131 residue by Ala did not abolish virus viability, but changes of this residue to Thr or Asn were not tolerated. No viable mutant could be isolated after transfection of cultured cells with transcripts mutated at the conserved Lys135, Ser136 or Asp177 residues. However, true revertants were selected from Arg135 and Ser135 mutants, from Glu177 and Gly177 mutants, and from Ala136 mutants. Thr136 mutants not only gave rise to true revertants, but also to two independent isolates of a suppressor mutant, Asn140→Tyr. All the lethal mutations resulted in severe inhibition of viral RNA synthesis in vivo, although no translational deficiency was detected in a cell-free system. This is the first direct evidence for the functional significance of the nucleoside triphosphatebinding pattern in the poliovirus 2C protein.

A region of the fusion protein of measles virus (residues 240 to 252) was predicted to contain a B and a T epitope. A synthetic peptide representing this sequence was shown to induce both T and B cell reactivity in several inbred strains of mice, but the responses were clearly major histocompatibility complex-restricted. Elongation of this peptide by six residues at the C terminus on the basis of predictions for B cell epitopes resulted not only in increased peptide immunogenicity in some strains of mice but also produced strain-related positive and negative effects on the recognition of the peptide. BALB/c and SWR mice were non-responders to the short version of the peptide but responded well to the elongated form. On the other hand, the injection of the elongated peptide into C57BL/6 mice resulted in a loss of both B and T cell responsiveness seen with the short version. These results indicate the importance of flanking sequences on the immunogenicity and antigenicity of synthetic B and T cell epitopes and highlight the necessity to determine the most appropriate size of peptide to be used as an immunogen.

Dugbe virus is a member of the nairovirus genus of the Bunyaviridae. Purified polyclonal anti-Dugbe virus IgG, which neutralized > 99.5% of virus, reduced attachment of virus to BSC-1 cell monolayers by only 36%. A 100-fold lower concentration neutralized virus by 88%, and had no effect upon attachment. Neutralizing IgG did not affect the ability of Dugbe virus to be internalized by or to fuse with BSC-1 cells. This suggests that IgG neutralization occurs largely at a stage subsequent to primary uncoating. Purified polyclonal anti-Dugbe virus IgM neutralized infectivity and had no effect on the attachment of virus to cells, but inhibited internalization of virus by about 50%. Thus IgM neutralizes partly by interfering with entry of virus and partly by a post-entry event. Neutralization by intermediate concentrations of IgM was enhanced 20-fold in the presence of complement. At high concentrations of IgM, complement-dependent neutralization declined. This is probably due to IgM binding in a planar rather than crab conformation, which does not expose the complement binding sites. Aggregation occurred only at relatively low concentrations of immunoglobulin. Electron microscopy and reactivation of infectivity by vortexing suggested that aggregation makes only a minor contribution to neutralization by IgG or IgM.

The established line of normal Fisher rat fibroblasts (FR3T3) is naturally resistant to the parvovirus minute virus of mice (MVM), and was used as a model system to study the influence of stepwise transformation on the susceptibility of cells to this virus. When transformed with genes encoding the class I nuclear oncoproteins large T antigen of polyomavirus (PyLT) or v-myc, cells retained a normal appearance, but acquired some ability to form colonies in soft agar. On the other hand, the class II transforming oncogenes encoding the middle T antigen of polyomavirus (PyMT) and c-Ha-ras-1 induced both morphological alterations and a high capacity for anchorage-independent growth in transfected cells. The concomitant expression of oncogenes from both classes (PyLT+PyMT; v-myc+c-Ha-ras-1) induced a supertransformed phenotype characterized by the piling-up of cells into poorly adherent foci, even in low density cultures. The progressive transformation of this cellular system was found to coincide with a gradual increase in its susceptibility to MVMp (MVM prototype strain) infection. Compared to parental cells, class I, class II and double transformants proved to be sensitized to killing by MVMp to a low, moderate and large extent, respectively. Thus, oncogenes from different functional classes appeared to cooperate in the responsiveness of cells to parvovirus attack. Interestingly, this cooperation exacerbated both the killing of infected cells and their capacity to produce viral non-structural (NS) proteins, in agreement with the reported cytotoxic activity of NS polypeptides. Therefore, in this system, parameters of the parvovirus life cycle may serve as indications of the overall progression of the transformation process.

Six isolates of erythromelalgia-related poxvirus (ERPV) were characterized with respect to host range, c.p.e. and inclusions, pock formation on chorioallantoic membrane (CAM), morphogenesis, serological reactivity, pathogenesis in animals and DNA restriction fragment profile. The results suggest that ERPV is either a new member of the Orthopoxvirus genus or a subspecies of ectromelia virus. Evidence is provided that (i) ERPV has a wide host range in vitro in which characteristic viral c.p.e. and inclusion bodies are induced; (ii) ERPV, unlike ectromelia virus, causes the formation of tiny greyish-white pocks on CAM both at 34 °C and 39 °C; (iii) eosinophilic A-type inclusions of ERPV do not contain viral particles; (iv) ERPV isolates are neutralized by both rabbit anti-vaccinia virus and mouse anti-ectromelia virus sera, but not vice versa; (v) young rabbits are not susceptible to ERPV by skin and/or corneal scratch infection even though ERPV is lethal for mice by intraperitoneal inoculation; (vi) the HindIII and SalI fragment profiles of ERPV P-4 DNA are similar to, but obviously different from, those of Chinese ectromelia virus. These biological and pathogenic characteristics of ERPV are distinguishable from those of other members of the genus Orthopoxvirus currently described in the literature.

Characterization of a tissue culture-adapted strain of murine cytomegalovirus (MCMV), the Vancouver strain, which demonstrated altered tissue tropism in mice was undertaken to help understand the mechanism of pathogenesis of cytomegaloviruses. The Vancouver strain grew to a limited extent in the spleen but failed to grow in the salivary glands of inoculated mice. This mutation probably arose during multiple in vitro passaging of the parental Smith strain. The Vancouver strain replicated more quickly and produced a greater yield of virus per cycle than the Smith strain in vitro, resulting in a larger plaque size. In addition to these phenotypic differences, the Vancouver strain was found to have a 9.4 kb deletion spanning the XbaI I/L junction of the parental Smith strain (0.960 to 0.995 map units), and a 0.9 kb insertion which mapped to the EcoRI K fragment (0.37 to 0.47 map units). Analysis of virus-induced proteins at various times post-infection identified only one major change in Vancouver strain-infected cells, the absence of a 42K protein found in Smith-infected cells at early and late times.

We examined a panel of monoclonal antibodies (MAbs) against bovine herpesvirus type 1 (BHV-1) glycoproteins gI, gIII and gIV for inhibition of virus attachment and interference with subsequent steps of infection. Attachment of radiolabelled virions was partially prevented by 600 to 700 µg/ml of IgM antibodies against gI and gIII and one IgG2A antibody against gIV, but not by the majority of MAbs against any of the three viral glycoproteins. Productive infection following attachment was prevented by lower concentrations of MAbs 5106 and 4807 against gI and by 0.7 to 5.5 µg/ml of all five MAbs against gIV. MAbs against gIV had almost the same activity whether added before or after BHV-1 was incubated with cells, suggesting that their principal activity is to prevent the penetration of virus through the cell membrane. The ability of polyethylene glycol to overcome neutralization by one anti-gIV MAb supported this concept, but an attempt to confirm this by direct electron microscopy failed. A bovine monospecific antiserum against gIV had approximately 10-fold more neutralizing activity against BHV-1 than did antisera against gI or gIII. Complement increased the activity of anti-gI and anti-gIII MAbs by 10- to 100-fold, but had little or no effect on neutralization by anti-gIV MAbs. Some antibodies against gI and gIV inhibited the enlargement of plaques in cell cultures. Taken together, these data suggest that MAbs against gIV are the principal agents of BHV-1 neutralization, and that these antibodies can be fully effective in areas such as the ocular and respiratory mucosae, from which complement is absent at the time of primary exposure to infection.

Using a solid-phase assay we have demonstrated specific competition between the preS1 sequence of hepatitis B virus and human IgA in their binding to isolated normal human liver plasma membranes, suggesting molecular mimicry. Monoclonal and polyclonal antibodies raised against virus and IgA epitopes were used to detect and map immunological cross-reactivity to the virus sequence involved in liver cell binding. These findings suggest the existence of a common receptor or of closely related receptors for the attachment of HBV and IgA to human liver cells.

Human papillomavirus (HPV) type 16 transcription was analysed by in situ hybridization using 125I-labelled subgenomic riboprobes, from 26 genital intraepithelial neoplastic (IN) lesions, in formalin-fixed biopsies from 18 different cases. Distinct transcription patterns separable by the presence or absence of late gene transcription were detected. In 12 lesions, late gene expression was absent; HPV transcripts corresponding to the E6 and E7 open reading frame (ORF) were detectable in all basal cells and were usually evenly distributed through all layers of the epithelium. Transcripts corresponding to the E1, E2 and E2/E4 ORFs were present in nine of 12 lesions and displayed a similar distribution. In 14 lesions late gene transcripts were present. E6 and E7 transcripts were detectable basally in all but one lesion. The levels of E7 but not E6 transcripts were markedly increased in the superficial cells of differentiating epithelia, with an identical distribution and at similar levels to those of the E2/E4 transcript. We propose that the most abundant transcript in genital IN lesions containing late gene expression is an E7/E1 ^ E2/E4 transcript corresponding to that reported in HPV-6/11 condylomata and which is derived from a similar promoter within the E7 ORF.