- Volume 71, Issue 5, 1990

The degradation of herpes simplex virus particles after uptake by phagocytes was studied, but, since lysis of the phagocyte also resulted in damage to the viral envelope, measurement of viral infectivity as a criterion of viral degradation after phagocytosis was not possible. Therefore we focused on later events in viral destruction, namely the degradation of macromolecules. We have demonstrated that polymorphonuclear leukocytes (PMN) and monocytes (MN) can rapidly degrade the membrane proteins of the phagocytosed herpesvirus virions. PMN and MN from a patient with chronic granulomatous disease showed a similar rate of degradation compared to PMN and MN from healthy donors, which excludes an important role for toxic oxygen species in viral protein degradation. Experiments using toxic oxygen species-generating systems supported this observation. In contrast to PMN, MN are also effective in the digestion of viral DNA. We conclude that PMN and MN are able to neutralize large amounts of phagocytosed HSV, so their role in antiviral defence has again been demonstrated.

Amino-terminal amino acid sequencing was carried out on proteins purified from herpes simplex virus type 1 capsids. The sequences of two capsid proteins (VP19C and VP23) showed them to be encoded by genes UL38 and UL18, respectively. The product of UL38 has been shown to be essential for capsid assembly, but no role has previously been assigned to the product of UL18.

Bovine MDBK cells were transfected with Rous sarcoma virus-based vectors for constitutive expression of the bovine herpesvirus type 1 (BHV-1) glycoprotein, gI. Cell lines stably expressing recombinant gI were cloned and characterized. Recombinant gI was localized intracellularly, predominantly in a perinuclear region, and on the cell surface. Cells expressing gI exhibited spontaneous polykaryon formation, thus confirming the fusogenic activity described previously in gI-expressing transfected murine LMTK- cells. The recombinant form of gI synthesized in transfected MDBK cells was similar in Mr to the form expressed in BHV-1-infected MDBK cells, unlike the recombinant form of gI expressed by LMTK− cells which is deficient in N -linked glycosylation. It was concluded that cell fusion associated with the expression of BHV-1 gI in transfected mammalian cells is a reproducible phenomenon in a number of cell types and is not due to species-specific factors or expression of abnormally glycosylated gI. Cell fusion is a useful in vitro marker for gI function and may contribute to the spread of BHV-1 infections in vivo.

To determine whether heparan sulphate residues on the cellular surface could serve as an attachment receptor for pseudorabies virus (PRV), the effect of heparin on PRV in plaque reduction and adsorption tests was investigated. PRV was significantly less sensitive to heparin than was herpes simplex virus type 1 (HSV-1). At concentrations of 500 µg/ml heparin the number of plaques formed by PRV was reduced to 7 % of the untreated control whereas the number of plaques formed by HSV-1 was reduced to below 0·1%. Adsorption of PRV to host cells was also less sensitive to heparin treatment than was adsorption of HSV-1. Experiments concerning the binding sites of PRV showed that heparin binds to the disulphide-linked glycoprotein complex gII (PRV gB), gIII (PRV gC) and probably gV.

Ten monoclonal antibodies have been raised against lysates of cells infected with cytopathic border disease virus (BDV). These antibodies all recognize non- cytopathic BDV and react with a number of different strains of bovine viral diarrhoea virus (BVDV). Studies with radiolabelled cell lysates show that all the antibodies precipitate two polypeptides of apparent M r 80000 and 130000 from cells infected with cytopathic virus and a single polypeptide of apparent M r 120000 from cells infected with non-cytopathic virus. Two of the monoclonal antibodies react on immunoblots and show the same pattern of reactivity indicating that these three polypeptides are antigenically related.

Mice infected with lactate dehydrogenase-elevating virus (LDV) developed antibodies reactive with a tumour cell surface antigen (TSA) of Moloney sarcoma virus (Mo-MSV)-transformed mouse cells (Sac). We demonstrate that STU mice infected with LDV were protected against growth of syngeneic Sac tumour cells as early as 23 days post-infection (p.i.) and up to 5 months p.i. Nine LDV strains, including the neurovirulent LDV-C, elicited production of anti-TSA antibodies, which were restricted to the IgM isotype. Monoclonal anti-TSA antibodies were raised 4 days after infection of STU mice with LDV. When tested against several transformants of STU and BALB/c mouse origin they were found to react with Mo-MSV transformants (PV-TC-77, STU mouse origin; MSV85 C1 3, BALB/c mouse origin), methylcholanthrene-transformed MethA cells (BALB/c origin) and L929 cells. We suggest that the well known tumour growth inhibition by LDV is due to LDV-induced anti-TSA antibodies.

We have identified the major immunogenic structural proteins of the human foamy virus (HFV), a distinct member of the foamy virus subfamily of Retroviridae. Radiolabelled viral proteins were immunoprecipitated from HFV-infected cells by foamy virus antisera of human and non-human primate origin. Precipitated viral proteins were in the range of 3IK to 170K. Labelling of proteins with [I4C]glucosamine or with [35S]methionine in the presence of tunicamycin, as well as endo- β - N -acetylglycosaminidase H and F treatment of [35S]methionine-labelled proteins, revealed three viral glycoproteins of approximately 170K, 130K and 47K, most likely representing the env gene-encoded precursor, the surface glycoprotein and the transmembrane protein of HFV, respectively.

We utilized the RNA polymerase chain reaction (PCR) to analyse the transcripts of the E6/E7 open reading frames of human papillomavirus type 16 (HPV-16). Total RNA was isolated from 14 cervical squamous carcinomas, nine cervical intraepithelial neoplasias and from human fibroblasts transformed with different HPV-16 constructs. In all specimens two spliced transcripts were detected. Sequence analysis of the cloned PCR products showed that both transcripts were generated by splicing out an intron in E6, from nucleotides (nt) 226 to 409 in one transcript and from nt 226 to 526 in the other. The major transcript present in all RNA specimens had the smallest intron in E6. The RNA PCR described here is the method of choice for analysing splice and donor sites in tissue specimens where a limited amount of RNA is available. Results obtained with transformed cells revealed no difference in splicing whether HPV-16 was controlled by its homologous promoter or by a heterologous promoter, the Rous sarcoma virus long terminal repeat.

The nucleic acids of many plant viruses encode proteins with one or more of the following properties: an M r of approximately 30000, localization in the cell wall of the infected plant and a demonstrated role in cell-to- cell transport of infection. A progressive alignment strategy, aligning first those sequences known to be similar, and then aligning the resulting groups of sequences, was used to examine further the relatedness of the amino acid sequences of putative transport proteins of caulimoviruses, of proteins similar to the putative transport protein of alfalfa mosaic virus (A1MV) and of those similar to the tobacco mosaic virus (TMV) 30K protein. The strategy first identified regions in which multiple dipeptides of one group were similar to those of another group. The regions of similarity were brought into alignment by the conservative introduction of gaps. The positions of the introduction of gaps were adjusted to optimize similarity.

Statistical significances of the resulting alignments, determined both by comparison with shuffled amino acid sequences and with the sequence alignment off-set by 1 to 15 residues in each direction, suggest that the amino acid sequences of the three groups of viruses are distantly related. Nevertheless, significant relationships between members of the caulimoviral group of sequences and members of each of the AIMV-like and TMV-like groups were found. These relationships and the analysis of the number of insertions/deletions between present sequences and a hypothetical common ancestor suggest that the sequences of the caulimoviral proteins are less diverged from the ancestor than either the AIMV-like or TMV-like proteins. The alignment identified common regions of predicted secondary structure and regions of similar hydropathy, regions possibly crucial for proper functioning of the proteins.

DNA complementary to Nicotiana velutina mosaic virus (NVMV) RNA was qloned and five segments larger than 0·9 kb were used in Northern blot hybridization analysis to identify two virus-specific RNAs, approximately 8 kb (RNA 1) and 3 kb (RNA 2) in size. The clones selected as probes did not hybridize with RNA from various tobamoviruses, or from beet necrotic yellow vein (BNYVV) and peanut clump furoviruses. In an attempt to determine the taxonomic position of the virus, about 75 % of the NVMV RNA 2 was sequenced and four open reading frames (ORFs) were identified. ORFs 1,2 and 3 encode proteins of M r 20K, 39K and 13K, whereas ORF 4 was incomplete. ORFs 2, 3 and 4 overlapped in an arrangement closely resembling the triple gene block identified in BNYVV RNA 2, barley stripe mosaic virus (BSMV) RNA 2, potato virus × and potato virus M RNA. The presumed coat protein gene of NVMV RNA 2 (ORF 1) is situated to the 5′ side of the triple gene block as for BNYVV and BSMV RNA 2. Amino acid homologies were detected among the 13K and 14K proteins of NVMV RNA 2, BNYVV RNA 2 and BSMV RNA 2. Significant homology was also detected between the 39K protein of NVMV RNA 2 and the 42K protein of BNYVV RNA 2, with a motif specific for ATP- and GTP-binding (NTP-binding motif), and a conserved viral DNA polymerase domain. The presence of a triple gene block in NVMV RNA 2 indicates that NVMV has affinities with members of the hordei-, furo-, potex- and carlavirus groups but not with the tobamovirus group. The divided RNA genome of NVMV, and the sizes of the two RNAs suggest that NVMV is most closely allied to the furoviruses, but the unique nature of its different biological properties and lack of any serological relationships with furoviruses lead us to conclude that NVMV has no clear relatedness to any taxonomic group of plant viruses.

Purified virus particles of potato virus S (PVS) when disrupted yielded only one readily resolved band of 7·5 kb in ethidium bromide-stained agarose gels. However Northern hybridization of viral RNA, probed with a clone specific to the viral coat protein gene, revealed a region of subgenomic RNAs of approximately 1·3 kb. Sucrose gradient fractionation of subgenomic RNA revealed that it coded for viral coat protein when translated in vitro in rabbit reticulocyte lysate. Virus particle lengths suggest that this subgenomic RNA may be encapsidated in 100 to 220 nm particles.