- Volume 71, Issue 8, 1990

A filamentous phage, øLf, which specifically infects Xanthomonas campestris pv. campestris was isolated. The phage particle measured 1000 (±200) × 8 nm. It formed turbid plaques of about 1 mm in diameter. During multiplication, the progeny virions extruded into the medium without retarding host cell growth. Stocks were stable for 6 months at 4 °C and survived treatment at 80 °C for 10 min. Treatment with chloroform, ethanol or acetone completely destroyed infectivity; ethyl ether and methanol inactivated 98 to 99% of the phage. SDS-polyacrylamide gel electrophoresis showed a major coat protein band of approximate Mr 4000 whereas an immunoprecipitation test detected the existence of two coat protein species. The phage genome was shown to be a single-stranded DNA molecule. A physical map was constructed and the DNA size was calculated to be 5·9 kb. Cells treated with Tris-HCl containing CaCl2 and polyethylene glycol 6000 were transfected by replicative form DNA at a frequency of 3·4 × 103 p.f.u./ µg.

The mRNA transcribed from the N gene of viral haemorrhagic septicaemia virus (VHSV) of salmonids has been cloned in Escherichia coli and expressed. Fusion proteins were recognized by monoclonal antibody directed against the N protein from the viral particle. A 1212 bp long open reading frame (ORF) coding for 404 amino acids with a calculated Mr of 44590 was deduced from the nucleotide sequence. The ORF was preceded by a 93 bp segment including in position 42 the AACAC pentanucleotide which is presumed to be the start signal for transcription by analogy with other rhabdoviral mRNAs. The upstream 41 bp region could correspond to the covalently linked positive polarity leader RNA as also found on the N mRNA from infectious haematopoietic necrosis virus (IHNV). This may be a characteristic of fish lyssaviruses. The AAACC sequence, which is part of the leader, was not found. Amino acids 44 to 359 from IHNV and 45 to 360 from VHSV are 45·3% homologous. A strong homology which could reflect functional importance was also found for potential phosphorylation sites and hydrophobic peaks despite the fact that the two viruses evolved on different continents.

Tumour necrosis factor α (TNF-α) elicited an antiviral response in some cell lines (MG-63 and HEp-2) but not in others (MDBK). Cell lines that generated an antiviral response to TNF-α also showed induction of a 15K protein which shared sequence homology with ubiquitin and reacted with an antibody to ubiquitin. This ubiquitin cross-reactive protein (UCRP) had been demonstrated previously to be induced by interferon. The TNF-α induction of UCRP occurred at the level of transcription. TNF-α induction of both the antiviral state and the 15K protein was blocked by either monoclonal or polyclonal anti-β-interferon (IFN-β) antibody. However no measurable increase in the mRNA specific for IFN-β was detected after TNF-α treatment. Nonetheless, in supernatants from cell cultures, the presence of an antiviral activity inhibitable by anti-IFN-β antibody indicates that these cells are making IFN-β already. We conclude that the TNF-α induction of antiviral activity and UCRP in cells is dependent upon the presence of constitutive low levels of IFN-β in the responding cells. Furthermore TNF functions to enhance the existing IFN-β activity.

A panel of monoclonal antibodies has been produced against a 1983 human influenza A (H1N1) virus that has been isolated and grown exclusively in MDCK cells. Several of these antibodies were used to select variants from MDCK-derived virus in cells and their epitopes were then located by determining the HA1 amino acid sequence. The operational antigenic map of the haemagglutinin indicates the presence of two distinct immunodominant antigenic regions which correspond but are not identical to antigenic sites Sa and Sb of the A (H1N1) virus A/PR/8/34. Also during this study, we characterized a unique group of antibodies for which variants require two distinct and specific HA1 amino acid substitutions to escape neutralization.

Human monoclonal antibodies to rabies virus were established by Epstein-Barr virus infection of peripheral blood lymphocytes collected from a rabies- vaccinated donor, and fusion with a heteromyeloma line. Two human monoclonal antibodies, HUM1 and HUM2, both IgG2, reacted with the envelope glycoprotein of the rabies virus. The antibody HUM1 neutralized rabies virus (lyssavirus serotype 1) and Mokola virus (lyssavirus serotype 3), but did not neutralize European bat lyssavirus, suggesting that some common antigenicity exists between the glycoproteins of serotypes 1 and 3. In addition, this antibody neutralized a series of viruses resistant to neutralization by antibodies recognizing, in a murine system, anti genic sites I, II and III; however, it failed to neutralize viruses altered at site VI, indicating that human monoclonal antibody HUM1 is directed against antigenic site VI. The other human anti-glycoprotein antibody, HUM2, neutralized the European bat lyssavirus in addition to serotypes 1 and 3, but none of the resistant variant viruses altered at the sites mentioned above. A third human monoclonal antibody, HUM3 (IgM), was reactive with the internal nucleoprotein of the rabies virus. This antibody contained a murine light chain corresponding to the cytoplasmic murine chain not secreted in the heteromyeloma line. The potential use of monoclonal antibodies in post-exposure treatment of rabies is discussed.

Recent evidence of a protective immune response to the flavivirus non-structural protein, NS1, has suggested its incorporation into possible recombinant vaccines. The region of the 17D yellow fever virus (YFV) genome encoding the C terminus of envelope glycoprotein and extending to the N terminus of non-structural protein NS3 (NSl-NS2a-NS2b; nucleotides 2030 to 4940) was expressed in vaccinia virus and physical and immunogenic properties of the NS1 moiety were studied. Recombinant NS1 protein, and native YFV NS1, was detected at the surface of infected cells by immunofluorescence and by immune cytolysis after treatment with anti-NSl antibody and complement. NS1 was also detected in the extracellular medium as a secreted form. Recombinant NS1 was immunoprecipi- tated as a single protein of approximately the same size as native 17D YFV NS1. Unboiled, both recombinant and native NS1 formed polymers of high M r. Immunization of mice with this recombinant vaccinia virus stimulated production of non-neutralizing, complement-fixing cytolytic antibody and conferred partial protection against lethal intracerebral inoculation of mice with live 17D YFV.

Foot-and-mouth disease virus (FMDV) cDNA cassettes containing sequences encoding the capsid precursor PI-2A with and without those encoding the proteases L and 3C were introduced into Autographa californica nuclear polyhedrosis virus (AcMNPV) expression vectors. Procapsid proteins 1AB, 1C and ID were produced in cells infected with recombinant baculoviruses, when L and 3C were present in the constructs, indicating that these FMDV proteases were active in insect cells. Unlike µl processing in poliovirus, which has been shown to be catalysed mainly by the 3CD gene product, the 3C protease of FMDV was able to process P1 independently of 3D. Cytotoxicity of the L protease for insect cells prevented the use of the optimized transfer vector, pAcRP23, for inserting L- containing cassettes into AcMNPV. By contrast, viable AcMNPV-FMDV recombinants could be made without restriction on choice of the transfer vector when the L gene was either not expressed or inactivated by an inframe deletion. In the latter case, normal cleavage at the L-Pl junction no longer occurred in cis, and a new processing event, probably catalysed by 3C, was observed within the C-terminal region of the residual L protein. Analysis of baculovirus-expressed products in sucrose gradients showed that a fraction of the capsid proteins is present in an aggregated form, migrating at 70S and possibly resembling FMDV empty capsid particles.

Using a simian virus 40/adenovirus 12 (Adl2)-recom binant virus, the Ad 12 early region IB (E1B) 19K protein has been produced at high levels after infection of Cos 1 cells. Expression of the 19K polypeptide reaches a maximum at about 48 h post-infection, declining at later times as host cell death occurs. Using two-dimensional isoelectric focusing/SDS-polyacryl- amide gel electrophoresis, we have shown that the Ad 12 E1B protein is a major species following infection of Cos 1 cells with recombinant virus. Two forms of 19K polypeptide can be distinguished following isoelectric focusing. Using subcellular fractionation of infected cells, it was found that a similar distribution of 19K protein occurred after recombinant virus and Ad 12 infection, with the polypeptide being most abundant in nuclear and membranous fractions. Similarly, as in Adl2-infected cells, a certain proportion of the protein is located on the outside surface of the cell after recombinant viral infection. Immuno- histochemical studies suggest that, at early times postinfection, the E1B 19K protein is located in the nuclear membrane, the Golgi apparatus and the endoplasmic reticulum. At later times, it can be seen to have spread to the cytoplasm as well as to the other organelles. These results are discussed in relation to the known functions of the 19K E1B protein.

The complete sequence of the genome of bovine polyomavirus (BPyV), formerly known as the CK isolate of the stump-tailed macaque virus, is presented. The genomic organization of BPyV is similar to that of the non-rodent polyomaviruses. With a genome size of 4697 bp, BPyV has the smallest polyomavirus genome known so far. When compared to simian virus 40 (SV40), the shortness of the BPyV genome is due mainly to differences in the coding capacity of the BPyV early region. The first exon of the proposed large T antigen encodes only 35 amino acids; also, a coding region corresponding to the C-terminal 64 amino acids of the SV40 large T antigen is absent in BPyV. It is proposed that the nucleotide sequence encompassing the small t antigen coding sequence contains an intron sequence of 71 nucleotides. Together the two exon sequences encode a 124 amino acid protein. We conclude that this may be the first example of a polyomavirus that has a small t antigen which is translated from two exon sequences. The enhancer region of BPyV does not show homology to the SV40 enhancer sequences. An agnogene is present with a coding capacity of 118 amino acid residues. The highest degree of homology to SV40 and PyV is present in the VP1 molecule.

The molecular cloning and characterization of an EcoRI fragment, 8·26 kb in size, of an Australian isolate of bovine leukaemia virus (pBLV-Al) is described. This fragment includes most of the proviral genome as well as 340 bp of flanking bovine DNA sequence at the 5′ end. Approximately 790 bp, including the 3′ long terminal repeat, was missing from this clone. At the level of restriction enzyme mapping, this isolate could be distinguished from American, Belgian and Japanese isolates. DNA sequencing of the entire clone demonstrated some variation at the amino acid level between pBLV-Al and the Japanese and Belgian isolates, particularly in the gag gene. In that gene there were 59 amino acid changes compared to the Japanese isolate and 24 compared to the Belgian isolate. The greater number in the case of the Japanese isolate was due to both single nucleotide changes and frameshift in a single region of the gene. This study also demonstrates that there are large tracts of amino acid sequence, particularly within the env andpol genes, that are highly conserved in different isolates. Some of these conserved sequences exist in regions containing epitopes important in virus infectivity.

We have determined the nucleotide sequence of two genes in the unique short region of the genome of pseudorabies virus (PRV). Near the internal repeat, upstream of the gene encoding glycoprotein gX, we identified an open reading frame (ORF) encoding a protein of 390 amino acids. We designated this gene PK because the predicted protein contains most of the conserved motifs of a eukaryotic protein kinase. The protein shares amino acid homology with the protein kinases encoded by gene US3 of herpes simplex virus type 1 (HSV-1) and gene 66 of varicella-zoster virus. Near the terminal repeat, downstream of a gene encoding an 11K protein, we identified an ORF encoding a protein of 256 amino acids. We designated this gene 28K, the Mr of the predicted protein. Part of the amino acid sequence of 28K is homologous to the predicted US2 protein of HSV-1. Northern blot analysis revealed a 2·7 kb mRNA encoding the putative protein kinase and a 1·2 kb mRNA encoding the 28K protein in PRV-infected cells. The 5′ ends of the mRNAs were mapped by primer extension. Two transcriptional start sites were identified for the PK mRNA: a minor start site immediately upstream of the ORF and a major start site (>95% of the mRNA) within the ORF, 64 nucleotides upstream of an internal ATG codon. A single transcriptional start site was identified for the 28K mRNA immediately upstream of the ORF. Immunoblot analysis with anti-peptide sera revealed that, in cells infected with PRV, the PK gene was translated into two proteins with Mr s of 53K and 4IK, and the 28K gene into a single protein with an M r of 28K.

We have cloned and determined the nucleotide sequence of a gene, pk, that lies immediately upstream from the gene encoding glycoprotein X in the short unique region of the alphaherpesvirus, pseudorabies virus (PRV). The gene has the potential to encode a protein of 334 amino acids, and is related to gene US3 of herpes simplex virus type 1 (HSV-1), which has been shown to encode a protein kinase. The predicted amino acid sequence encoded by the PRV pk gene is homologous to the corresponding sequence encoded by the HSV-1 US3 gene in the C-terminal catalytic domain, but diverges markedly in the N-terminal domain. As with HSV-1, the mRNA for the pk gene appears to be 3′ coterminal with that for the glycoprotein downstream. An antiserum was raised against a protein generated from the fusion of part of the PRV pk catalytic domain with Escherichia coli β-galactosidase. This specifically reacted with a previously described physically homogeneous protein kinase, PRV-PK, isolated from hamster fibroblasts lytically infected with PRV. Although the majority of the PRV-PK is found in the cytoplasm, some was also detected in purified PRV virions by using the same antibody; a similar distribution was found for the HSV-1 protein kinase, using an antiserum raised against the corresponding HSV-1 fusion protein. When presented with heat- inactivated virions, purified PRV-PK (in common with certain cellular protein kinases also present in the virion) was able to phosphorylate in vitro the major virion phosphoprotein phosphorylated in vivo.

Mice were immunized with synthetic peptides covering the first 56 amino acids of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) and a fusion protein, produced in Escherichia coli, containing the first 55 amino acid residues of gD. It was found that mice immunized with peptides composed of amino acid residues 1 to 13, 18 to 30, 22 to 38 and 38 to 56 of gD were not significantly protected against a lethal challenge with HSV-1. Immunization with peptide 9–21 and the gD fusion protein resulted in significant protection. Antisera, from mice immunized with HSV-1, were investigated for reactivity with a series of 57 overlapping gD peptides covering the entire amino acid sequence, except for the membrane-spanning region. All antisera reacted with peptides 9–21, 10–24, 151–165, 216–232, 282–301 and with peptide 340–354 located in the anchoring region of gD, and 15 other peptides were recognized by at least one antiserum. Twelve peptides (10–24, 151–165, 216–232, 244–267, 260–274, 270–284, 260–284, 282–301, 300–314, 340–354, 348–362 and 355–369) reacted most frequently with the hyperimmune sera from mice and were selected for further study. These were conjugated to bovine serum albumin and used to immunize rabbits. Only antisera against peptide 10–24, which covers the same epitope as peptide 9–21, neutralized HSV-1 in vitro.

Herpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) genes of which at least three are involved in the transcriptional regulation of later classes of viral genes. Perhaps the most important of these regulatory proteins is Vmw175, a nuclear phosphoprotein of 1298 predicted amino acid residues. In the absence of functional Vmw175 the virus fails to activate early or late genes or to repress IE gene expression. All viruses of the sub-family alphaherpesvirinae encode polypeptides that are closely related to Vmw175. Mutational studies have shown that regions of homology within this family of gene regulators are generally of functional importance. One of the most striking conserved stretches of amino acid sequence is a run of serine residues followed by a highly acidic region in the amino-terminal fifth of the polypeptide. We have constructed an HSV-1 virus which lacks this serine-rich run within Vmw175. Surprisingly, the virus was viable in tissue culture cells and expressed apparently normal amounts of viral polypeptides. In plaque assays it was very slightly temperature-sensitive and, depending on the state of the host cells, could generate plaques with a syncytial morphology. The mutant protein was able to bind to DNA in a manner indistinguishable from that of the wild-type polypeptide. We conclude that despite its conservation in all of the alphaherpesvirinae so far sequenced, the serine-rich homology is not important for virus growth in tissue culture.

The herpes simplex viruses (HSV-1 and HSV-2) encode a ribonucleotide reductase consisting of two non-identical subunits (RR1 and RR2) which associate to form the active holoenzyme. To facilitate the purification and subsequent biochemical characterization of this enzyme, we have cloned the small subunit 2 of the HSV-2 ribonucleotide reductase (RR2 HSV-2) in a helper-independent adenovirus type 5 vector under the control of the adenovirus type 2 major late promoter. After infection of 293 cells with the recombinant virus, the amount of RR2 HSV-2 protein produced was eightfold higher than in HSV-2-infected cells. The specific activities of the RR2 HSV-2 recombinant subunit and the RR2 HSV-2 protein in HSV-2-infected cells were determined by their mixing with saturating amounts of isolated RR1 HSV-1 subunit. By comparison of the relative amount of each RR2 HSV-2 subunit with its specific activity, we calculated that the recombinant protein intrinsic activity was similar to that of the protein produced in HSV-2-infected cells. These results demonstrated that the adenovirus expression vector is a good system to produce an active RR2 HSV-2 subunit in fairly high amounts.

The equine herpesvirus 4 (EHV-4) gene glycoprotein H (gH) gene homologue was localized by virtue of the conserved genomic position of this gene throughout members of the herpesvirus family. The gene maps immediately downstream of the thymidine kinase gene at approximately 0.49 to 0.51 map units within genomic fragment BamHI C. The EHV-4 gH primary translation product is predicted to be a polypeptide of M r 94100, 855 amino acids long, which possesses features characteristic of a membrane glycoprotein, namely an N-terminal signal sequence, a large hydrophilic domain containing 11 putative N-linked glycosylation sites, a C-terminal transmembrane domain, and a charged cytoplasmic tail. Comparison to other herpesvirus glycoproteins revealed identities of 85%, 26% and 32% with the gH counterparts of the alphaherpesviruses EHV-1, herpes simplex virus 1 and varicella-zoster virus, respectively, and of 17% and 18% with those of human cytomegalovirus, herpesvirus saimiri and Epstein-Barr virus. The EHV-4 gH exhibits features previously reported to be conserved throughout the gH polypeptides of herpesviruses of all three subgroups. A region of direct repeat elements and a possible origin of DNA replication are located immediately downstream of the gH gene.

We have determined the genomic location and nucleotide sequence of the equine herpesvirus 4 thymidine kinase (TK) gene. The gene is positioned at approximately 0·48 map units within the long unique component of the genome and is flanked by genes encoding a herpes simplex virus 1 (HSV-1) UL24 homologue and glycoprotein H. The predicted protein is composed of 352 amino acids, has an M r of 38 800 and exhibits 36 % identity to the predicted TK of HSV-1.

The post-translational events leading from the precursor to the processed forms of a glycoprotein with an M t of 28K to 32K (gp28/32) of Marek’s disease virus (MDV) serotype 2 were examined with pulse-chase experiments and treatment with tunicamycin and monensin. Cell-free translation of infected cell mRNA followed by immunoprecipitation analysis suggested that a polypeptide with a size of 22K is the initial precursor. Experiments with endo-β-N-acetylglucosa- minidase H and endo-β-N-acetylglucosaminidase F indicated that gp28/32 contains mostly N-linked oligosaccharides of the complex type. These studies showed that 22K, the initial product, is then processed through intermediates to the 28K to 32K form.