- Volume 75, Issue 2, 1994

A ribosomal protein S2 kinase was purified 6000-fold from cytoplasmic extracts of HeLa cells infected with vaccinia virus, using 80S ribosomes or 40S ribosomal subunits as a substrate. Although the preparation was not homogeneous, a 34K component was identified, the chromatographic behaviour of which correlated with enzyme activity. During its purification the ribosomal protein S2 kinase was resolved from a less abundant ribosomal protein S13 kinase, demonstrating the two to be different entities. A second protein kinase activity against a 43K ribosomal protein comigrated with the ribosomal protein S2 kinase activity during all five chromatographic procedures employed, and we conclude that the two activities are properties of a single species. Two-dimensional gel electrophoresis demonstrated that this second substrate was the acidic ribosomal protein Sa, of isoelectric point approximately 5·2, previously shown to be phosphorylated during infection with vaccinia virus. Another substrate for the ribosomal protein S2/Sa kinase in vitro was the 36K viral ssDNA-binding protein, of isoelectric point approximately 5·0, which is also known to be phosphorylated in vivo. The 34K protein correlating with the catalytic activity in the most purified preparations of the ribosomal protein S2/Sa kinase was recognized by an antibody specific for a protein expressed in Escherichia coli from vaccinia virus gene B1R. This and other evidence suggest strongly that the ribosomal protein S2/Sa kinase is the product of this gene.

The potential for in vivo genetic recombination has been suggested for Aujeszky’s disease virus (ADV) stemming from the use of modified-live vaccines carrying vaccine- specific deletion mutations. This scenario serves as a model for the study of biological factors that affect in vivo herpesviral recombination and, ultimately, the natural evolution of herpesviruses. This report describes experiments evaluating the efficiency of ADV recombination under in vitro conditions of low multiplicity infection which were felt to represent in vivo conditions more closely than previous in vitro measurements of ADV recombination. A series of experiments were performed to measure in vitro recovery of recombinant viral progeny following co-infection of cell monolayers with nonsaturating concentrations of two parental strains of ADV. A simple statistical model was generated to estimate the rate of cellular co-infection. After a single replicative cycle infectious viral progeny were recovered and their genotypes determined at two alleles by characterization of two gene loci using a battery of PCR assays. Recovery rates of parental and recombinant progeny genotypes were calculated from the PCR data. The observed frequency of recovery of recombinant viral progeny closely approximated the values projected by the model calculations. The data from our system suggest that at low multiplicity of infection, the rate of genetic recombination is a function of the probability of cellular co-infection.

Human cytomegalovirus (HCMV) is a common cause of congenital infection leading to birth defects and a leading cause of serious illness in patients with immunodeficiencies. Studies in this laboratory have focused on a molecular analysis of the immune response to glycoprotein B (gB) of HCMV. This protein has been shown to elicit B cell, helper T cell (Th), and cytotoxic T cell responses, suggesting that it may be useful as a subunit HCMV vaccine. However, previous studies showed that although peripheral blood mononuclear cells (PBMC) from all HCMV-seropositive donors proliferate in response to stimulation with whole HCMV, not all donors respond to purified recombinant gB. In the present study, PBMC from HCMV-seropositive donors homozygous for HLA-DR were tested for proliferative responses to whole HCMV and to purified gB expressed in vaccinia virus. PBMC from all donors proliferated in response to HCMV, but those from multiple donors expressing the HLA-DR3Dw3 and -DR4Dw4 specificities, and single donors expressing the -DR15Dw2, -DR13Dw19 and -DR14Dw9 specificities, failed to respond to gB. These results suggested a possible HLA-DR association with low proliferative responses to gB. In further studies, PBMC from donors expressing both putative gB-high responder and low responder HLA-DR alleles were stimulated multiple times with gB to generate gB-specific T cell lines. These cells were then tested for proliferative responses to gB presented by irradiated PBMC sharing only one DR allele with the responder cells. Cells from the gB-specific lines proliferated only when antigen was presented in the context of a responder DR allele but not when presented in the context of a low responder DR allele. Analysis of immune sera revealed that those from donors with PBMC proliferative responses always contained antibodies reactive with B cell epitopes on both the N-terminal gp93 and C-terminal gp55 portions of gB. In contrast, many of the sera from donors with low gB-specific proliferative responses had gp55-specific antibodies but lacked antibodies to gp93. These results suggest that immunogenetic differences in Th responsiveness to gB may lead to lack of antigen-specific help for antibody responses to gp93 in some cases. The prevalence of these low responder HLA alleles in the population, and the central importance of the T cell response to the generation of antibodies suggest that native gB alone may not be an attractive candidate for an HCMV subunit vaccine.

We describe the analysis of the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter using an HSV-1-based vector system. Sequences under investigation for LAT promoter activity were analysed for their ability to direct chloramphenicol acetyltrans-ferase gene expression, either in transfection assays or following their insertion into an HSV-1 vector from which the endogenous LAT promoter sequences had been removed. The analysis mapped the main determinants of LAT promoter activity during lytic infection of tissue culture cells to a 277 bp region between −279 and − 2 relative to the recognized 5′ end of the primary 8·3 kb transcript. The LAT promoter constructs behaved similarly in the context of the virus genome and in the plasmid-based transfection assays. Comparison of the relative activities following infection of fibroblast and neuroblastoma cell lines indicates that sequences upstream from −279 are important for LAT promoter activity in neurons.

The proteins predicted to be encoded by varicella-zoster virus (VZV) genes 47 and 66 display sequence similarity to the serine/threonine family of protein kinases. Homologues of gene 47 exist in α-, β and γ-herpesviruses but homologues of gene 66 are specific to the α- herpesviruses. Monospecific rabbit antisera were raised against two separate fusion proteins constructed from a portion of each protein fused to the carboxy terminus of β-galactosidase. These antisera were used to characterize the 47 and 66 proteins in VZV-infected cells and in cells infected with vaccinia virus recombinants expressing each protein. The 47 protein is a 54K phosphoprotein which is distributed between the cytoplasmic and nuclear compartments of VZV-infected cells and is associated with the capsid/tegument fraction of purified VZV particles. Gene 66 encodes a 48K phosphoprotein when expressed by VZV or a vaccinia virus recombinant, and, in the latter case, the 66 protein was located exclusively in the cytoplasm. The 47 protein immuno- precipitated from VZV-infected cells could be phosphorylated in vitro, but the same protein produced by in vitro transcription and translation could not. This and other evidence indicates that additional proteins induced or encoded by VZV may be involved in the phosphorylation of the 47 protein.

The association of hepatitis B virus (HBV) infection with hepatocellular carcinoma (HCC) is well established. Insertional mutagenesis, trans-activation by truncated X or preS2/S regions and activation of growth regulatory genes or oncogenes have all been suggested as possible mechanisms for this carcinogenesis. However, no consensus regarding the mechanism or region of the HBV genome involved has been established. Of the 36 HCC tissues analysed for the presence and extent of the HBV genome, using multiple overlapping PCR, 22 (61%) were found to be positive. Twenty of these showed the presence of a fragment (nucleotides 636 to 746) that covered part of the surface antigen gene. The recognized trans-activators, X and preS2/S, were present in only seven (31·8%) and 12 (54·5%) cases, respectively. In two cases the entire viral genome was detected. The trans-activation potential of the cloned S fragment (nucleotides 426 to 851) covering the frequently detected fragment (nucleotides 636 to 746) was investigated in co-transfection experiments. This fragment was able to trans-activate the HBV enhancer-X promoter target. To define the specificity of the trans-activation and the sequences involved, frameshift and deletion mutants of this fragment were constructed and analysed. The transactivation activity was lost in the frameshift mutants. The deletion mutants that retained nucleotide sequences 436 to 679 showed trans-activation activity whereas the other ones (nucleotide sequences 436 to 611) did not show any activity. It is suggested that the frequently detected HBV genome fragment belonging to the S gene frame has a trans-activation potential. This may explain the mechanism for pathogenicity of HBV-associated HCC.

One-day-old ducklings experimentally infected with duck hepatitis B virus (DHBV) were found to be immunologically tolerant to virus antigens (DHBsAg, DHBcAg), with no humoral or cellular immune responses being detected. When immunized with virus antigens in Freund’s complete adjuvant, no immune responses could be induced. Rabbit anti-DHBs sera were complexed to a solid matrix (Staphylococcus aureus Cowan A strain) and purified DHBsAg was bound to this complex to form a solid matrix antibody-antigen (SMAA) complex. This SMAA was used as an immunogen to immunize the tolerant ducks. After three injections, in 12 of 17 ducks serum DHBV DNA became absent and serum DHBsAg was cleared. In eight of 16 ducks, low titres of anti-DHBs could be detected. The SMAA approach shows potential for application in immunoregulatory treatment for chronically infected hepatitis B patients.

High level resistance to 3′-azido-3′-deoxythymidine (AZT, zidovudine or Retrovir) is conferred by the presence of four or five mutations (Met-41→ Leu; Asp- 67→Asn; Lys-70→ Arg; Thr-215→Tyr or Phe; Lys- 219→Gin) in the human immunodeficiency virus (HIV) reverse transcriptase. The order of appearance of these five mutations in asymptomatic patients during therapy has been studied. This has enabled us to propose a model for the acquisition of zidovudine resistance mutations during the treatment of high-risk asymptomatic HIV-infected individuals. A consistent acquisition pattern of mutations at codons 41, 70 and 215 was observed in 17 individuals. Complex mixtures of HIV species containing different combinations of single and linked double resistance mutations were present early in zidovudine therapy in isolates from two patients studied in detail. From these mixtures the linked Leu-41/Tyr- 215 genotype outgrew all others initially. The development of each new virus population is likely to be mediated primarily by the increase in the level of drug resistance rather than changes in the growth kinetics of the virus. This leads us to conclude that one major driving force in the outgrowth of different mutant viruses is the selective advantage conferred by higher levels of drug resistance.

In the presence of lauric acid (C12), the production of infectious vesicular stomatitis virus (VSV) was inhibited in a dose-dependent manner. The inhibitory effect was reversible: after removal of C12 the antiviral effect disappeared. In addition, the chain length of the monocarboxylic acids proved to be crucial, as those with shorter or longer chains were less effective or had no antiviral activity. Concomitant with the C12-induced inhibition was the stimulation of triacylglycerol synthesis, increasing the amount up to ninefold. Analysis of the antiviral mechanism of C12 revealed that the correct assembly of the viral components was disturbed, but viral RNA and protein synthesis remained unimpaired. By cell fractionation and Western blot analysis the amount of viral M protein located in the plasma membrane was found to be markedly reduced after treatment with Cl2, whereas in the cytoplasm the quantity of M protein was similar to that in untreated cells. C12 did not influence M protein synthesis, but prevented the binding of M protein to the host cell membrane, where the protein plays an essential role in virus assembly. Thus, treatment of VSV-infected cells with C12 resulted in inhibition of virus release. It is suggested that the newly synthesized triacylglycerols might interact with the host cell plasma membrane and interfere with virus maturation.

A panel of 23 monoclonal antibodies (MAbs) specific for the attachment (G) glycoprotein of bovine respiratory syncytial virus (BRS virus), recognizing seven antigenic areas on the G protein, was used to determine the antigenic heterogeneity among 19 BRS viruses isolated over a 20 year period from various parts of the world. The pattern of reactivity of the isolates, as determined by ELISA, identified two major subgroups of BRSV. This finding was confirmed by radioimmunoprecipitation of the G protein by the MAbs and was also demonstrated using polyclonal sera obtained from calves hyper- immunized with BRS virus strains from each subgroup. The subgroups could also be differentiated by differences in the apparent M r of the fusion (F) glycoprotein and its cleavage products. The apparent M rs of the F0, F1 and F2 polypeptides were 73K, 46K and 17K for subgroup A strains and 77K, 46K and 23K for subgroup B strains. These studies provide evidence for two major lineages of BRS virus, similar to the situation with human RS virus.

To investigate the possible mechanism of maintenance of old human influenza A (H3N2) viruses in pigs, the haemagglutinins (HAs) of seven isolates from swine were studied by analysis of nucleotide and deduced primary amino acid sequences, as well as reactivity of the HA molecule to chicken antisera and monoclonal antibodies. The swine HAs were closely similar to the HA of the A/Victoria/3/75 human variant as regards antigenic and molecular characteristics. These findings are consistent with the hypothesis that the swine HA genes were transmitted from an early human H3 virus to pigs, where they survived with limited mutations over a period of 5 years. The sequence data were also compared with swine H3 sequences to investigate genetic relationships between the H3 genes from swine viruses isolated in different geographical areas. An evolutionary tree, constructed from the nucleotide sequences of viruses isolated from pigs in China and in Italy, illustrated that, depending on the country of their isolation, the HA genes of swine influenza A (H3N2) viruses have different origins, e.g. human and avian, and evolved independently in different lineages. The study provides direct support for the hypothesis that pigs might serve as a ‘mixing vessel’ for the generation of pandemic strains of human influenza viruses.

The activity and stability of influenza vims neuraminidase is known to depend on the presence of calcium ions. The atomic structure of the tetrameric neuraminidase head shows two distinct Ca2+ binding sites, one with low affinity on the molecular fourfold symmetry axis and one with high affinity close to the active site in each of the monomers. Here we show that Ca is essential for the thermostability of the isolated neuraminidase tetramer. Inactivation of Ca-free neuraminidase at high temperatures is accompanied by changes in protein structure leading to protease sensitivity. More than one Ca ion per tetramer is involved in stabilization, suggesting a role for the high affinity Ca binding site and the cooperative stabilization of the subunits. Sites which are located close to the fourfold axis of the neuraminidase tetramer and which are able to bind a variety of different metal ions are also described.

The mutation rates to a viable mar (monoclonal antibody-resistant mutant) genotype of wild-type influenza (A/Victoria/3/75; H3N2) virus or its mutator variant strains have been previously determined. In order to estimate the mutation rates per nucleotide position, the sequence alterations present in 44 mar mutants isolated from either the wild-type or the mut43 mutator strain have been determined. These mar mutants were selected with either of two non-overlapping, haemagglutinin-specific, monoclonal antibodies (2G10 and p7). Most of the protein changes were identified as substitutions of large, charged amino acids for glycine residues, as the result of G to A transitions. Particularly interesting amino acid changes, not previously reported, were observed in the p7 monoclonal antibody-specific mutants, in which only Gly to Ser and Gly to Asp at position 226 were detected. The identification of the nucleotide substitutions responsible for the mar phenotype has allowed the calculation of approximate values for the total mutation rates at these positions.

The fusion kinetics with erythrocyte ghosts of two influenza A virus strains, A/Aichi/2/68 (X:31) and A/PR/8/34 (PR/8), were compared and correlated with the kinetics of haemagglutinin (HA) conformational change. Previously it had been shown that X:31 fuses with liposomes or erythrocytes at 4 °C, pH 5 after a lag time of 5 to 10 min whereas PR/8 displayed no fusion with liposomes at that temperature. We have confirmed the absence of cold fusion by PR/8 with erythrocyte ghosts. In contrast to X:31, PR/8 could not be committed to fuse at neutral pH and 37 °C by a preincubation at low pH and 4 °C. To examine whether the lack of commitment and cold fusion were due to a failure of PR/8 HA to undergo conformational changes at low temperature and pH, we analysed susceptibility of HA to proteinase K digestion, liposome binding to the virus, and immunoprecipitations of HA with conformation-specific antibodies. Although there was little binding of PR/8 to liposomes at 4 °C and pH 5, we did observe exposure of the fusion peptide. This study reveals a low temperature intermediate in membrane fusion exhibited by the HA of influenza virus strain PR/8, which involves low pH-induced conformational changes including exposure of the fusion peptide with little interaction of HA with the target membrane.

Respiratory syncytial virus (RSV) infects humans and cattle causing serious respiratory tract disease in both. The genome of the human and bovine RSV (HRSV and BRSV) codes for two non-structural and eight structural proteins. RSV has also been isolated from naturally infected sheep, but the genome of the ovine RSV (ORSV) has not been characterized and nor has the virus host range been identified. Here we report on the cloning and sequencing of the two non-structural 1C and IB genes and of the small hydrophobic (SH) protein gene of the ORSV. The nucleotide identity of the ORSV 1C gene to those of subgroups A and B of HRSV was 70% and 65 % respectively whereas the predicted amino acid identity was 68 % and 69 % respectively. The ORSV IB gene had a 70 % and 72 % nucleotide identity with that of subgroups A and B of the HRSV respectively, and 79% predicted amino acid identity with both HRSV subgroups. The identity level of these two ORSV 1C and IB genes to those of the BRSV could not be determined since these two BRSV genes have not been sequenced. The SH protein of RSV is a structural protein expressed on the surface of infected cells. In common with HRSV and BRSV subgroups, the ORSV SH had the three proposed domains with the C-terminal domain least conserved among the viruses. In the latter domain, the ORSV SH gene had a nucleotide identity of 68 to 69 % and 47 to 51 % with those of the BRSV and HRSV, respectively, and a predicted amino acid identity of 56% and 33 to 47% with those of BRSV and HRSV, respectively. Defining the genes and their products should help determine which genes and gene products are most suitable for use as diagnostic tools or as vaccine candidates.

An experimental scheme was developed for direct sequence analysis of Puumala virus-containing specimens from wild rodents (Clethrionomys glareolus). Total RNA isolated from rodent lung tissues was reverse-transcribed in the presence of a universal 11 nucleotide primer complementary to all three viral RNA segments followed by amplification in a PCR with gene-specific primers. A full-length PCR product of approximately 1800 bp from the S segment encoding the viral nucleo-protein and a product of approximately 900 bp from the M segment (encoding the C-terminal two-thirds of the G2 protein and including the 3′ non-coding region) of Puumala virus (from C. glareolus trapped in Udmurtia) were prepared and sequenced. No pronounced differences to Vero cell-grown viruses were seen. The Udmurtia/894Cg/91 strain was more closely related to the Bashkiria/CG 18–20/84 strain than to the Finnish prototype strain of Puumala virus, Sotkamo/V-2969/81. Thus there is a correlation with the geographic origin of the three strains. The results indicate the occurrence of genetic drift and different selection pressures leading to (i) clustering of mutations, (ii) a lower frequency of nucleotide substitutions in the coding than in the 3′ noncoding regions and (iii) a higher frequency of amino acid substitutions in G2 than in the N protein.

Infection of cultured vertebrate cells by Sindbis virus, an alphavirus, results in a reduction in the overall rate of protein synthesis and in selective termination (shutoff) of host-specified protein synthesis. The shutoff of host protein synthesis by Sindbis virus has been temporally correlated with a decrease in intracellular K+ concentration (and an increase in intracellular Na+ concentration) which occurs as a consequence of virus- mediated inhibition of the plasma membrane-associated Na+/K+ ATPase. Incubation of Sindbis virus-infected cells in medium containing an elevated concentration of K+ resulted in an increase in the intracellular concentration of K+, an increase in the overall rate of protein synthesis, and in partial reversal of the virus- induced termination of cell-specified protein synthesis. These results suggest that the virus-induced decrease in intracellular K+ concentration is required for efficient shutoff of host protein synthesis by Sindbis virus.

We have compared the nucleotide sequence of an envelope protein gene fragment encoding amino acids 291 to 406 of 22 yellow fever (YF) virus strains of diverse geographic and host origins isolated over a 63 year time span. The nucleotide fragment of viral RNA was examined by direct sequencing of a PCR product derived from complementary DNA. Alignment with the prototype Asibi strain sequence showed divergence of 0 to 2·15 % corresponding to a maximum of 5·2 % divergence in the amino acid sequence. Taking 10% nucleotide divergence as a cut-off point, the 22 YF virus strains fell into three topotypes which corresponded to different geographical areas, namely West Africa, Central-East Africa, and South America. Two subgroups were defined in West Africa, a genotypic group circulating in the sylvatic zone of the western part of Africa, from western Ivory Coast-Mali to Senegal, and a group responsible for large outbreaks from eastern Ivory Coast-Burkina Faso to Cameroon. Strains from Central-East Africa showed a low ratio of transition: transversion of about 1 instead of 8 to 10 for other strains, when their nucleotide sequences were compared with those of other African strains. This may reflect a more distant relationship between the former strains and the others. No change was observed in the highly conserved amino acid domain encompassing the TGD sequence, an important determinant of flavivirus tropism and pathogenesis. Our results support earlier observations on the genetic relationships between YF isolates established by T1 oligonucleotide fingerprinting and offer a useful tool for the understanding of YF virus distribution and evolution.

We have recovered five infectious molecular clones of the lentivirus equine infectious anaemia virus (EIAV). The clones were recovered from fetal equine kidney (FEK) cells infected with a virulent, cell culture-adapted virus stock (designated PV) and have been characterized at a molecular level. Each clone has unique envelope and long terminal repeat (LTR) sequences. We further investigated LTR sequence variation in the PV stock using PCR amplification to obtain additional LTR clones from infected FEK cells and from peripheral blood mononuclear cells (PBMCs) from animals experimentally infected with PV. Sequence analysis of resulting clones indicates a selection for different LTR populations in pony PBMCs compared to FEK cells. Finally, we observed that the cloned EIAV proviruses did not remain infectious when maintained in a derivative of pBR322. However, two proviruses have been stably maintained in a low copy number vector (PLG338-SPORT).