- Volume 81, Issue 4, 2000

It was recently shown that the class II membrane glycoprotein G of bovine respiratory syncytial virus (BRSV) is integrated into the envelope of recombinant bovine herpesvirus-1 (BHV-1) virions in the correct orientation. To verify the hypothesis that the membrane anchor of BRSV G might be suitable to target heterologous polypeptides into the membrane of recombinant BHV-1 particles, an open reading frame encoding a fusion protein between amino acids 1 to 71 of the BRSV G glycoprotein and the green fluorescent protein (TMIIGFP) was recombined into the genome of BHV-1. The resulting recombinant BHV-1/eTMIIGFP had growth properties similar to those of wild-type BHV-1. Live-cell analysis of cells infected with BHV-1/eTMIIGFP indicated that the fusion protein localized to the cell surface. Immunoprecipitations and virus neutralization assays using a GFP-specific antiserum proved that TMIIGFP was incorporated as a class II membrane protein into virions.

The programme of Epstein–Barr virus (EBV) gene expression that leads to virus-induced growth transformation of resting B lymphocytes is initiated through activation of the BamHI W promoter, Wp. The factors regulating Wp, and the basis of its preferential activity in B cells, remain poorly understood. Previous work has identified a B cell-specific enhancer region which is critical for Wp function and which contains three binding sites for cellular factors. Here we focus on one of these sites and show, using bandshift assays, that it interacts with three members of the CREB/ATF family of cell transcription factors, CREB1, ATF1 and ATFa. A mutation which abrogates the binding of these factors reduces Wp reporter activity specifically in B cell lines, whereas a mutation which converts the site to a consensus CREB-binding sequence maintains wild-type promoter function. Furthermore Wp activity in B cell, but not in non-B cell, lines could be inhibited by cotransfection of expression plasmids expressing dominant negative forms of CREB1 and ATF1. Increasing the basal activity of CREB/ATF proteins in cells by treatment with protein kinase A or protein kinase C agonists led to small increases in Wp activity in B cell lines, but did not restore promoter activity in non-B cell lines up to B cell levels. We conclude that CREB/ATF factors are important activators of Wp in a B cell environment but require additional B cell-specific factors in order to mediate their effects.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that is implicated in the pathogenesis of Kaposi’s sarcoma. The nucleotide sequence of the KSHV open reading frame (ORF) 36 predicts a polypeptide with significant sequence homology to known protein kinases. In this paper, we show that KSHV ORF36 mRNA is expressed during lytic growth and that ORF36 protein is localized in the nucleus. To determine whether the KSHV ORF36 protein is a protein kinase, we expressed it as a glutathione S-transferase (GST) fusion protein (GST–ORF36). Affinity-purified preparations of the GST–ORF36 fusion protein revealed that the protein is autophosphorylated. Mutation of lysine-108 to glutamine dramatically decreased the protein kinase activity of the purified protein, supporting the hypothesis that the protein kinase activity is inherent to the ORF36 protein. Phosphoamino acid analysis showed that the KSHV ORF36 fusion protein is phosphorylated on a serine residue, implying that KSHV ORF36 encodes a serine protein kinase.

P37 (F13L gene product), the most abundant protein in the envelope of the extracellular virus form of the prototype poxvirus, vaccinia virus (VV), is a crucial player in the process leading to acquisition of the envelope, virus egress and transmission. We have cloned and sequenced a swinepox virus (SPV) gene homologous to VV F13L. The SPV gene product, termed P42, was 54% identical to P37, the VV F13L gene product, and, among the poxviruses, was most similar (73% identity) to the myxoma virus homologue. The SPV P42 gene contained late transcription signals and was expressed only at late times during infection. The protein was palmitylated, and showed an intracellular distribution similar to that of VV P37, both by immunofluorescence and by subcellular fractionation. As with VV P37, SPV P42 was incorporated in extracellular enveloped SPV particles, but was absent from the intracellular mature virus form. To check the ability of SPV P42 to function in the context of VV infection, we inserted the SPV gene into a VV deficient in P37, which is severely blocked in virus envelopment and cell-to-cell transmission. Despite correct expression of SPV P42, the resulting recombinant VV showed no rescue of extracellular virus formation or cell-to-cell virus spread. The lack of function of SPV P42 in the VV genetic background suggests that specific interactions between SPV P42 or VV P37 and other viral proteins is required to drive the envelopment process.

Ectromelia virus (EV) virulence factor p28 (EVp28) is a member of a family of poxvirus proteins that are defined largely by the presence of a C-terminal RING finger motif and localization to virus factories within the cytoplasm of infected cells. Previously, overexpression of the Shope fibroma virus (SFV) homologue, N1R, in vaccinia virus (VV)-infected BGMK cells was found to inhibit virus-induced apoptosis. Here, we report that both EVp28 and overexpression of SFV N1R in poxvirus-infected HeLa cells protect specifically from UV light-induced apoptosis, but not from apoptosis induced by Fas or TNF. Further, we report that both VV and EV protect from apoptosis induced by UV, Fas and TNF. Immunoblot analysis indicates that EVp28 acts upstream of caspase-3, blocking activation of the protease in response to UV irradiation. Although no difference was found in replication of an EVp28− mutant virus, which expresses a truncated p28 protein lacking the RING motif, compared to EV wild-type in HeLa cells, UV irradiation of infected HeLa cells reduced the replication of the EV mutant compared with wild-type EV.

DNA synthesis within the hepatitis B virus (HBV) nucleocapsid appears to be coupled to nucleocapsid envelopment. The nature of the envelopment signal is unknown, but is thought to involve a conformational change at the surface of the capsid that facilitates interaction with HBV envelope proteins. In binding assays in vitro, it was found that empty HBV core particles bound synthetic peptides corresponding to HBV envelope protein domains with the same affinity as did HBV DNA-containing core particles. This suggests that the selection of replication-competent nucleocapsids for envelopment is not related to the capacity of DNA-containing core particles to bind specifically to HBV envelope proteins, and that there must be an alternative mechanism.

We have identified a novel protein that promotes Bombyx mori nucleopolyhedrovirus (BmNPV) replication in vitro. This protein was purified from heat-treated haemolymph of B. mori larvae by gel filtration and ion exchange chromatography, and designated as promoting protein (PP). The molecular mass of native PP estimated by column chromatography and that of denatured PP estimated by SDS–PAGE were 9600 Da and 15200 Da, respectively, suggesting that native PP is composed of a single polypeptide and may behave in the column as if it is a smaller protein because of its conformation and/or adsorptive nature. Addition of the PP to the culture medium of SES-BoMo-15A cells derived from B. mori embryos resulted in the strong promotion of BmNPV replication. The promoting activity positively correlated with the amount of PP in the culture medium up to 1 μg/ml, above which maximum virus replication occurred and resulted in the highest budded virus production and polyhedrin promoter-mediated luciferase gene expression of 10000-fold and 6000-fold higher than those without PP, respectively. A cDNA encoding the PP precursor (prePP) was successfully cloned and sequenced. Comparison between the amino acid sequence deduced from the nucleotide sequence of prePP cDNA and the N-terminal 18 amino acids determined for the purified PP indicated that the prePP (154 amino acids) consisted of a mature PP polypeptide (136 amino acids) with a signal sequence at the N terminus. Recombinant PP expressed from the cDNA using a baculovirus vector was similar in molecular mass, immunoreactivity and promoting activity to the native PP.

Nucleotide sequence analyses of cDNAs derived from the double-stranded RNA genome segments 6 and 7 (S6 and S7) of Bombyx mori cypovirus 1 (BmCPV-1) have revealed that they consist of 1796 and 1501 nucleotides encoding putative proteins of 561 and 448 amino acids with molecular masses of 63604 and 49875 (p64 and p50), respectively. The amino acid sequence of p64, which has a high leucine residue content (10%), contains a leucine zipper motif. Antiserum raised against p64 specifically bound to a viral structural protein of ca. 68 kDa (V4), while antiserum against p50, which specifically bound to a protein of ca. 56 kDa in BmN4 cells infected with BmCPV-1, reacted with a cluster of four viral structural proteins ranging from ca. 34 to 40 kDa (V5). These observations indicate that p50 might be cleaved to V5 during the formation of virus particles.

Triatoma virus (TrV) is the only virus described to date that infects triatomines, and has previously been considered to be a member of the family Picornaviridae on the basis of physico-chemical properties. The genome of TrV was sequenced completely (9010 nt). Analysis of the sequence revealed the presence of two large open reading frames (ORFs). The predicted amino acid sequence of ORF1 (nt 549–5936) showed significant similarity to the non-structural proteins of several animal and plant RNA viruses. This ORF product contains sequence motifs characteristic of RNA-dependent RNA polymerases (RdRp), cysteine proteases and RNA helicases. ORF1 is preceded by 548 nucleotides of non-coding RNA and the two ORFs are separated by 172 nucleotides of non-coding RNA. Direct N terminus sequence analysis of two capsid proteins showed that ORF2 (nt 6109–8715) encodes the structural proteins of TrV. The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, Rhopalosiphum padi virus and Himetobi P virus and to a partial sequence from the 3′ end of the cricket paralysis virus genome. All of these viruses have a novel genome organization and it has been proposed that they are not members of the Picornaviridae, as previously thought, but belong to a new virus family. On the basis of similarities of genome organization, we propose that TrV also belongs to this new virus family.

The previously reported (Partridge et al., Nature 247, 391–392, 1974 R9 ) glycosylation of the capsid proteins of cowpea mosaic virus (CPMV) has been reinvestigated. In initial studies, a preparation of purified CPMV particles was hydrolysed with HCl and amino acids and sugars were derivatized with o-phthalaldehyde (OPA). No glucosamine or galactosamine, amino sugars previously reported to occur in significant quantities in CPMV capsids, could be detected by reverse-phase high-performance liquid chromatography (RP-HPLC) of the derivatized hydrolysates. A complete analysis of all sugars potentially present was carried out by hydrolysing a sample of purified CPMV capsid proteins and derivatizing the sugars with 1-phenyl-3-methyl-5-pyrazolone. RP-HPLC analysis demonstrated that the capsids do not contain significant quantities of any sugar. The results show that, contrary to the previous report, the coat proteins of CPMV are not glycosylated.

The genes encoding the helper component (HC) proteins of two strains of Potato virus Y (PVY) were cloned and the proteins expressed from a vector derived from Potato virus X (PVX). The expressed HC contained six N-terminal histidine residues to facilitate purification by metal affinity chromatography. Approximately 2–4 μg/g of purified HC was obtained from leaves of Nicotiana benthamiana plants systemically infected by recombinant PVX. Preparations of the HC protein derived from PVY ordinary strain (PVYo) assisted aphid transmission of purified particles of PVYo and PVY strain C (PVYc; a naturally occurring non-aphid transmissible strain of PVY which contains a defective HC), as well as Potato aucuba mosaic virus. The HC derived from PVYc contained the Glu-Ile-Thr-Cys (EITC) motif, and mutation of Glu (E) to Lys (K) enabled the mutant PVX-expressed preparations to assist virus transmission by aphids. Expression of HC protein from the PVX vector produced biologically active protein. This approach should facilitate further studies to elucidate more precisely the molecular mechanism of virus transmission by aphids.

When expressed in transgenic tobacco plants, transgene mRNA that includes the 3′ untranslated region (3′ UTR) of Lettuce mosaic virus served as template for synthesis of complementary (−)-strand RNA following an infection by Tobacco etch virus, Tobacco vein mottle virus or Pepper mottle virus, but not when infected with Cucumber mosaic virus. Deletion of the 3′ UTR from the transgene abolished the synthesis of (−)-strand transcripts. Similar results were obtained in transgenic tobacco plants expressing mRNA that includes the RNA3 3′ UTR of Cucumber mosaic virus when infected with Tomato aspermy virus. These results show that the viral RNA-dependent RNA polymerase of several potyviruses and Tomato aspermy virus have the ability to recognize heterologous 3′ UTRs when included in transgene mRNAs, and to use them as transcription promoters.