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Human adenovirus type 5 infection causes the disruption of structures in the cell nucleus termed promyelocytic leukaemia (PML) protein nuclear domains or ND10, which contain the PML protein as a critical component. This disruption is achieved through the action of the viral E4 Orf3 protein, which forms track-like nuclear structures that associate with the PML protein. This association is mediated by a direct interaction of Orf3 with a specific PML isoform, PMLII. We show here that the Orf3 interaction properties of PMLII are conferred by a 40 aa residue segment of the unique C-terminal domain of the protein. This segment was sufficient to confer interaction on a heterologous protein. The analysis was informed by prior application of a bioinformatic tool for the prediction of potential protein interaction sites within unstructured protein sequences (predictors of naturally disordered region analysis; PONDR). This tool predicted three potential molecular recognition elements (MoRE) within the C-terminal domain of PMLII, one of which was found to form the core of the Orf3 interaction site, thus demonstrating the utility of this approach. The sequence of the mapped Orf3-binding site on PML protein was found to be relatively poorly conserved across other species; however, the overall organization of MoREs within unstructured sequence was retained, suggesting the potential for conservation of functional interactions.

Theorf3a(also called X1 or U274) gene is the largest unique open reading frame in the severe acute respiratory syndrome coronavirus genome and has been proposed to encode a protein with three transmembrane domains and a large cytoplasmic domain. Recent work has suggested that the 3a protein may play a structural role in the viral life cycle, although the mechanisms for this remain uncharacterized. Here, the expression of the 3a protein in variousin vitrosystems is shown, it has been localized to the Golgi region and its membrane topology in transfected cells has been confirmed. Three potential caveolin-1-binding sites were reported to be present in the 3a protein. By using various biochemical, biophysical and genetic techniques, interaction of the 3a protein with caveolin-1 is demonstrated. Any one of the potential sites in the 3a protein was sufficient for this interaction. These results are discussed with respect to the possible roles of the 3a protein in the viral life cycle.

p7 protein is a small protein encoded by Hepatitis C virus (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny in vitro) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcripts. Confocal microscopy revealed that the tagged p7 protein was localized in the endoplasmic reticulum (ER) but not associated with mitochondria. Immunoelectron microscopy confirmed the p7 localization data and, moreover, showed that intracellular virus-like particles formed in the cells transfected with the wild-type, but not the recombinant, transcripts. Following a few passages of the transfected cells, the recombinant genome with the HA tag reverted to wild-type and the entire tag was deleted. Therefore, in this study, it has been demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation.

Hepatitis C virus (HCV) often causes persistent infection in humans. This could be due in part to the effect of viral proteins on cellular gene expression. Earlier observations suggest that the HCV core protein expressed from genotype 1a modulates important cellular genes at the transcriptional level, affects programmed cell death (apoptosis) and promotes cell growth. Recently, different groups of investigators have reported the translation of an ∼16 kDa protein (named F/ARFP/core+1 ORF) from an alternate open reading frame of the HCV core-encoding genomic region. The functional significance of this F protein is presently unknown. Thus, whether the F and core proteins have both shared and distinct functions was investigated here. The experimental observations suggested that the F protein does not significantly modulate c-myc, hTERT and p53 promoter activities, unlike the HCV core protein. Interestingly, the F protein repressed p21 expression. Further studies indicated that the F protein does not inhibit tumour necrosis factor alpha-mediated apoptosis of HepG2 cells or promote rat embryo fibroblast growth. Taken together, these results suggest that the F protein does not share major properties identified previously for the HCV core protein, other than regulating p21 expression.

The human cytomegalovirus (HCMV) tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection. Protein pp71 stimulates expression from a range of promoters in a sequence-independent manner, and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0. The intracellular localization of pp71 was investigated after its expression from transfected plasmids or from HSV-1 mutants constructed to produce pp71 transiently. The protein colocalized with the cell promyelocytic leukaemia (PML) protein at nuclear domain 10 (ND10) structures but, unlike ICP0, pp71 did not induce disruption of ND10. The activity of pp71 in mouse sensory neuronsin vivowas investigated after co-inoculation of animals with pairs of HSV-1 mutants, one expressing pp71 and the second containing theE.coli lacZgene controlled by various promoters. In this system, pp71 stimulated β-galactosidase expression from a range of viral IE promoters when mice were analysed at 4 days postinoculation. At later times, expression of pp71 resulted in a reduction in numbers of neurons containing β-galactosidase, indicating cytotoxicity or promoter shutoff. The HSV-1 latency-active promoter was not responsive to pp71, demonstrating specificity in the activity of the protein. Pp71 was as active in mice lacking both copies of the PML gene (PML−/−) as in control animals, and in PML−/− fibroblasts pp71 stimulated gene expression as effectively as in other cell types. Therefore, neither the PML protein nor the normal ND10 structure is necessary for pp71 to stimulate gene expression.

Vaccinia virus (VV) gene A41L encodes an acidic protein with amino acid similarity to the 35 kDa protein of VV strain Lister, a soluble protein called vCKBP that binds CC chemokines, and to a protein from orf virus, called GIF, that binds GM-CSF and IL-2. However, despite the similarity, recombinant A41L protein was found not to bind these ligands or a variety of other chemoattractant molecules when tested using surface plasmon resonance. The A41L gene is expressed early and late during infection and encodes a 30 kDa protein that contains bothN- andO-linked carbohydrate and is secreted from the infected cell. All 16 strains of VV and 2 strains of cowpox virus that were tested express the A41L protein, implying it has an important function for orthopoxviruses. Nonetheless, a VV strain Western Reserve deletion mutant lacking the A41L gene (vΔA41L) formed normal sized plaques and replicated to the same titre as wild-type and revertant viruses. The importance of the A41L proteinin vivowas demonstrated in a mouse intradermal model in which infection with vΔA41L caused more severe lesions compared to wild-type and revertant viruses. Further examination in this model revealed that deletion of A41L enhanced clearance of infectious virus, suggesting that A41L expression reduces immunopathology. Consistent with this, histological examination of infected rabbit skin showed that the A41L protein could reduce the infiltration of inflammatory cells into the infected area. Together, these data suggest that the A41L protein constitutes a novel immunomodulatory protein.

The herpes simplex virus type 2 (HSV-2) gene UL14 encodes a 32 kDa protein which is a minor component of the virion tegument and is expressed late in infection. The UL14 protein shows varied localization patterns in HSV-2-infected and singly expressing cells, suggesting the possibility that it is multifunctional. We have investigated the influence of the UL14 protein on the intracellular localization of capsid proteins and DNA cleavage and packaging proteins in coexpressing cells. VP26 is the minor capsid protein; it binds to hexons of the outer capsid shell and is predominantly cytoplasmic upon sole expression. We have found that VP26 coexpressed with the UL14 protein showed mutual and predominant relocation into the nucleus. At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32 and UL33) that are required for DNA cleavage and packaging. We have found that the UL33 protein, which was also cytoplasmic by sole expression, was relocated to the nucleus upon expression with the UL14 protein, which again seemed to be a result of mutual influence. Coexpression experiments also suggested the possibility of a mutual influence between the UL14 and UL17 proteins, and the UL17 protein and VP26. Our results suggest that the UL14 protein can influence the intracellular localization patterns of a number of proteins belonging to the capsid or the DNA encapsidation machinery.

The UL34 gene of herpes simplex virus type 2 (HSV-2) is highly conserved in the herpesvirus family. The UL34 gene product was identified In lysates of HSV-2-infected cells as protein species with molecular masses of 31 and 32·5 kDa, the latter being a phosphorylated product. Synthesis of these proteins occurred at late times post-infection and was highly dependent on viral DNA synthesis. Immunofluorescence assays revealed that the UL34 protein was localized in the cytoplasm in a continuous net-like structure, closely resembling the staining pattern of the endoplasmic reticulum (ER), in both HSV-2-infected cells and in cells transiently expressing UL34 protein. Deletion mutant analysis showed that this colocalization required the C terminus of the UL34 protein. The UL34 protein associated with virions but not with A, B or C capsids. We treated virions, HSV-2-infected cells and cells expressing the UL34 protein with a protease in order to examine the topology of the UL34 protein. In addition, we constructed UL34 deletion mutant proteins and examined their intracellular localization. Our data strongly support the hypothesis that the UL34 protein is inserted into the viral envelope as a tail-anchored type II membrane protein and is significant for virus envelopment.

Human papillomavirus type 16 (HPV-16) is the dominant risk factor for the development of cervical cancer. The virus encodes three oncoproteins, of which the E7 oncoprotein is the major protein involved in cell immortalization and transformation. E7 is a multi-functional protein. It binds the retinoblastoma tumour-suppressor protein (pRb), which regulates progression through the G(1) restriction point in the cell cycle. The E7 protein interacts with transcription-regulatory proteins such as the TATA box-binding protein and with proteins of the AP1 transcription factor family. To identify additional proteins regulated by E7, differential-display PCR was used to identify differentially expressed mRNAs in cells containing an inducible E7 protein. It is reported that E7 expression leads to regulation of the genes encoding the calcium-binding protein S100P and the mitochondrial ADP/ATP carrier protein. These data identify new functions of the E7 protein and thus expand the number of routes by which HPV-16 influences cell growth control, although the function of S100P has still to be elucidated.

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.

The envelope of hepatitis B virus (HBV) consists of three related proteins known as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg). L-HBsAg has a 108-119 amino acid extension at the N terminus compared with M-HBsAg and contains the preS1 sequence of the HBV envelope. Previous research has identified this region as the likely virus attachment protein which is thought to interact with the cellular receptor for the virus. However, as the receptor has still not been identified unequivocally, we used the preS1 region of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system. Several positive clones were isolated which encoded cellular proteins that interacted with the HBV preS1 protein. The specificity was examined in an independent manner in experiments in which baculovirus-derived glutathione S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by in vitro translation from the positive clones. The intensity of the interactions using this alternative approach mirrored those observed in the yeast two-hybrid system and two proteins (an unidentified protein and a mitochondrial protein) were selected for further study. The specificity of the binding reaction between the preS1 protein and these two proteins was further confirmed in a competition assay; HBV purified from serum, but not purified HBsAg, was able to compete with preS1 and thus block GST-preS1 binding to the unidentified protein but not to the mitochondrial protein. The unidentified protein was then expressed as a fusion protein with GST and this was able to bind HBV virions in a direct manner.

Adenovirus protein V is associated with the DNA-containing virus core and functions as a bridge between the capsid and the core. A yeast two-hybrid analysis performed with a human cDNA library using protein V as 'bait' selected a cellular protein, p32 --described previously as associated with the splicing factor ASF/SF2. By expression and purification of p32 and preparation of an antibody we confirmed the binding of p32 to V by a variety of methods including immune precipitation. We demonstrated that p32 was primarily located in the cytoplasm in association with mitochondria but could also be detected in the nucleus as distinct granules and tubules. By examining infected cells using confocal microscopy and immunofluorescence we were able to follow the intracellular locations of protein V and p32 and it is postulated that p32 is part of a system which imports proteins to the nucleus and that adenovirus hijacks this process to deliver its genome to the nucleus.

The spike (S) protein of murine coronavirus JHMV, variant cl-2, comprises two polypeptides, N-terminal S1 (with an N-terminal signal peptide) and C-terminal S2 (with a C-terminal transmembrane domain). In order to express these subunits, we constructed three different vaccinia virus transfer vectors (VV-TVs) containing cDNAs encoding the S1 protein without a transmembrane domain (pSFS1utt), the S1 protein with a C-terminal transmembrane domain derived from S2 (pSFS1tmd) or the S2 protein with an N-terminal signal peptide derived from S1 (pSFssS2). The S1 and S2 proteins were expressed in DBT cells by infection with vaccinia virus and transfection of these VV-TVs. In cells transfected with the pSFS1utt and pSFS1tmd, 96K and 106K proteins, respectively, were detected by Western blotting. The ssS2 protein expressed by pSFssS2 was 96K, which was slightly larger than the authentic S2 protein. The S1utt and S1tmd proteins were shown by binding studies using a panel of monoclonal antibodies to be antigenically indistinguishable from the authentic S1 protein. The S1tmd and ssS2 proteins were detected on the cell surface by immunofluorescence, whereas the S1utt protein was not. However, when the S1utt protein was expressed together with the ssS2 protein, the S1utt was detected on the cell membrane. This suggested that the S1utt was associated with ssS2 on the cell membrane. These observations indicate that the expressed S1 and S2 proteins associated in a similar manner to the authentic S1 and S2 proteins produced in DBT cells infected with cl-2. However, cell fusion was not observed in cells expressing either S1 or S2 nor in cells coexpressing both S1 and S2, although the whole S protein expressed by VV-TV did induce fusion.

By immunoprecipitating protein products from virus-infected baby rat kidney (BRK) cells with specific antibodies, we found that the smaller, 243 residue (243R) E1A protein of human adenovirus 5 (Ad5) activated expression of the virus genes for E1B 55K, E2A 72K, E3 19K, hexon, fibre and penton base and the cellular gene for PCNA. The 243R protein also activated the E2A 72K gene in several rodent cell lines. In transient expression assays, this protein trans-activated the E2 early and major late promoters, suggesting that its effect was at least partially transcriptional. Similar assays with mutants of the E2 early promoter suggested that the ATF- and distal E2F-binding sites were required for this activation. Using mutant viruses with deletions in E1A, we found evidence for three separate pathways by which the 243R protein activated gene expression: one depended on sequences in exon 1 required for this protein to bind to p300, a second depended on sequences in exon 1 required for the protein to bind to pRb and the third appeared to be independent of exon 1 altogether and to depend on exon 2. The relative importance of these pathways for activation varied with the gene and cell. We conclude that a major role of E1A in the transformation of BRK cells by Ad5 is to activate specific genes by at least the first two pathways.

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 (greater than 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 Mrs of 53K and 41K, and the 28K gene into a single protein with an Mr of 28K.

We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68,000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection. Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.