- Volume 79, Issue 9, 1998

Sequences from 420 to 70 from the ICP0 transcriptional start site of herpes simplex virus type 1 are dispensable for reactivation from latency. A putative cAMP-response element (CRE) outside of this region was non-functional in both murine neuroblastoma (NB41A3) and rat pheochromo- cytoma (PC12) cells. Also, poor binding of cAMP- response element binding protein (CREB) was observed. Sequences from 95 to 37 are important for constitutive activity in NB41A3, PC12 and baby hamster kidney (BHK) cells. The TATA box and Sp1 site were also shown to be major contributors to constitutive activity. Finally, high constitutive activity of a deleted construct (N420 to 1) in NB41A3 and BHK cells suggests transcription initiates upstream of 420 in the absence of VP16. The implications of these observations regarding ICP0 expression during the virus life-cycle are discussed.

Cytomegalovirus (CMV) infection in the period of temporary immunodeficiency after haematoablative treatment and bone marrow transplantation (BMT) is associated with a risk of graft failure and multiple-organ CMV disease. The efficacy of immune system reconstitution is decisive for the prevention of CMV pathogenesis after BMT. Previous data in murine model systems have documented a redundancy in the immune effector mechanisms controlling CMV. CD8 T cells proved to be relevant but not irreplaceable as antiviral effectors. Specifically, in a state of long-term in vivo depletion of the CD8 T-cell subset, CD4 T cells were educed to become deputy effectors controlling CMV by a mechanism involving antiviral cytokines. It is of medical importance to know whether one can trust in this 'flexible defence’ in all clinical settings. It is demonstrated here that reconstitution of CD8 T cells is crucial for the prevention of fatal multiple- organ CMV disease under the specific conditions of BMT.

In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5′ fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protein and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs. The results indicated the presence of an N-terminal region within pUL97 which changed the intracellular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matrix fraction. Expression of all pUL97 mutants could be confirmed by Western blot analysis. pUL97-associated ganciclovir (GCV) phosphorylation in rVV-infected cells, determined quantitatively by HPLC analysis, was abolished completely using individual UL97 deletion mutants. Phosphorylation of full-length and some of the mutated pUL97 was detected in cells infected with the rVVs. The UL97 constructs carrying point mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V, and the 4 aa deletion 590AACR593, also resulted in decreased but not abolished phosphorylation of GCVinthe rVVsystem, whereas the phosphorylation of pUL97 itself was not influenced. The rVV system is a suitable method for quantitatively testing the functional relevance of pUL97 mutations.

Multiplex RT-PCR analysis of human cytomegalovirus (HCMV) replication in human fibroblasts showed transcription of the natural killer (NK) cell decoy gene, UL18, from 72 h onwards. Transcription of glycoprotein B (gpUL55; a late gene) occurred from early time-points and peaked at 24 h post-infection. UL18 mRNA was also detected in the peripheral blood mononuclear cells of organ transplant recipients with HCMV viraemia, especially those with HCMV DNA virus loads greater than 105 genomes/ml whole blood. Thus, UL18 is produced via a low abundance transcript late during the infectious cycle at a time coincidental with the increased risk of NK cell lysis as a consequence of class I HLA down-regulation.

The latent membrane protein 1 (LMP1) of Epstein- Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumori- genic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-κB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-κB induction in the up-regulation of the B cell activation markers ICAM-1 andCD71 byLMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel-responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IκB mutant. ICAM-1 and CD71 are nevertheless up- regulated by LMP1 in primary B cells and cell lines expressing the dominant IκB. Furthermore, LMP1- induced cell size increase of primary B cells was unaffected by IκB expression. It was concluded that even when LMP1 is unable to activate NF-κB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-κB.

Enzootic haematuria and urinary bladder cancer in cattle are associated with feeding on bracken fern and bovine papillomavirus (BPV) infection. An increased rate of chromosomal aberrations in peripheral blood lymphocytes from chronically affected haematuric cows raised in bracken fern pastures has been reported, suggesting the presence of BPV in the peripheral blood of afflicted animals. The purpose of the present investigation was to examine the role of peripheral blood as a potential BPV-transmitting agent and search for clastogenic effects in experimentally infected animals kept on a bracken fern-free diet. Healthy cows were inoculated with blood samples of haematuric animals every two weeks for 18 months. Recipient cows, their off-spring, donor animals and a control group were kept on a bracken fern-free diet throughout the experiment. Clinical and molecular analyses for detection of BPV infection were carried out periodically in all groups. Short-term lymphocyte cultures were performed to assess chromosomal aberration levels. The donor cows, the recipient cows and their offspring presented increased levels of chromosomal aberrations. BPV-2 DNA was identified by Southern blotting, PCRand cycle-sequencing of PCR products in peripheral blood of donor and recipient animals and in the progeny of recipient animals. Data support both the concept that BPV can be transmitted through blood and the hypothesis that infection with the virus causes the clastogenic alterations observed in the present experimental model. The presence of BPV-2 DNA and chromosomal alterations in peripheral blood of offspring at the moment of birth is evidence for vertical transmission of BPV.

Recently, the L1 capsid protein of canine oral papillomavirus (COPV) has been used as an effective systemic vaccine that prevents viral infections of the oral mucosa. The efficacy of this vaccine is critically dependent upon native L1 conformation and, when purified from Sf9 insect cells, the L1 protein not only displays type-specific, conformation-dependent epitopes but it also assembles spontaneously into virus-like particles (VLPs). To determine whether VLP formation was coupled to the expression of conformation-dependent epitopes, we generated a series of N-and C-terminal L1 deletion mutants and evaluated their ability to form VLPs (by electron microscopy) and to react with conformation-dependent antibodies (by immunofluorescence microscopy). We found that (a)deletion of the 26 C-terminal residues generated a mutant protein which formed VLPs efficiently and folded correctly both in the cytoplasm and in the nucleus; (b)further truncation of the L1 C terminus (67 amino acids) resulted in a capsid protein which formed VLPs but which failed to express conformational epitopes; (c)deletion of the first 25 N-terminal amino acids also abolished expression of conformational epitopes (without altering VLP formation) but the native conformation of this deletion mutant could be restored by the addition of the human papillomavirus type 11 N terminus. These results demonstrate that VLP formation and conformational epitope expression can be dissociated and that the L1 N terminus has a critical role in protein folding. In addition, it appears that correct L1 protein folding is not dependent upon the nucleoplasmic environment.

The multifunctional protein of papovaviruses, T-antigen, regulates the virus lytic cycle partly by exerting transcriptional control over viral and cellular gene expression. In this study, the ability of the T-antigen of human neurotropic JC virus (JCV) to enhance expression from the virus late promoter has been further examined. By deletion analysis, a T-antigen-responsive region was mapped within the JCV 98 bp enhancer/promoter between nucleotides 139 and 168. Interestingly, T-antigen appears to mediate transactivation by increasing expression from a basal transcriptional initiation site and through a novel T-antigen-dependent initiation site (TADI). The TADI element contains a region homologous to initiator (Inr) sequences and is sufficient to confer T-antigen responsiveness to a heterologous minimal promoter. Electrophoretic mobility shift and UV crosslinking analyses demonstrate that multiple cellular proteins interact with both single-and double-stranded forms of this sequence. Mutations within the TADI element which abolish T-antigen-mediated transcriptional activation also prevent the formation of specific nucleoprotein complexes. These data suggest that the ability of JCV T-antigen to regulate JCV late gene expression may be partly due to the formation of specific nucleoprotein complexes and transcriptional initiation from the TADI site on the viral promoter.

While Rep proteins are required for adeno-associated virus (AAV) replication, little is known about cellular proteins that interact with Rep. We demonstrate here that transcription-positive cofactor 4 (PC4, p15) fused to Gal4-activating domain interacted with both AAV-2 and AAV-3 Rep proteins fused to Gal4 DNA-binding domain, leading to reporter activation in the yeast two-hybrid system. In addition to its coactivating function, PC4 recently has been shown to be involved in replication of simian virus 40. To study a functional role for the PC4-Rep protein interaction, 293–31 cells were cotransfected with a PC4 expression plasmid and an infectious clone of AAV-3, followed by superinfection with helper adenovirus. A significantly increased number of AAV-3 genomes were rescued in PC4 transfected cells. Our results support a possible involvement of PC4 in AAV replication and may be used in efficient production of AAV vectors for gene therapy.

Bovine parvovirus (BPV), an autonomous parvovirus, haemagglutinates human type O erythrocytes and infects certain bovine cells in culture. Little is known about the receptor to which it attaches, either on nucleated host cells or on erythrocytes. Haemagglutination assays and radiolabelled virusbinding tests measuring the effects of trypsin, chymotrypsin, neuraminidase, phospholipase C and sodium periodate on attachment of BPV to receptors indicated that BPV interacted with N-acetyl-neuraminic acid-containing (sialyl) glycoproteins. SDS-polyacrylamide gel separation of erythrocyte ghost proteins and virus overlay protein-binding revealed BPV binding to glycophorin A. Confirmation testing showed BPV binding to purified glyco-phorin A on dot blots and on gels containing membrane glycophorin A and purified glycophorin A. Further, in competition assays, purified glycophorin A completely inhibited the BPV haemagglutination reaction. The results of this study indicate that BPV binds to sialated membrane glycoproteins, one of which is the major erythrocyte membrane glycoprotein, glycophorin A.

Porcine circovirus (PCV) was initially recognized as a contaminant of continuous pig kidney cell lines and was not thought to be pathogenic. Antibodies reactive to the cell culture isolate of PCV (PCV PK-15) are prevalent in the swine population worldwide. Recently, PCV PK-15-like antigen and nucleic acid were demonstrated in lesions associated with wasting syndromes in pigs in North America and Europe. Monoclonal antibodies raised to circo-viruses isolated from pigs with wasting syndromes highlighted differences between these circoviruses and the PCV PK-15 cell culture isolate. This has led to speculation that a new pathogenic PCV may have emerged in the swine populations of several countries. We report the cloning and characterization of novel circovirus DNAs purified from virus isolates made from tissues of North American and European pigs with wasting syndromes. These North American and European circoviruses form a closely related group at the nucleotide sequence level (> 96% intra-group nucleotide sequence identity) but exhibit < 80% nucleotide sequence identity with the PCV PK-15 cell culture isolate. This report provides evidence for a new type of possibly pathogenic PCV. We propose that these new circoviruses should be referred to as PCV2 as opposed to the original PK-15 cell culture isolate, which should be referred to as PCV1.

Hepatitis B virus (HBV) polymerase (P) gene is translated from the bicistronic pregenomic RNAwith the core (C) gene in the first cistron. The P ORF is preceded by the C AUG and three AUG codons within the C region, where a minicistron of 7 amino acids can potentially be translated. Our results indicate that the efficiency of the P gene translation initiation was about 10% of that of the C gene when both genes were fused in-frame to a lacZ reporter in an mRNA similar in structure to the pregenomic RNA. By mutational analysis, about 74% of the translation initiation of HBV P gene was shown to be by ribosomes that reinitiated after terminating translation of this minicistron, while the rest was by two mechanisms: one by ribosomes leaky scanning through every upstream AUG and the other by ribosomal backwards scanning to the P AUG after finishing the translation of the C gene. The efficiency of termination-reinitiation depended on the size of the minicistron, i.e. the reinitiation efficiency decreased about 50% when the size increased from 24 nt to 57 nt. When a 44 nt HBV sequence comprising the minicistron was inserted at the 5′ untranslated region of the cat gene, CAT expression was regulated in a similar way to that of the HBV P gene. Moreover, when transfection occurred with an HBV expression plasmid containing an inactivated minicistron, production of virus-like particles dropped to about one-third of the wild-type level, suggesting that the termination-reinitiation mechanism is indeed important for HBV P gene expression.

HLA-B57 has been shown to be strongly associated with slow disease progression in human immunodeficiency virus type 1 (HIV-1)-infected patients from the Amsterdam Cohort. Since HIV-1-specific CTL can control and eliminate virus-infected cells, we sought to characterize the dominant HLA-B57-restricted CTL responses at the epitope level. It was found that HLA-B57-restricted CTL responses were targeted at multiple proteins of HIV-1, with CTL specific for Gag and RT being the most pronounced. Gag-specific CTL recognized peptides ISPRTLNAW (aa 147–155) and STLQEQIGW (aa 241–249), which had previously been reported as HLA-B57-restricted. The RT-specific CTL response in one longterm survivor studied in great detail persisted for > 10 years and was dominated by HLA-B57-restricted CTL that recognized the newly defined epitope IVLPEKDSW (RTLAI, aa 244–252). This epitope could be recognized in the context of both HLA-B*5701 and HLA-B*5801. Interestingly, three epitope variants of IVLPEKDSW were observed, which coincided with the strongest detectable CTL response to RT. One variant (T2E7) was not recognized by IVLPEKDSW-specific CTL despite the fact that this variant bound to HLA-B*5701 with asimilar affinity as the index peptide. Finally, only viruses which contained the epitope index sequence were obtained suggesting efficient virus control by CTL. In conclusion, we report the characterization of dominant HIV-1 Gag- and RT-derived, HLA-B57-restricted CTL epitopes which are associated with longer time to AIDS. Further characterization of CTL responses restricted by HLA-B57 and other protective HLA alleles may contribute to the development of effective AIDS vaccines.

The simian immunodeficiency virus (SIV) mnd(GB-1) strain, isolated from a mandrill, replicates in a human T cell line, CEM cells, and is inhibited by the CXC-chemokines, stromal cell-derived factor 1α and 1β (SDF-1α/SDF-1β),the natural ligands for CXCR4. The IC50 was around 70–80 ng/ml, which corresponds to the IC50 of SDF-1α/SDF-1β for T-tropic human immunodeficiency virus type 1 (HIV-1) and HIV-2. The specific anti-CXCR4 MAb 12G5 inhibited replication of SIVmnd at an IC50 of 1 μg/ml. Also, the IC50 of 8 ng/ml for SIVmnd of the bicyclam AMD3100, a specific CXCR4 antagonist, is comparable with its IC50 for T-tropic HIV-1 and HIV-2 strains. Two other SIV strains, SIVagm3 and SIVmac251, were insensitive to SDF-1α/SDF-1β, anti-CXCR4 MAb and AMD3100. SIVmnd replicates only in HOS.CD4 cells expressing CXCR4 and not in HOS.CD4 transfectants expressing CCR1, CCR2b, CCR3, CCR4 or CCR5. This is, to our knowledge, the first SIV strain found to use CXCR4 and not CCR5 as a main coreceptor for entering human cells.

Unspliced mRNA from RNA segment 6 of influenza C virus contains a single open reading frame that potentially encodes a polypeptide of 374 amino acids. This polypeptide, which has not been identified as yet, is predicted to contain the complete amino acid sequence of the matrix protein, M1, as well as that of a small integral membrane protein, CM2. Here, we found that small amounts of two previously unrecognized proteins with apparent molecular masses of 42 (P42) and 44 kDa (P44) were immunoprecipitated with immune serum against CM2. The electrophoretic mobilities of P42 and P44 varied depending on virus strain, indicating that they are virus-coded. Treatment of infected cells with tunicamycin and digestion of immuno-precipitated proteins with various endoglycosidases revealed that P42 is modified by the addition of a high-mannose oligosaccharide chain to generate P44. A monoclonal antibody against M1, like anti-CM2 serum, was able to immunoprecipitate both the P42 and P44 proteins. Furthermore, the tryptic peptide map of either P42 or P44 was indistinguishable from the map of the mixture of M1 and CM2. These results, taken together, suggest strongly that P42 and P44 correspond to the 374 amino acid protein encoded by unspliced RNA segment 6 mRNA and its N-glycosylated form, respectively.

Neutralization of human respiratory syncytial virus (HRSV) by monoclonal antibodies specific for the G protein was evaluated in a microneutralization test. Only certain antibodies showed some degree of neutralization, reflected in a reduction of virus titre (10–20 times maximum), compared with negative controls. In contrast, a pool of antibodies that recognized conserved, group-specific and strain-specific epitopes showed a significant increase in virus neutralization (up to 500–1000 times). By testing binary, tertiary and quaternary combinations, four antibodies were identified which showed maximal effect in the neutralization test. These findings are discussed in terms of the location of antibody binding sites in the G protein primary structure and their relevance for HRSV neutralization and immunobiology.

Human respiratory syncytial virus (HRSV) is classified into two major groups, A and B, each of which contains multiple variants. To characterize the molecular epidemiology of HRSV strains over time, sequencing studies of a variable region of the attachment protein gene from a single community in the United States during 5 successive years were performed. Phylogenetic analysis revealed distinct clades (genotypes) that were further classified in subtypes based on ⩾ 96% nucleotide similarity. Five genotypes and 22 subtypes among 123 group A HRSV isolates, and four distinct genotypes and six subtypes among 81 group B HRSV isolates were identified. One to two genotypes or subtypes accounted for ⩾ 50% of isolates from a given year. A shift in the predominant genotype or subtype occurred each year such that no genotype or subtype predominated for more than 1 of the 5 study years. The consistency in the displacement of the predominant strain suggests that a shift, even within the same group, is advantageous to the virus. It was hypothesized that the ‘novel’ strain is better able to evade previously induced immunity in the population and consequently either circulates more efficiently or is more pathogenic. The yearly shift in HRSV strains may contribute to the ability of HRSV to consistently cause yearly outbreaks of HRSV disease. These results also suggest that isolates may need to be characterized as to both group and genotype to fully understand protective immunity after natural infection and efficacy studies of candidate vaccines.

The complete nucleotide sequence of a functional clone of the large polymerase (L) gene of bovine respiratory syncytial virus (BRSV) strain A51908 was determined by analysis of cloned cDNAs obtained from genomic and mRNAs. The BRSV L gene is 6573 nt in length and the derived polypeptide has 2162 aa. Alignment of the sequences of the BRSV L gene, and its encoded protein, with sequences of the L gene and protein of human respiratory syncytial virus strain A2 showed 77% identity at the nucleotide level and 84% identity at the amino acid level. By comparison, the L gene and protein of avian pneumovirus showed only 50% identity at the nucleotide level and 64% identity at the amino acid level. A minigenome was constructed to encode a BRSV vRNA analogue containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative BRSV transcription motifs and flanked by the BRSV genomic termini. Transfection of plasmids encoding the BRSV minigenome, nucleocapsid protein (N), phosphoprotein (P) and L protein, each under the control of T7 promoter, into cells infected with a vaccinia virus recombinant expressing the T7 RNA polymerase gave rise to CAT activity and progeny with the minigenome. This result indicates that the N, P and L proteins are necessary and sufficient for transcription and replication of the BRSV minigenome and are functional. Further, inclusion of small amounts of the M2 protein along with the N, P and L proteins greatly augmented minigenome transcription.

Induction of the cellular stress response elevates cytoplasmic levels of heat shock proteins (HSPs) belonging to multiple families. When infected with canine distemper virus or measles virus (MV), cells containing elevated HSPs support increased viral gene expression and cytopathic effect. The present work tests the hypothesis that increases in the major inducible 70 kDa HSP (hsp72) are sufficient to mediate the effect of stress response induction on infection phenotype. Human astrocytoma cells (U373) were stably transfected with the human hsp72 gene under control of the β-actin promoter. Constitutive overexpression of hsp72 was demonstrated in multiple clones by Western blot analysis of cytoplasmic total protein. Southern blot analysis of cell DNA confirmed the recovery of genetically distinct clones. Infection of these clonal populations with MV resulted in increased viral transcript production relative to infected control cell lines. Increased transcript production was associated with increased viral membrane glycoprotein expression and cytopathic effect (i.e., mean plaque area). Increases in cytopathic effect were due to the emergence of a large plaque phenotype from a small plaque-purified inoculum, mimicking the effect of cellular stress response induction upon viral infection phenotype. Large plaque phenotypic variants reported in the literature are associated with enhanced neurovirulence, a fact that highlights the potential significance of physiologic elevations in hsp72 (e.g., fever-induced) that accompany in vivo viral infection.