- Volume 78, Issue 2, 1997

In order to locate amino acid residues involved in the formation of feline calicivirus (FCV) neutralizing epitopes, we analysed the capsid protein gene of monoclonal antibody neutralization-resistant variants of FCV. Amino acid substitutions in the variants were identified in the two hypervariable regions of the capsid protein. Four linear and two conformational epitopes were located in the regions from residues 426 to 460 and 490 to 520, respectively. The relative positions of individual epitopes agreed with our previous antigenic analy- sis.Two antigenic sitescomposed of the neutralizing epitopes were mapped in the hypervariable regions of the capsid protein, demonstrating that a relationship exists between the genetic variability and antigenic differences in the neutralization of FCV.

We have sought evidence of enteroviral persistence in humans. Eight individuals with chronic fatigue syndrome (CFS) were positive for enteroviral sequences, detected by PCR in two serum samples taken at least 5 months apart. The nucleotide sequence of the 5′ non-translated region (bases 174–423) was determined for each amplicon. Four individuals had virtually identical nucleotide sequences ( > 97%) in both samples. The sequence pairs also each had a unique shared pattern indicating that the virus had persisted. In one individual (HO), it was clear that there had been infection with two different enteroviruses. In the remaining three individuals, the lack of unique shared features suggested that re-infection had occurred, rather than persistence. With the exception of HO, the sequences fell into a subgroup that is related to the Coxsackie B-like viruses.

The causative agent of Taura syndrome (TS) was recognized in 1994 to be viral in nature and tentatively classified as belonging to either the family Picornaviridae or Nodaviridae. The work reported here has led to a more definitive classification of this new penaeid virus. Located within the cytoplasm of infected cuticular epithelial cells of penaeid shrimp, the virus is a 31 to 32 nm icosahedral particle with a buoyant density of 1·338 ± 0·001 g/ml. Three major (55, 40 and24 kDa) and one minor (58 kDa) polypeptides constitute its proteinic capsid. Its genome contains a single molecule of ssRNA, which is polyadenylated at the 3′ end and approximately 9 kb in length. Based on these characteristics, we believe that TS virus should be included in the family Picornaviridae. Ecuadorian and Hawaiian TS virus isolates were found to be identical in their biophysical, biochemical and biological characteristics, and should be considered as the same virus.

For many RNA viruses, relatively recent times of origin of extant viruses are implied by the high rate of substitution observed in longitudinal studies. However, extrapolation of short-term rates of substitution can give misleading estimates of times of divergence. We show here that the common ancestor of different types of hepatitis C virus (HCV) is older than previously thought. The rate of HCV sequence change was measured amongst a cohort of individuals infected following administration of anti-D immunoglobulin. Virus sequences were obtained in the E1 and NS5B genes and compared with each other and with sequences from an infective batch. Taking account of the bias towards synonymous transition substitutions, the time of divergence of variants of subtype 1b is estimated to have occurred 70–80 years ago. The numerous subtypes of HCV are proposed to derive from more than 300 years of endemic infection in certain geographical regions, with recent spread of some subtypes to other parts of the world. Estimation of the time of origin of the major HCV genotypes (types 1–6) is problematic, but our data and analogy with other viruses suggest that divergence occurred at least 500–2000 years ago.

We recently established a cell culture system for the replication of hepatitis C virus (HCV) by using a human T cell leukaemia virus type I-infected cell line, MT-2, and showed that the quasi-species of the hypervariable region 1 observed in the original inoculum became homogeneous in MT-2 cells 10 days after inoculation of HCV. In this study, we obtained HCV cDNA clones covering the whole viral genome by RT-nested PCR using RNA from HCV- infected cloned MT-2C cells, which support viral replication more efficiently, at 12 days after inoculation. A total of 41 distinct HCV cDNA clones covering almost the whole viral genome were also isolated from a cDNA library derived from the original inoculum. Molecular evolutional analyses comparing the sequences of the HCV clones obtained from both sources revealed that the HCV populations became homogeneous in more than half of the compared regions. This finding suggests that limited HCV populations are able to replicate in MT-2C cells. In addition, we isolated cDNA clones containing a 3′ X-tail sequence, which was recently identified as a bona fide 3′ terminus of the HCV genome, in the HCV-infected MT-2C cells and confirmed that the nucleotide sequence of the 3′ X-tail was highly conserved, suggesting its implication in HCV replication. Finally, on the basis of the sequences of HCV cDNA clones obtained from HCV- infected MT-2C cells, we determined the entire nucleotide sequence of the HCV genome containing the 3′ X-tail as a candidate for an infectious HCV molecular clone.

The NS3 protein of flaviviruses is a multifunctional polypeptide required for virus replication. Enzymic activities that have been demonstrated or predicted from the presence of sequence motifs include protease, NTPase, helicase and RNA triphosphatase. Both full-length and truncated forms of NS3 have been identified in infected cells. To examine internal cleavage of the NS3 protein of dengue virus 2 (DEN-2), infected cells or COS cells transfected with cDNA encoding NS2B/3 were radiolabelled and immunoprecipitated with antiserum against NS3 or hyperimmune mouse ascitic fluid. The polypeptides detected were NS2B/3 (M r 83000), NS3 (M r 69000), NS3′ (M r 50000) and NS3" (M r 19000). The latter polypeptide has not been previously identified. For DEN-2, it has been proposed that NS3′ results from cleavage at the site …R457R| GR460… within an RNA helicase sequence motif of NS3. Our results demonstrated that cleavage occurred at this site, and that prior cleavage between NS2B/NS3 was not necessary.

A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins. nsP2, used for immunization, was expressed as a histidine fusion protein in Escherichia coli and purified by metal affinity chromatography. Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones. Of these, 30 MAbs recognized nsP2 in Western immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which are the sites of viral RNA synthesis in alphavirus-infected cells. MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum. Most of the MAbs (20/33) recognized the nuclear form of nsP2, which may be associated with SFV neurovirulence. Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other. None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating thatthey were directed to non-conserved sequences specific for SFV. Interestingly, these epitopes were located mostly within the N-terminal half of nsP2. Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and insect cells. This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.

The genome of influenza A virus consists of eight negative-stranded RNA segments which have partially complementary non-coding terminal sequences. Previous transcription studies of the virion RNA promoter in vitro have shown that the 5′ terminus forms an integral part of the promoter and an ‘RNA-fork’ model has been proposed for the initiation of transcription. According to this model part of the promoter is formed by an RNA-duplex which involves complementary residues 10 to 12 of the 3′ end and residues 11′ to 13′ of the 5′ end. With a reverse genetics system, based on the chloramphenicol acetyltransferase (CAT) gene, we have now tested this part of the promoter in vivo. Single mutations of the conserved residues at positions 11′ and 12 of the 3′ terminus and at positions 12′ and 13′ of the 5′ terminus abolished promoter activity. The introduction of complementary mutations into both termini partially restored activity. On the other hand, mutations at positions 10 of the 3′ terminus and 11′ of the 5′ terminus inhibited activity independently of whether a base- pair was formed or not. Thus, at these positions, the nature of the residues is apparently more important than their ability to form base-pairs. These results extend our previous virion ‘RNA-fork’ model and are consistent with in vitro findings that the 5′ terminus is involved in the initiation of transcription.

Antigenic variation among eight bovine respiratory syncytial virus (BRSV) isolates was determined using monoclonal antibodies (MAbs) specific for the attachment (G) protein. Two major (and one intermediate) subgroups were identified, as well as one strain that did not fit any pattern. The subgroups could also be differentiated on the basis of the M r of the F protein cleavage product, F2. The nucleotide sequence of the G gene of seven of the BRSV strains was determined and compared with published G gene sequences. Subgroups A and A/B were more closely related in protein sequence than subgroups A and Bor subgroups A/B and B. These results could not be correlated with those obtained by the determination of the M r of the F2 polypeptide. Multiple sequence alignments showed a high level of amino acid identity at the inter-subgroup level (85% identity between subgroup A and subgroup B strains), similartothe intra-subgroup human (H)RSV identity, suggesting that the BRSV isolates form a continuum rather than distinct subgroups. However, unusual variability was observed within the immunodominant domain (amino acids 174–188) in contrast with the situation in HRSV strains belonging to the same subgroup.

To characterize the variability of recent field isolates of canine distemper virus (CDV) from different hosts and geographical areas, we conducted nucleotide sequence analysis of the gene encoding the haemagglutinin (H), the attachment protein of this virus. Pronounced differences between field isolates were revealed in comparison to the Convac and Onderstepoort vaccine strains. The diversity of CDV appeared to exceed that determined for measles virus. Phylogenetic analysis also separated the field isolates of CDV from the vaccine strains and provided evidence for the existence of different contemporary genotypes of CDV. Isolates from a Greenlandic sledge dog and a Siberian seal formed a distinct lineage. The remaining isolates formed a group. This group contained two European isolates from mink and ferret, a single lineage comprising three European dog isolates, and another separate lineage of North American isolates from dog, javelina, raccoon and captive leopards.

We isolated three strains of canine distemper virus (CDV)-the Ueno, Hamamatsu, and Yanaka strains -from dogs in Japan and analysed the molecular properties of their haemagglutinin (H) proteins. Immunoprecipitation of all three strains with a monoclonal antibody revealed H proteins with molecular masses of 84 kDa, which differs from the molecular mass (78 kDa) of the H protein of the Onderstepoort vaccine strain. However, after tuni- camycin treatment immunoprecipitation identified H proteins of identical molecular mass (68 kDa) for all three field isolates and the vaccine strain. Sequence analysis showed nine potential sites for asparagine-linked glycosylation in the H proteins of the new isolates, in contrast to four in the H protein of the Onderstepoort strain. Thus, variation in glycosylation of the H proteins of the isolates and the vaccine strain may cause differences in antigenicity of the viruses. Sequences of the H genes showed that the new Japanese isolates have 99% identity with each other, 95% with other European and American isolates (from seals, a German dog, a ferret and large felids) and 90% with the vaccine strain. Phylogenetically, the new Japanese isolates form one cluster which is separate from recent European or American isolates, all of which are distinct from vaccine strains.

The nef gene product of both human and simian immunodeficiency viruses is critically important for virus replication and disease progression in vivo. However, the precise biological function of Nef remains poorly characterized in vitro, with previous reports suggesting that Nef might be either cytotoxic or cytostatic. As a result of difficulties encountered by several groups in establishing cell lines constitutively expressing Nef, we have developed two inducible systems resulting in stable Nef expression in various mammalian cell lines. Tetracycline-regulated Nef expression was achieved in HeLa cells but could not be established in human T cell lines. Jurkat E6-1 T cell and RAW264.7 murine macrophage cell lines expressing a regulated nef gene were generated using a system in which Nef expression was controlled by a mutated version of the heavy metal-inducible human metallothionein IIA promoter. Induction of high levels of Nef expression in HeLa-Nef and Jurkat-Nef cells resulted in a moderate (2-fold) and a dramatic (10-fold) retardation of cell growth respectively, supporting the contention that Nef may be a cytotoxic or cytostatic factor. This property was also observed at low basal levels of Nef expression in RAW264.7-Nef macrophage clones (5-fold reduction in growth) and was associated with an altered morphological phenotype suggesting that different cell types may be more susceptible to the cytostatic activity of Nef. The regulated Nef-expression systems provide tools for investigating the molecular basis of Nef function, including Nef-mediated cytopathogenicity, CD4 down-regulation and enhancement of virus infectivity.

Little sequence information is available for human immunodeficiency virus type 1 (HIV-1) vif genes of African origin. Here we describe 37 new complete vif genes of 18 AIDS patients from Uganda and showthat vif hasa high in vivo genetic variability. vif proviral DNA sequences of peripheral blood cells were determined by direct sequencing of PCR products. Only 52% of the deduced Vif amino acids were absolutely conserved; when Vif sequences previously analysed were considered, only 32% of the Vif consensus sequence comprised conserved and as such possibly functionally important motifs. The high inter-individual vif variability was in contrast to a very low intra-individual variability. One patient carried a vif gene with a stable C-terminal deletion, but N-terminal truncations were not found in patients’ predominant vif sequences. The vif genes analysed comprised subtypes A, D and an A/D mosaic. Phylogenetic analyses additionally showed that HIV-1 in Uganda has spread across the boundaries of ethnic groups.

E6 and E7 oncoproteins of human papillomavirus (HPV) play significant roles in the pathogenesis of cervical cancer. However, the pattern of E6/E7 expression during the productive virus life cycle in differentiating epithelia of the uterine cervix remains unclear. In addition, little is known about the cellular factors regulating E6/E7 expression in differentiating epithelia. In the present study, using transient expression assays and DNA binding assays, we demonstrated that E6/E7 transcription is critically regulated by the cellular factor API, a Jun/Fos heterodimer complex. Immunohistochemical analyses of various uterine cervical lesions showed API expression in lower cell layers of normal cervix and low-grade cervical intraepithelial neoplasia (CIN), while it was detected throughout all layers in high-grade CIN and invasive cancer. In situ RNA-RNA hybridization analyses of organotypic raft culture specimens of an HPV-31-containing cell line revealed that E6/E7 transcripts were expressed in most cell layers, with reduced expression in differentiated cells. This pattern of HPV expression correlated with the pattern of API expression detected by immunohistochemical analyses. These findings suggest that E6/E7 expression in differentiating epithelia is dependent on AP1, which appears to be associated with proliferative activity of the cells. Since E6/E7 expression induces cell proliferation, co-expression of AP1 and E6/E7 in undifferentiated cell layers might create a positive regulatory loop, probably contributing to maintenance of initial HPV infection and subsequent activation in basal and suprabasal cell layers.

Early region 1A (E1A) of adenoviruses (Ad) codes for potent activator and repressor molecules which are involved in the regulation of viral and cellular gene expression. Gene regulatory functions of E1A proteins are mainly located in their conserved regions (CR) 1 to 3. In addition to the CRs, specific amino acids (aa) of the N-terminal end play an important role in some gene regulatory functions. We describe here the identification and characterization of a novel trans-activation domain which is located in the non-conserved N-terminal end of Ad12 E1A, namely aa 1–29. Fusion of this region to the DNA-binding domain of the yeast transcription factor Gal4 generates a strong trans-activator which induces gene expression of reporter constructs in dependence on Gal4 DNA-binding sites. Furthermore, transient expression assays using the physiological E1A-responsive adenoviral E2 early promoter revealed that the N terminus is involved in its activation. The gene regulatory function of the N terminus is specific for E1A proteins of the highly oncogenic serotype Ad12, as the respective E1A N terminus of the non-oncogenic serotype Ad2 is unable to activate the expression of the reporter gene as Gal4 fusion protein. Moreover, deletion mutant analyses demonstrate that Ad12 E1A proteins carry three independently acting activation domains: (1) aa 1–29, (2) CR1 and (3) CR3.

The cellular transcription co-activators p300 and the CREB-binding protein CBP are cellular targets for transformation by the E1A proteins of nononcogenic adenovirus 5 (Ad5). In this study, we show that the E1A proteins of oncogenic Ad12, like those of Ad5, can also bind to CBP and that this interaction is direct. In addition, we show that the Ad12 E1A proteins can also bind directly to p300. These results suggest that E1A can modulate the function of proteins of the p300 family via direct protein-protein interactions.

Salmonella typhimurium strains expressing foreign antigens of various pathogens are capable of eliciting antigen-specific humoral and cellular immune responses. Attenuated S. typhimurium strain χ4550 (∆cya ∆crp ∆asd) was used as an expression vector for herpes simplex virus (HSV) antigens. Genes encoding glycoprotein D (gD) and the immediate- early protein ICP27 of HSV-1 were cloned and expressed in plasmid pYA292 (asd+ ) and subsequently placed into χ4550 . Following two oral immunizations, the protective efficacy of recombinant strains against zosteriform challenge with HSV-1 was measured in 3–4-week-old BALB/c mice. Levels of protection observed were 77% with the ICP27 construct but only 31 %with the gD construct. Zosteriform protection correlates with a CD4+- mediated delayed-type hypersensitivity (DTH) reaction against HSV. Accordingly, significant DTH was observed only in mice immunized orally with the S. typhimurium ICP27 construct. ELISA analysis of antigen-specific humoral responses failed to detect serum antibody responses following oral administration although recombinant S. typhimurium were isolated from spleens of orally dosed mice up to day 30. Intravenous (i.v.) immunization with the gD- expressing construct did, however, induce detectable serum antibody responses. Some humoral IgA responses against gD in faecal samples were detected as early as 3 weeks post-oral immunization while those induced by the i.v. route were slightly lower. These data suggest that recombinant S. typhimurium HSV antigens are capable of inducing immunity against HSV, some aspects of which are protective against HSV challenge.

Because synthesis of rRNA persists late during herpes simplex type 1 infection and because S6 phosphorylation is always correlated with efficient translation of ribosomal protein mRNA, we tested the hypothesis that ribosomal protein synthesis and ribosome biogenesis could persist after infection. At different times after infection, proteins were labelled with 35S for 1 h before harvesting and ribosomes were purified. Measurement of radioactivity incorporated into individual ribosomal proteins separated by two-dimensional PAGE demonstrated that ribosomal proteins are still synthesized and assembled into mature ribosomes up to late times during infection, while synthesis of β-actin is severely inhibited. During expression of late genes, ribosome biogenesis was estimated to be 58% of that of the control as judged by [3H]uridine incorporation into rRNA. As for β-actin mRNA, the level of ribosomal protein mRNA decreased progressively from the beginning of infection, reaching about 30% of the control level during expression of late genes. Taken together, these results demonstrate that ribosomal proteins are still synthesized up to the late time of infection and efficiently assembled into mature ribosomes, while there is a severe shutoff of the synthesis of other cellular proteins.

Expression from a short human cytomegalovirus (HCMV) major immediate early (IE) promoter- enhancer was tested in three different virus vectors: recombinant adenovirus (Ad), recombinant herpes simplex virus type 1 (HSV-1) and HSV-1-derived amplicon vectors. The HCMV major IE promoter- enhancer within a replication-deficient recombinant Ad vector was shown to produce cell-specific expression in rat nervous system cell cultures. Recombinant Ad entered all cell types examined but the HCMV major IE promoter was silent in primary cultures of neocortical neurons and Schwann cells, although it drove transgene expression in astrocytes and fibroblasts. Moreover, in neurons and Schwann cells, expression from the HCMV major IE promoter-enhancer in the replication-deficient Ad vector was activated by superinfection with HSV-1, replication-competent Ad and HCMV. The HCMV major IE promoter-enhancer was active in neurons when inserted into HSV-1 recombinant vectors. Further experiments with HSV-1-derived amplicons strongly suggested that an IE protein was responsible for the activation of HCMV major IE-induced expression in neurons. This demonstrates that the activity of the HCMV major IE promoter-enhancer element can depend on the expression of other genes encoded in the virus vector backbone within which it is inserted, and that it can function in a neuronal cell type-specific manner when inserted into a replication-deficient Ad vector.