- Volume 79, Issue 3, 1998

Human immunodeficiency virus type 1 (HIV-1) envelope vaccines can now be evaluated for efficacy in macaques by challenging with chimeric viruses in which the env, tat and rev genes of simian immunodeficiency virus (SIV) have been replaced by those of HIV-1. Most experiments have so far been conducted using gp120 molecules derived fromT-cell- adapted LAI or MN strains of HIV-1, which predominantly use the CXCR-4 co-receptor. These vaccines protect against infection by apathogenic chimeric virus carrying the same envelope sequences. In the experiment described here, four macaques were vaccinated with W61D gp120 derived from a low passage Dutch isolate and capable of inhibiting the binding of MIP1 β to the co-receptor CCR-5. This vaccine was potent, inducing high titres of binding and neutralizing antibodies against the homologous HIV-1 and tenfold lower titres against a heterologous challenge virus (SHIVSF33) in which the env, tat and rev genes of SIV had been replaced by those of a San Francisco isolate, HIV-1SF33. Despite strong immune responses to the vaccine there was no evidence that it protected against challenge with this chimeric virus. The antigenic divergence between vaccine and challenge virus or the increased virulence of the challenge virus may be responsible for the inability of this vaccine to protect against infection by SHIVSF33.

The modulation of MHC class I molecule expression on the surface of cells as a consequence of foot- and-mouth disease virus (FMDV) infection has been examined. On cells infected with FMDV, class I expression was reduced to approximately 70% of the initial value 3 h after the infection and to 53% after 6 h. On cells depleted of surface class I complexes by acid treatment, the appearance of newly assembled class I-peptide complexes on the cell surface of non-infected cells increased immediately upon neutralization and original class I levels were recovered in about 20 h. In contrast, the appearance of new peptide-bound class I molecules on the cell surface was inhibited as early as 30 min after FMDV infection. Since the shut-down of FMDV- mediated host protein synthesis occurs approximately 2–3 h post-infection, this result suggests that an earlier event, which prevents the surface expression of newly synthesized complexes, is induced following FMDV infection. Thus, FMDV- infected cells rapidly become unable to present viral peptides in association with MHC class I molecules to T lymphocytes. Such a mechanism would assist virus evasion of the cytotoxic immune response of the host.

A genomic-length cDNA clone corresponding to the RNA of dengue virus type 2 (DEN-2) New Guinea C strain (NGC) was constructed in a low copy number vector. The cloned cDNA was stably propagated in Escherichia coli and designated pDVWS501. RNA transcripts produced in vitro from the cDNA using T7 RNA polymerase yielded infectious virus (MON501) upon electroporation into BHK-21 cells. When compared with parental NGC virus, MON501 replicated to similar levels in Aedes albopictus C6/36 cells and showed similar neurovirulence in suckling mice. In contrast, a second genomic-length cDNA clone (pDVWS310) used as an intermediate in the construction of pDVWS501 produced virus (MON310) that replicated well in C6/36 cells but was not neurovirulent in mice. MON310 contained the prM and E genes of the non-neurovirulent PUO- 218 strain in an NGC background. There were seven amino acid differences between the prM and E proteins of MON310 and MON501. The differences were generally conservative, with the exception of E residue 126, which was Glu in MON310 and Lys in MON501. To examine the role of this residue in mouse neurovirulence, substitutions of Glu → Lys and Lys → Glu were made in MON310and MON501, respectively. The properties of these mutants clearly demonstrated that Lys at E residue 126 is a major determinant of DEN-2 mouse neurovirulence.

A variant of hepatitis E virus (HEV), designated HEV US-1, was identified in a hepatitis patient in the United States (US); the patient had no history of travel to areas where HEV is endemic. Nucleotide sequences were obtained from the 5′ end of open reading frame (ORF) 1 (1418 nt), the 3′ end of ORF1 (1359 nt), the entire ORF2 and ORF3 regions, and the 3-untranslated region (2127 nt). The HEV US-1 strain is significantly divergent from other human HEV isolates with nucleotide identities ranging from 76·8 to 77·5%. Phylogenetic analyses indicate that HEV US-1 and a recently discovered HEV variant from swine may represent separate isolates of a new strain of HEV, significantly divergent from the Mexican and Burmese strains. Synthetic peptides derived from the carboxyl amino acids of ORF2 and ORF3 were shown to be useful for detecting exposure to HEV. In addition, IgM class antibodies directed against HEV US-1 synthetic peptides were detected in the US patient infected with HEV US-1, but were absent using synthetic peptides from the Burmese or Mexican strains of HEV. A preferential reactivity to HEV US-1 specific peptides has lead to the identification of a second isolate of this virus also from a patient with acute hepatitis from the US. The discovery of these HEV variants may be important in understanding the worldwide distribution of HEV infection.

The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vitro polymerase assay based on purified viral cores and synthetic oligoribonucleotides corresponding to the 3′ and 5′ ends of cRNA. In vitro polymerase activity relied on an interaction between the 3′ and 5′ ends of cRNA and was ApG primer-dependent. Mutational analysis of the promoter showed that interstrand base-pairing of residues 11 and 12 of the 3′ promoter arm with residues 10 and 11 of the 5′ promoter arm, respectively, was essential for polymerase activity. These data provide the first clear evidence for a cRNA panhandle in an orthomyxovirus. No evidence was obtained for the presence of a 5′ or 3′ hook structure in the cRNA promoter, and transcription could not be primed with rabbit globin mRNA or synthetic cap analogues. This demonstrates that cap snatching activity relies on the presence of the vRNA terminal sequences.

The MAb IIB4 recognizes an immunodominant epitope on influenza virus haemagglutinin (HA) which is shared by different strains of the human subtype H3. This epitope includes amino acids 198, 199 and 201, as determined by selection of IIB4 escape mutants, and is involved in haemagglutination- inhibition (HI) and virus-neutralization (VN). We have developed anti-idiotypic MAbs (Ab2) that mimic the IIB4 epitope. Two Ab2, 78 and 464, completely inhibited binding of MAb IIB4 to the virus. Nucleotide sequences of VL- and VH-encoding regions were determined for IIB4 and both Ab2. VH of IIB4 and 78 belong to the same IgG family (VII) and show high nucleotide identity (89%). Conversely, VH and VL sequences of both internal image-bearing Ab2 revealed lower degrees of identity (61 and 50%, respectively). Ab2 were used for syngeneic immunization to elicit polyclonal Ab3 responses. Like Ab1, Ab3 immunoprecipitated viral HA and displayed HI and VN activity. The different VN activity of anti-78 and anti-464 in vitro correlated with the affinities of their corresponding Ab2 to IIB4. In vivo immunization with either Ab2 protected 15–37% of mice against lethal influenza infection or delayed dying. In contrast to VN activity of Ab3 in vitro, there was no significant difference between the protection of mice induced by Ab2 78 and 464. We demonstrate, for the influenza model, that active immunization with a single influenza virus HA epitope in the form of its internal image leads to partial protection in vivo.

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by twodimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]ortho- phosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic α subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PAsubunit of the influenza virus RNA polymerase is a phosphoprotein.

Five monoclonal antibody (MAb) neutralization escape mutants of respiratory syncytial virus (RSV) were produced by growing the Long strain RSV (group A virus) in the presence of a neutralizing, group cross-reactive MAb specific for the attachment protein (G). Four viruses (RSV-2, −6, −14 and −15) had amino acid replacements clustered within a highly conserved centrally located 13 amino acid region (position 164–176). Reactivity with group A- specific MAbs and with polyclonal anti-G serum was maintained and growth kinetics were unaffected. An additional virus (RSV-3) had four amino acid substitutions in the cytoplasmic tail and transmembrane region of G, and had restricted growth and formed small syncytia. Immunofluorescent and Western blot analysis indicated that G protein was not membrane associated and had reduced incorporation into the virion, thereby escaping neutralization by L9 and polyclonal anti-G serum. The predominant form of G produced by RSV-3 was found in infected cell supernatants, consistent with the size of secreted G.

Human papillomavirus (HPV) 16 E6 induces the degradation of the tumour suppressor protein p53 by the ubiquitin-dependent proteolysis pathway. In vitro, this process involves the formation of a trimolecular complex between E6, p53 and a cellular protein E6-associated protein (E6-AP). However, an analysis of their potential interactions in vivo has not been carried out. We have established a model for the expression and analysis of the interactions of these three proteins in insect cells, a eukaryotic system where potentially crucial modifications of the proteins will occur. In baculovirus-infected cells the degradation of p53 can occur. However, p53 is only degraded early in the infectious cycle due to a lack of ATP at later times. Consequently, substantial quantities of material can be produced in this system for further analysis. Evidence is also provided that, in vivo, E6 can interact with p53 in the absence of E6-AP and that E6-AP can interact with p53 in the absence of E6. Furthermore, analysis of the subcellular localization of the proteins using both biochemical fractionation and indirect immunofluorescence suggests that the degradation of p53 occurs in the perinuclear region of the cell.

Bovine papillomavirus 4 (BPV-4) is a mucosal epitheliotropic papillomavirus. It encodes a transcriptional regulator, E2, which acts on the BPV-4 transcriptional control region (the long control region or LCR) to regulate transcription. The distribution of E2 binding sites within the LCR of BPV- 4 is identical to that of the human papillomaviruses HPV-16 and HPV-18, indicating that the mechanism of transcriptional control by E2 of mucosal epitheliotropic papillomaviruses is conserved. In this study it has been shown that E2 activates transcription through the BPV-4 LCR promoter in primary bovine palate keratinocytes but not in primary bovine palate fibroblasts. The epithelial specific transcriptional activation of the BPV-4 LCR by E2 is promoter-specific because following binding to the BPV-4 LCR placed in an enhancer mode, E2 can activate transcription from heterologous promoters, such as SV40, in both keratinocytes and fibroblasts. Chimaeric VP16-E2 molecules suggest that the epithelial specific transcriptional activation of the BPV-4 LCR promoter is mediated by the E2 transactivation domain. Although low to intermediate levels of E2 can activate transcription from the BPV-4 LCR promoter, high levels of E2 result in down-regulation of transcription from this promoter in keratinocytes. Mutation of E2 binding site 1 (BS1), which is 3 bp upstream from the TATA box, abrogates down-regulation of transcription by high levels of E2. The results present a model system for studying transcriptional regulation of mucosal epi- theliotropic papillomavirus LCRs by E2.

Ovine adenovirus OAV287 (OAV) is the prototype of a virus group which is phylogenetically distinct from the mastadenoviruses and aviadenoviruses. The genome arrangement of OAV showed that virus- associated (VA) RNA genes were not located between the reading frames for p52/55K and terminal protein as these overlapped. To determine whether VA genes were located elsewhere, several approaches were used. Nuclear extracts containing RNA polymerase III activity were used to transcribe OAV genome fragments in vitro. A product of ~ 120 bp was produced from two widely separated coding regions of the genome. However, when these were subcloned and used as radiolabelled probes to analyse RNA from OAV-infected cells, no VA-like RNA was detected, although late mRNAs that were transcribed from the regions were identified. In addition, 5′ radiolabelling of small RNA species in control- and OAV-infected cells followed by gel analysis did not identify candidate VA RNAs. Radiolabelling of proteins in control- and OAV-infected cells showed that there was little preferential translation of viral proteins compared with host polypeptides, in contrast to the situation for adenovirus 5 (Ad5). In addition, the kinetics of OAV infection were slower than observed for human adenoviruses. Collectively, the data suggest that OAV is unique in that it does not produce VA RNA during infection. This conclusion is supported by a comparison of the genomes of the phylogenetically related OAV and egg drop syndrome viruses which shows that the VA gene identified in the latter is located in a region absent from OAV.

Previous analyses using transient transfection assays indicated that the promoter for the gene encoding the herpes simplex virus type 1 (HSV-1) neurovirulence protein ICP34.5 can be divided into an essential core region of approximately 80 bp and two potent upstream silencer domains corresponding to the DR2 and DR6 repeat arrays. In order to examine the potential role of transcriptional silencing during productive HSV-1 infection, recombinant viruses were generated in which wild-type or mutant ICP34.5 promoters controlling the expression of a chloramphenicol acetyltransferase reporter gene were inserted into the thymidine kinase gene of the viral genome. The intact promoter in the virus HSV- Δ1CAT exhibited delayed-early kinetics of expression that were comparable to those of the ICP34.5 gene promoter at its native site in the genome. Deletion of the core promoter domain eliminated promoter activity in the virus HSV- Δ5CAT, indicating that this region was required for expression not only in transient transfections assays but also in the context of the viral genome. However, deletion of the DR2 repeat array from the ICP34.5 promoter in the virus HSV- Δ7CAT was found to increase promoter activity only minimally at late times, and even to reduce activity at early times. Thus, in marked contrastto its behaviour in transient expression assays, the DR2 repeat array does not appear to act as a transcriptional silencer in the context of the HSV-1 genome during productive infection.

Herpes simplex virus type 1 (HSV-1) variant 1716 is deleted in the gene encoding ICP34.5 and is neuroattenuated after intracranial inoculation of mice. Although the mechanism of attenuation is unclear, this property has been exploited to eliminate experimental brain tumours. Previously, it was shown that infectious 1716 was recoverable for up to 3 days after intracranial inoculation suggesting that there may be limited replication in the central nervous system (CNS). Here it is demonstrated that 1716 replicates in specific cell types (predominantly CNS ependymal cells) of BALB/c mice, using im- munohistochemical, immunofluorescence, in situ hybridization and virus titration studies. While 1716-infected mice exhibited no overt signs of encephalitis, histological analysis showed a persistent loss of the ependymal lining. Thus, although ICP34.5-deficient viruses are neuroattenuated, they do retain the ability to replicate in and destroy the ependyma of the murine CNS. A detailed understanding of the mechanism(s) of neuroattenuation and limited replication could lead to the rational design of safe HSV vectors for cancer and gene therapy in the CNS.

The herpesvirus regulatory protein IE110k possesses a cysteine-rich, RING finger motif required for its role in transactivation and virus replication. IE110k also localizes to subnuclear compartments termed PODs (PML oncogenic domains). Localization to PODs induces redistribution of the proteins associated with this nuclear compartment, including the cellular RING finger protein, PML. Here we construct a series of deletions, RING domain swaps and point mutations to analyse specific requirements within the IE110k RING finger for subnuclear localization, redistribution of PML and transactivation and we examine the relationship between these activities. We find that IE110k localizes to distinct nuclear subdomains that are more numerous than the cellular PODs and that mutation of two residues within a predicted loop of the RING finger, or replacing the IE110k RING finger with a RING finger from a cellular gene abrogates the ability of IE110k to localize to these extra compartments and traps IE110k in the original PODs. We further demonstrate that RING fingers from the cellular genes mdm-2 and Bmi I, when placed within IE110k, alter the nuclear distribution of IE110k, do not transactivate, and do not redistribute PML. We also demonstrate that the majority of wild-type IE110k, like PML, is associated with the nuclear matrix. Although substitutions and deletions within the RING finger abolish transactivation, these mutant proteins remain tightly associated with the matrix. These results further dissect the determinants involved in different aspects of nuclear compartmentalization of IE110k and are discussed in relation to PML, PODs and the IE110k RING finger.

Herpes simplex virus (HSV) causes many disease states including mucosal lesions, encephalitis or disseminated infection in the immunocompromised host. These diverse clinical manifestations reflect the capacity of the virus to infect both epithelial and neuronal cell types. Determining the requirements for virus entry into both cell types may provide insights into the pathogenesis of HSV. Previous studies have focused on identifying viral and cellular requirements for entry using epithelial cells. However, little is known about the requirements for binding and entry into neuronal cells. The purpose of the studies reported here wasto identify viral and cellular components involved in entry of HSV-1 into primary neuronal cells. Heparan sulfate glyco- saminoglycans were found to serve as a receptor for entry of HSV-1 into primary neuronal cells. Evidence to support this includes the findings that heparin (an analogue of heparan sulfate) competitively inhibited virus binding and expression of immediate early virus gene products. In addition, heparitinase removed viral receptors and inhibited virus entry. In epithelial cells, deletion of HSV-1 glycoprotein C (gC) results in virions that have reduced specific binding activity (virus particles bound per cell) and specific infectivity. However, in neuronal cells, it was found that deletion of gC resulted in no loss in specific binding activity, but did result in significant impairment of virus entry as measured by expression of immediate early viral gene product. Taken together, these findings suggest cell-type differences in virus binding and entry and a different role for gC in neuronal cell infection.

Clearance of herpes simplex virus (HSV) from spinal ganglia of experimentally infected mice is known to be dependent on CD8 T-cells but not on destruction of infected neurons, consistent with a non-cytolytic Tc2 response in the peripheral nervous system. Here, we demonstrate the striking rapidity of such a response in C57BL/10 mice. The number of neurons containing viral DNA and viral antigens increased until 136 h after inoculation of virulent HSV type 1 (strain SC16) into flank skin. Subsequent disappearance of HSV DNA and antigens from infected ganglia was virtually complete only 8 h later. A consistent and unexpected observation was detection of viral antigens in sensory nerve axons for at least 8 h after their disappearance from neuronal somas, raising the intriguing possibility that virus or viral proteins may be transported distally after infection has been terminated.

Double infection with two interferon (IFN)-sensitive strains of herpes simplex virus (HSV), HSV-1(17syn) and HSV-2(UW268), showed reduced inhibition of virus growth by IFN. Intertypic recombinants with IFN resistance were obtained from the doubly infected cultures. These results indicate that HSV IFN resistance is controlled by at least two genetic regions. Restriction endonuclease analysis demonstrated that the recombinants were similar to HSV-2 in their genomic structure but the BamHI-A, BglII-I and BglII-N fragments of HSV-2 were commonly lost in the recombinants, suggesting that any of these fragments could be associated with HSV-2 IFN resistance. We cloned these fragments and BamHI-E, which overlaps BglII-N, from an IFN-resistant HSV-2 strain, HSV-2(G), and examined each fragment for its ability to rescue IFN resistance of HSV- 2(UW268) by co-transfecting with the HSV- 2(UW268) genome. Of the HSV-2(G) fragments, only BglII-N increased plating efficiency of progeny viruses in IFN-treated cells. An IFN-resistant HSV-2 clone was obtained from the BglII-N of HSV-2(G) and HSV-2(UW268) genome co-transfected culture, and a part of BglII-N of HSV-2(UW268) was replaced with that of HSV-2(G) in the HSV-2 clone. Thus, it was concluded that one of the HSV regions encoding IFN resistance is located on the BglII-N fragment of HSV-2.

Germ cell tumours (GCT) of the testis are the most common malignant tumours occurring in young adults. In view of the young age of patients, the increasing incidence of GCT and the overexpression of wild-type p53 observed in a majority of tumours, the possibility of the involvement of a virus in the development of this cancer was considered. Testicular GCT were analysed for the presence of cytomegalovirus and Epstein-Barr virus (EBV), which are known to cause overexpression of wild- type p53 protein, and parvovirus B19. The testicular tissue of 39 patients with testicular GCT and 12 patients with healthy testicular tissues was tested for presence of viral DNA by PCR. Neither cytomegalovirus nor EBV DNAs were detected in the 39 tumours analysed, but parvovirus B19 DNA sequences were demonstrated in the testicular tissue of 85% (33/39 cases) of patients with GCT. The sera of 16 of the 39 patients with GCT were tested for the presence of parvovirus B19 IgM and IgG. B19-specific IgG was detected in the sera of 11 patients (69%). Only one case was positive for parvovirus B19 IgM, which was also shown to have B19 genome sequences in the serum by PCR, indicating that in a majority of cases an acute B19 infection can be excluded as being the source of the B19 DNA sequences in the testis. B19 DNA could not be detected in normal testicular tissue and thus parvovirus B19 could play a role, direct or indirect, in the development of testicular GCT or have tropism for the tumour cells.

Two strains of minute virus of mice (MVM) show different host-cell specificities. MVM(i) grows in T lymphocytes whereas MVM(p) is fibroblast-specific. By constructing recombinant viral DNAs between the genomes of the two strains, we have shown that two segments of the MVM(i) genome are required for lytic viral growth in T lymphocytic EL4 cells. One segment (iE) was found between nucleotides 1084 and 2070, in a region encoding the early viral proteins and containing mRNA splice signals and the late P39 promoter. The other (iL) was between nucleotides 3523 and 4339 in the region coding for capsid protein. The P39 promoters within the E segment from MVM(i) or MVM(p) were equally active in transfected EL4 cells. However, pE-con- taining MVM DNA produced more NS2 mRNA than iE-containing DNA, apparently the result of virus- strain-specific differences in the regulation of splicing.