- Volume 74, Issue 8, 1993

We have compared the promoter/enhancer structure of human polyomavirus JC (JCV) isolates from 11 progressive multifocal leukoencephalopathy brains. The duplications and deletions of the regulatory region were different in each patient, and usually only one sequence was found in each. The sites of strand breakage in the promoter were not random; four or five preferred sites or areas exist. Alignment of the JCV prototype Mad-1 regulatory region with the unduplicated archetypal structure defines six blocks of sequence, A to F. The preferred sites of strand breaks delineate these regions, although Mad-1 is an unusual promoter containing a break site not observed in other isolates, and an additional site is targeted in several promoters. Region A, containing the TATA box, and the first half of region C, containing several enhancer elements, and region E are consistently retained. Region B, the 23 bp insertion in the archetypal structure (relative to Mad-1) was also retained in all 11 isolates. Region D, the 66 bp insertion, was retained in isolates from three patients. Regions A and D were never duplicated, whereas regions C and E usually were duplicated or triplicated. Variation in the exact point of breakage within the preferred sites, alternative use of the sites in individual promoters and occasional short deletions at other sites result in sequences that are unique in each case. At the same time, the limited choice of break sites and the characteristic fates of the regions themselves result in three broad patterns of repeat sequences. The patterns do not correspond to the viral genotypes 1 and 2 defined by coding region base changes, and do not appear to be a stable feature of the virus. Rather, rearrangements appear to be generated in the host from a basic archetypal sequence.

Differences in the antigenic structure of the major glycoproteins, gI, gIII and gIV, of bovine herpesvirus type 1∙1 (BHV1∙1) and the neurovirulent BHV1∙3 were demonstrated with a panel of monoclonal antibodies (MAbs) prepared against the BHV1∙1 glycoproteins. Glycoprotein gIII of BHV1∙3 was the most dissimilar, reacting with only four of 15 gIII-specific MAbs. Glycoproteins gI and gIV of BHV1∙3 reacted with eight of 11 and eight of 12 specific MAbs, respectively. Monospecific bovine antisera to the two viruses supported findings from the MAb analysis in that gI and gIV glycoproteins were cross-recognized, but gIII was not. Virus-neutralizing MAbs reactive to each glycoprotein and which reacted with both viruses also neutralized both viruses. Previously undescribed glycoproteins which were antigenically related to the intact gIII glycoproteins, but of reduced sizes and lacking at least one gIII epitope, were found for both viruses. Tunicamycin inhibition experiments and immunoprecipitation data suggested that these proteins were intracellular degradation products. Comparisons of the peptide footprints of the glycoproteins from the two viruses using protease V8 digestion after immunoprecipitation with cross-reactive MAbs revealed distinctive footprint patterns for the respective glyco-proteins.

All herpes simplex virus (HSV) infected cell-specific polypeptides (ICSPs) were synthesized in the presence of lithium at a concentration (60 mm) inhibitory to the production of infectious virus. Yields of certain ICSPs were increased and others, in particular glycoprotein C, decreased. HSV DNA synthesis was completely inhibited; synthesis and in vitro activities of HSV DNA polymerase and thymidine kinase were decreased but to a degree insufficient to account for the complete inhibition of HSV DNA synthesis. HSV DNA synthesis was inhibited to an equivalent degree by either incubation with 60 mm-lithium or by potassium starvation; both procedures decreased intracellular potassium by an equivalent amount as adjudged by X-ray microanalysis. We conclude that lithium inhibits HSV DNA synthesis by displacement of potassium from a potassium-dependent biochemical reaction or by other physiological changes brought about by the loss of cellular potassium. The possibility that lithium also directly inhibits a virus replicative event cannot be excluded.

The cellular factors that regulate infection and replication of respiratory syncytial virus (RSV) in human alveolar macrophages were examined. RSV-exposed alveolar macrophages demonstrated a time-dependent expression of viral glycoproteins, maximal by 24 h post-infection resulting in infection of approx. 38% of the cells. Essentially all (33%) of these freshly isolated alveolar macrophages replicated RSV as shown by infectious centre assays. This RSV-permissive subpopulation of alveolar macrophages consisted primarily of major histocompatibility class II-expressing cells as determined by fluorescence-activated cell sorting. Reinfection of alveolar macrophages did not significantly alter the number of cells infected or capable of replicating RSV. However, in vitro differentiation of alveolar macrophages prior to infection resulted in a significant (P < 0·05), time-dependent decrease (approx. sevenfold) in the number of cells that replicated virus. The mechanism by which cellular differentiation restricted RSV replication is unknown. Production of defective interfering particles did not account for this decrease. Alveolar macrophages infected with RSV produce a variety of cytokines potentially contributing to this restricted viral replication. Pretreatment with several of these cytokines did not affect viral infection or replication. However, tumour necrosis factor (TNFα) significantly (P < 0·05) decreased viral replication but only by 30 to 60%. Thus RSV replication is reduced by in vitro differentiation of alveolar macrophages and, to a lesser degree, by pretreatment with TNF.

The core of a unique linear neutralization epitope (G5) on the glycoprotein of rabies virus, recognized by a virus-neutralizing mouse monoclonal antibody (MAb 6-15C4), was determined by Pepscan analysis. The G5 epitope was defined as an octapeptide (LHDFRSDE). The contribution of the individual amino acids of the G5 epitope to the binding of MAb 6-15C4 was analysed with a set of synthetic peptides in which the individual amino acids had been replaced in turn by each of the other 19 naturally occurring amino acids. Five amino acids of the octapeptide proved to be essential for the binding of MAb 6-15C4. The conservation of the G5 epitope within the glycoprotein of the different rabies virus strains sequenced to date proved to be absolute at the amino acid level. Studies concerning the immunodominance of the G5 epitope were carried out by determining the presence of G5 epitope-specific serum antibodies in vaccinated humans and mice, and by determining the frequency of G5 epitope-specific B lymphocytes in the blood of vaccinated humans. These studies indicated that antibodies to the G5 epitope constitute a minor population of the rabies virus-specific serum antibodies induced by rabies vaccination.

A monoclonal antibody (C3) produced against foot-and-mouth disease virus type O1Caseros was found to neutralize quadrivalent monoclonal antibody escape mutant (G67) of foot-and-mouth disease virus type O1Kaufbeuren. This mutant had been characterized at the sequence level as having distinct changes affecting four non-overlapping neutralizable sites. The C3 monoclonal antibody was used to prepare a quintuple escape mutant from the G67 and a single escape mutant from the parental O1Kaufbeuren viruses. Polyclonal postvaccinated and infected cattle sera as well as polyclonal mouse and guinea-pig sera, which neutralized the quadrivalent mutant, no longer neutralized the quintuple mutant, indicating that a fifth site had been identified and that changing the fifth site eliminated all neutralization. The site was characterized using serological techniques and found to be conformationally dependent, trypsin-sensitive and independent of sites previously characterized by monoclonal antibodies. Amino acid sequencing comparing parental, single C3 and quintuple mutants showed that a single change from a glutamine to a histidine, at amino acid 149 in the structural protein VP1, (1D) characterized the C3 mutation. The fifth site probably represents a conformational epitope which is formed due to the interaction of the VP1 loop region with other surface amino acids.

P and W2 viruses are pathogenic in two crustacéans of the Mediterranean Sea, Macropipus depurator and Carcinus mediterraneus, respectively. Investigation of virus, virus density and genome structure leads us to propose their classification in a genus similar to aquareovirus of the Reoviridae family. They differ from aquareoviruses by the number of dsRNA segments forming the genome (12 instead of 11), their electrophoretic pattern in PAGE (1/5/6 instead of 3/3/5), and the absence of virus replication in fish cell lines.

The genomic region coding for the antigenic structure responsible for the induction of neutralizing antibodies was localized in the central variable region of the VP2 gene by comparing the nucleotide sequence of five escape mutants derived from the standard infectious bursal disease virus strain Cu-1. Exchange of a single amino acid at one of the prominent hydrophilic parts of this region proved to be sufficient for altering the neutralizing properties. The reactivity of neutralizing antibodies with peptides expressed in vitro encompassing both hydrophilic areas suggests that the entire variable region is engaged in the formation of this conformation-dependent antigenic site. VP2-specific, non-neutralizing monoclonal antibodies directed against the sequence-dependent epitope of the serotype I strain Cu-1 and the serotype II strain 23/82 cross-reacted with peptides located towards the carboxy terminus of VP2; no reaction occurred with peptides derived from the amino-terminal side adjacent to the variable region.

Sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted and the basal promoter activity of the LTR was measured by the chloramphenicol acetyltransferase (CAT) assay. The activity of the FIV LTR was reduced in Felis catus whole foetus 4 (fcwf-4) cells and Crandell feline kidney cells by this deletion. Cotransfection of murine c-Fos or c-Jun expression plasmids with the FIV LTR-CAT reporter plasmid into fcwf-4 cells revealed that FIV LTR could be activated by c-Fos but not c-Jun in the cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild-type in two feline CD4-positive T lymphoblastoid cell lines. It was found that the rate and activity of the mutant were almost the same as those of the wild-type. From these data, we conclude that the 31 bp fragment is important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.

The human immunodeficiency virus (HIV) type 1 nef gene product was expressed as an N-terminal fusion protein with glutathione-S-transferase (GST) in the baculovirus system. The resulting nefGST fusion protein was found to be authentically myristylated at the N terminus and could be purified to homogeneity by one-step affinity chromatography on immobilized glutathione. The high affinity of nefGST for glutathione was exploited to develop an assay to identify cellular proteins capable of interacting with nef. Several such proteins were identified in extracts from the Jurkat human T cell line. The interaction between nef-binding proteins and immobilized nefGST could be specifically competed by the addition of soluble nef. Cell fractionation showed that nef-binding proteins were present in both cytosolic and membrane-associated fractions. A non-myristylated derivative failed to bind to the membrane-associated proteins but was able to bind to the cytosolic group, albeit with reduced affinity. In addition, a single protein present in both soluble and membrane-associated fractions exhibited myristylation-independent binding to nef. By analogy with other myristylated proteins such as MARCKS (myristylated alanine-rich C kinase substrate) and the Rous sarcoma virus transforming protein, src, the membrane-associated proteins that bind only to myristylated nef may represent a specific membrane target for nef. The cytosolic proteins that interact with nef may constitute soluble components of an as yet unidentified signal transduction pathway which is the target of nef action in the HIV-1-infected cell.

A new gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) has been identified that encodes a highly basic 8.9K protein. The gene called p8.9 is expressed as a 0.5 kb transcript by 1 h post-infection and initiates at an early gene motif. The promoter of the 0.5 kb transcript has two upstream elements, repeats I and II, which are similar to motifs previously characterized in the OpMNPV IE-2 gene and the Autographa californica nuclear polyhedrosis virus IE-N and PE38 genes. A second p8.9 transcript expressed from 8 to 72 h post-infection was shown to initiate 634 bp upstream from the early gene motif in a region that has no similarity to any previously described baculovirus promoter or initiation site. Transient assays utilizing a reporter gene have shown that the p8.9 early promoter is active in a Lymantria dispar (LD652Y) and Spodoptera frugipeda (Sf9) cell lines in the absence of other viral factors. In addition, it was also demonstrated that the p8.9 promoter is trans-activated by the OpMNPV IE-2 and p34 genes, but not by IE-1.

Several primary cell lines that support the complete replication of Cydia pomonella granulosis virus have been established from one culture of C. pomonella embryonic cells. Virus passaged three times in cells and once in larvae showed no change in restriction enzyme fragment patterns. Stages in virus replication observed by electron microscopy resembled those from in vivo studies. Cell lines that were maintained at or below 21 °C retained susceptibility to virus over a period of 4 years whereas the same cell lines maintained at 27 °C gradually lost their susceptibility and eventually could not be infected at all.

A portion of the genome of the Buzura suppressaria (Lepidoptera) single nucleocapsid nuclear polyhedrosis virus (BsSNPV) containing the polyhedrin gene was sequenced. An open reading frame of 738 nucleotides encoded a protein of 246 amino acids and represented the polyhedrin gene. A conserved TAAG motif, associated with transcriptional start sites in other polyhedrin genes, was identified 51 nucleotides upstream of the BsSNPV polyhedrin gene. A putative polyadenylation signal, AATAAA, was found immediately downstream of the polypeptide termination codon. Comparison of the amino acid sequence of BsSNPV polyhedrin with other NPV polyhedrins and granulosis virus granulins showed that the BsSNPV polyhedrin was most closely related to the polyhedrin of Orgyia pseudotsugata (Lepidoptera) SNPV and most distantly related to the polyhedrin of Neodiprion sertifer (Hymenoptera) SNPV.

The virus-associated (VA) RNA gene regions of the human subgroup F adenoviruses (types 40 and 41) were amplified using primers corresponding to flanking open reading frames of human and simian (SA7) adenovirus sequences previously published. The subgroup F adenoviruses, like human Ad12 (subgroup A) and SA7, were found to have only one VA RNA gene at this locus (map unit 30). The type 40 and type 41 VA RNA genes have primary sequence characteristics in common with other known VA RNA genes, have high cross-identity (93%), and show appreciably higher identity to the single VA RNA gene present in SA7 (77% and 81%, respectively) than to any of the VA RNA genes of Ad2, Ad7 or Ad12 (< 61%). These findings may have implications for explaining the evolution and growth peculiarities of human subgroup F adenoviruses.

The complete genome of a hepatitis B virus (HBV) from Brazil that expressed the subtype adw4 of HBV surface antigen (HBsAg) was cloned and sequenced. The genome, termed w4B, consists of 3215 bp. The overall genetic organization of typical hepadnaviruses with four open reading frames including the preC region was found to be conserved. When comparing the w4B sequence with 19 complete HBV genomes it was, however, found to be more divergent (15%) than any other HBV sequence thus far reported. Until now, no more than 11% divergence has been reported. Distinct from the five known HBV genotypes A to E, w4B made up a new, sixth genotype. The importance of the conserved third start codon in the HBV X gene became apparent in isolate w4B. By mutation, this ATG was out of frame, and by what appears to have been a linked mutation, a new start site two codons downstream was re-established. The significance of several other mutations is discussed.

A putative thymidylate kinase gene of African swine fever virus has been identified at the left end of the SalI I′ fragment of the virus genome. The gene, designated A240L, has the potential to encode a protein of 240 amino acids with an M r of 27754 and is transcribed early after infection. Primer extension analysis indicates that transcription is initiated a short distance from the first ATG codon of open reading frame A240L. The deduced amino acid sequence of this open reading frame shows significant similarity with the human, yeast and vaccinia virus thymidylate kinases, the degree of identity being 23·7, 25 and 23·5%, respectively. The putative African swine fever virus thymidylate kinase sequence is essentially collinear with the other thymidylate kinase sequences, but contains a carboxy-terminal extension of 37 amino acids rich in glutamic and aspartic acids. The A240L protein conserves the ATP-binding and nucleotide/nucleoside-binding domains characteristic of thymidylate kinases.

The size and genomic location of viral transcripts expressed in simian varicella virus (SVV)-infected Vero cells were determined. Total cellular RNA and polyadenylated RNA were isolated from SVV-infected and mock-infected Vero cells. Viral transcripts were detected by Northern blot hybridization analysis using overlapping SVV DNA probes representative of the entire SVV genome. The results indicated that all regions of the SVV genome are transcribed during SVV infection in vitro. At least 53 distinct viral RNA species ranging in size from 9·2 to 0·8 kb were detected. DNA probes derived from the SVV DNA long (L) and short (S) components hybridized to 44 RNAs (9·2 to 0·8 kb) and nine RNAs (4·9 to 0·8 kb), respectively. A transcript map of the SVV genome was constructed. The comparison made between the transcript maps of SVV and varicellazoster virus (VZV) provides further support that the SVV and VZV genomes have an analogous gene organization.

Following amplification by PCR of a portion of the matrix phosphoprotein pp150 gene, electrophoretic analysis revealed the simultaneous presence of two viral variants of human cytomegalovirus in the blood of a heart transplant recipient. Repeated denaturation-annealing cycles during the amplification reaction led to the formation of heteroduplex molecules with altered electrophoretic mobility. Sequence analysis of the amplification products showed the presence of a viral variant carrying an in-frame three nucleotide deletion, which caused the absence of an aspartic acid in the corresponding protein. Attempts to plaque-purify the deletion mutant were unsuccessful, suggesting that the variant was growth-defective.