Showing papers in "Journal of General Virology in 1993"

Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region

Abstract: Hepatitis C virus (HCV) showed substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68 to 79% overall sequence similarity to one another. Phylogenetic analysis of nucleotide sequences derived from part of the gene encoding a non-structural protein (NS-5) has provided evidence for six major genotypes of HCV amongst a worldwide collection of 76 samples from HCV-infected blood donors and patients with chronic hepatitis. Many of these HCV types comprised a number of more closely related subtypes, leading to a current total of 11 genetically distinct viral populations. Phylogenetic analysis of other regions of the viral genome produced relationships between published sequences equivalent to those found in NS-5, apart from the more highly conserved 5' non-coding region in which only the six major HCV types, but not subtypes, could be differentiated. A new nomenclature for HCV variants is proposed in this communication that reflects the two-tiered nature of sequence differences between different viral isolates. The scheme classifies all known HCV variants to date, and describes criteria that would enable new variants to be assigned within the classification as they are discovered.

Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay

Abstract: A reverse-hybridization assay, the line probe assay (LiPA), based on variations found in the 5′ untranslated regions of the different hepatitis C virus (HCV) genotypes was developed, permitting simple and fast determination of four HCV genotypes and their subtypes. Using this assay, 61 PCR-positive Brazilian HCV sera were typed. Of the sera, 33% had a type 1 HCV infection, 38% had type 1b (related to HCV-J), 1.5% had type 2a (related to HC-J6), 24.5% had type 3 (related to E-b1 and HCV-T), and 3% of the sera were co-infected. This assay format was further evaluated using 13 sera from Belgium and the Netherlands, and all of these could be classified. Two pools of Japanese sera were classified as either type 2a or were co-infected with types 1b and 2a, but no type 2b sequences were detected. Another eight PCR-positive sera were obtained from Burundi and Gabon. The sequence of the 5′ untranslated region of these African viruses was strongly divergent from the three previously described types. Therefore, these isolates were tentatively classified as type 4. These and some of the other non-type 1 sera often demonstrated weaker reactivities than type 1 isolates in currently used second generation antibody confirmation assays.

Sequence variability in the 5' non-coding region of hepatitis C virus: identification of a new virus type and restrictions on sequence diversity.

Abstract: We have analysed the pattern of nucleotide sequence variability in the 5' non-coding region (5' NCR) of geographically dispersed variants of hepatitis C virus (HCV). Phylogenetic analysis of sequences in this region indicated the existence of a new virus type, provisionally termed type 4, the identity of which was confirmed by further analysis of the more variable part of the HCV core protein coding region. The geographical distribution of HCV type 4 was distinct from that of other HCV types, it being particularly widespread in Africa and absent or rare in Europe and the Far East. Much of the variability in the 5' NCR appears to be constrained by a requirement for specific secondary structures in the viral RNA. In one of the most variable regions of the 5' NCR (positions -169 to -114), most of the nucleotide changes that are characteristic of different HCV types were covariant, with complementary substitutions at other positions. According to the proposed secondary structure of the 5' NCR, such changes preserved base pairing within a stem-loop structure, whereas the nucleotide insertions found in a proportion of 5' NCR sequences, including those of type 4, localized exclusively to the non-base-paired terminal loop. The specific nucleotide substitutions in the 5' NCR that differentiate each of the four HCV types can be detected by restriction enzyme cleavage, providing a rapid and reliable method for virus typing.

Characterization of the genomic sequence of type V (or 3a) hepatitis C virus isolates and PCR primers for specific detection

Abstract: We have identified four new hepatitis C virus (HCV) isolates whose genomic RNA could be amplified by PCR using primers from the 5′ untranslated region (UTR), but the RNA could not be detected with genotype I to IV (or types 1a, 1b, 2a and 2b respectively)-specific core region-derived primers. We compared the nucleotide sequences of the new isolates from positions 65 to 1850 (3′ end of 5′ UTR, C, E1 and 5′ end of E2/NS1) and 8276 to 9394 (3′ end of NS5 and 3′ UTR) with those for genotypes I to IV. The four isolates had the following characteristics: (i) the overall nucleotide sequence similarity between the four isolates was 95 to 96%, compared to 73 to 74%, 73%, 70% or 69 to 70% against genotypes I, II, III or IV, respectively; (ii) the sequence similarity to other reported ‘type V (3a)’ isolates was 88 to 100%; (iii) the hypervariable region 1 [(HVR)-1] was present but HVR-2 was absent within the E2/NS1 region; (iv) only one in-frame termination codon was present for the presumed polyprotein; (v) the 3′ UTR preceding a terminal poly(U) stretch was significantly shorter than in genotype I to IV isolates. We classified the four isolates as genotype V (3a), and searched for uniquely conserved nucleotide sequences that could be used for type-specific PCR. A core region-derived primer pair (no. 104V: 5′ CGTAAAACTTCT GAACGGTC, sense and no. 339: 5′ GCTGAGCCCA GGACCGGTCT, antisense) was identified and successfully used to diagnose genotype V (3a) HCV infection.

Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen

Abstract: A 681 nucleotide fragment of the hepatitis B virus (HBV) genome was sequenced that corresponded to the complete gene for hepatitis B surface antigen (HBsAg) in 80 HBsAg- and hepatitis B e antigen (HBeAg)-positive sera of diverse geographical origins. These and 42 previously published HBV sequences within the S gene were used for the construction of a dendrogram. In this comparison, each of the 122 HBsAg genes was found to be related to one or other of the six previously identified genomic groups of HBV, A to F. The HBV strains within each genomic group showed a characteristic geographical distribution. Group A genomes were represented by 23 strains mainly originating in northern Europe and sub-Saharan Africa. The group B and C genomes, represented by 17 and 28 strains respectively, were confined to populations with origins in eastern Asia and the Far East. The group D genomes, represented by 38 strains, were found worldwide, but were the predominant strains in the Mediterranean area, the Near and Middle East, and in south Asia. Group E genomes, represented by nine strains, were indigenous to western sub-Saharan Africa as far south as Angola. There were indications that the F group, made up of six strains, represented the genomic group of HBV among populations with origins in the New World. Thus, HBV has diverged into genomic groups according to the distribution of mankind in the different continents. As well as giving information on the genetic relationship of HBV strains of different geographical origin, this study also provides information on the primary structure of HBsAg in different regions of the world. Such data might prove valuable in explaining the reported failures to obtain protection with current HBV vaccines.

Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells

Abstract: The process of cell death caused by influenza virus infection in cultured MDCK and HeLa cells was analysed. This infection gave rise to nuclear fragmentation and chromatin condensation accompanied by chromosomal DNA fragmentation into oligonucleosomes. Chromosomal DNA fragmentation progressed concomitantly with cell lysis of MDCK cells and HeLa cells, producing high and low yields of virus particles, respectively, indicating that the extent of cell lysis was not proportional to the virus production. The endonuclease inhibitor zinc blocked DNA fragmentation in MDCK cells. Cycloheximide inhibited DNA fragmentation as well as cell lysis. Inhibition occurred when the drug was added to the medium within 2 h after infection but not efficiently at 4 h or later. Infection induced the Fas Ag gene, which encodes a possible apoptosis-mediating molecule, in the early infectious stage followed by the expression of Fas Ag on the cell surface. These results suggested that influenza virus infection causes apoptotic death of cultured cells, and their fate might be determined at an early stage of the infection by induction of an apoptotic gene.

Modification of discrete nuclear domains induced by herpes simplex virus type 1 immediate early gene 1 product (ICP0)

Abstract: The outcome of herpes simplex virus type 1 (HSV-1) infection depends upon the interplay of both host and viral factors. During lytic infection, HSV-1 causes a loss of immunofluorescent staining of discrete nuclear domains (ND10). This elimination of the host's ND10 staining occurs under conditions that allow only HSV-1 immediate early viral gene expression. Western blot analysis indicates that the loss of ND10 staining is due to ND10 redistribution, rather than protein degradation or turnover. When deletion mutants of all of the HSV-1 immediate early genes were tested, only infection with an immediate early gene 1 product (ICP0) deletion mutant, d11403, was unable to eliminate ND10 antigen staining. Also, ICP0 transiently colocalized with ND10 antigens, after which ND10 antigens became undetectable. At late times during infection with d11403, the host ND10 antigens were retained in virus-induced structures which were never observed during wild-type HSV-1 infection. These results suggested that ICP0 may be directly involved in the modification of the host nuclear domain. Infection with an adenovirus recombinant that expressed ICP0 demonstrated that in the absence of other HSV-1 proteins ICP0 was sufficient for the change in nuclear distribution of host antigens located at ND10. We postulate that the trans-activation function of ICP0 during viral replication may be mediated by replacing, modifying or reorganizing nuclear host factors.

RNA editing in Newcastle disease virus.

Abstract: The co-transcriptional editing of the Newcastle disease virus (NDV) P gene has been studied by sequence analysis of cloned viral genomic RNA and mRNA. Evidence has been obtained for the specific insertion of non-templated G nucleotides, the consequence of which is the generation of three populations of P gene-derived mRNAs. The three populations encode proteins (P, V and W) which have a common N-terminal region, but which utilize three different reading frames at their C termini. Paradoxically, NDV edits its P gene mRNA by the insertion of non-templated G residues in a manner similar to Sendai and measles viruses (P → V editing) despite its apparent closer evolutionary relationship to the simian virus type 5, mumps and related group of viruses which edit a V genomic sequence to generate an mRNA to encode a functional P protein (V → P editing).

Multiplication of tomato spotted wilt virus in its insect vector, Frankliniella occidentalis.

Abstract: The accumulation of two proteins, the nucleocapsid (N) protein and a non-structural (NSs) protein both encoded by the S RNA of tomato spotted wilt virus (TSWV), was followed in larvae during development and in adults of Frankliniella occidentalis after ingesting the virus for short periods on infected plants. The amounts of both proteins increased, as shown by ELISA and Western blot analysis, within 2 days above the levels ingested, indicating multiplication of TSWV in these insects. Accumulation of these proteins and of virus particles was further confirmed by in situ immunolabelling of the salivary glands and other tissues of adult thrips. The accumulation of large amounts of N and NSs protein, the occurrence of several vesicles with virus particles in the salivary glands and the massive numbers of virus particles in the salivary gland ducts demonstrate that the salivary glands are a major site of TSWV replication. The occurrence of virus particles in the salivary vesicles is indicative of the involvement of the Golgi apparatus in the maturation of the virus particles and its transport to the salivary ducts.

Peripheral blood leukocytes serve as a possible extrahepatic site for hepatitis C virus replication

Abstract: To study possible extrahepatic sites for the replication of hepatitis C virus (HCV), we examined fresh and cultured peripheral blood mononuclear leukocytes (PBML), as well as different subpopulations of PBML of HCV-infected patients, for the presence of viral genomic and antigenomic RNA. Sense and antisense oligonucleotide primers derived from HCV sequences were used for reverse transcription (RT) followed by an amplification with the polymerase chain reaction assay (PCR). Using antisense primers for RT, genomic viral RNA could be detected in serum, liver, total PBML and B lymphocytes of chronically infected patients. However, only liver tissue and PBML specimens were positive when a sense primer was used. To demonstrate further the specificity of these findings, total PBML were stimulated using pokeweed mitogen and synthesis of HCV RNA was determined by incorporation of [3H]uridine into nascent viral RNA molecules using a hybrid release assay. Additionally, total PBML from an uninfected person could be infected in vitro using an HCV RNA-positive serum. The PCR products obtained from serum, liver and PBML specimens of an HCV-positive individual were found to have nearly identical sequences. Our findings suggest that PBML could be a site for viral replication of HCV during the natural course of infection and may represent a reservoir for hepatitis C virions.

Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus.

Abstract: A sequence motif that is conserved in a number of S-adenosylmethionine (SAM)-utilizing methyltransferases and is implicated in SAM binding was identified in the N-terminal portion of NS5 proteins of flaviviruses and in lambda 2 protein of reovirus. An additional conserved motif was shared by these viral proteins and two distinct groups of methyltransferases including as the prototypes Rhodobacter capsulatus hydroxyneurosporene methylase (crtF gene product) and yeast 3,4-dihydroxy-5-hexaprenylbenzoate methylase (COQ3 gene product), respectively. Statistically significant similarity was revealed between the region of flavivirus NS5 containing the SAM-binding motif and a newly characterized family of putative methyltransferases from bacteria, yeast and plants, which is related to the Coq3 group. Amino acid sequence signatures were derived that are unique for NS5 proteins and different subsets of (putative) cellular methyltransferases. It is hypothesized that the N-terminal domain of NS5 is a methyltransferase involved in viral RNA capping. Thus NS5 may be a two-domain protein, with its C-terminal domain comprising the RNA-dependent RNA polymerase. The putative methyltransferase domain of flaviviruses is unrelated to the methyltransferase domain previously characterized in positive-strand RNA viruses of the alphavirus-like supergroup. The lack of sequence similarity and different location of the putative methyltransferase domain underscores the drastic difference in the genome layout of flaviviruses and alphaviruses. The identification of the putative methyltransferase domain in reovirus lambda 2 protein is compatible with the available evidence that this protein is the viral capping enzyme.

Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome.

Abstract: We have expressed the full-length coding region and selected domains of the hepatitis C virus (HCV) cDNA in mammalian cells by transfection. Using HCV antibody-positive human sera and monospecific antibodies the proteins encoded by the putative structural and non-structural regions of the open reading frame of HCV were identified as core (p22), E1 (gp32–35), E2 (gp68–72), NS2 (p23), NS3 (p72), NS4a and b (p10 and p27) and NS5a and b (p56 and p70). We have also defined the subcellular localizations of the HCV proteins using indirect immunofluorescence assays.

Nucleotide sequence and genome organization of tomato leaf curl geminivirus

Abstract: The genome of tomato leaf curl virus (TLCV) from Australia was cloned and its complete nucleotide sequence determined. It is a single circular ssDNA of 2766 nucleotides containing the consensus nonanucleotide sequence present in all geminiviruses. It has six open reading frames with an organization resembling that of certain other dicotyledonous plant-infecting monopartite geminiviruses, i.e. tomato yellow leaf curl and beet curly top viruses. The regulatory sequences present indicate a bidirectional mode of transcription. A dimeric TLCV DNA clone was constructed in a binary vector and used to agroinoculate three different host species. Typical virus infections were produced, confirming that the single DNA component is sufficient for infectivity.

Classification of tospoviruses based on phylogeny of nucleoprotein gene sequences

Abstract: The nucleotide sequences of the nucleoprotein (N) genes of seven tospovirus isolates representing three serogroups were determined and used to establish phylogenetic parameters to delineate species within the Tospovirus genus of the Bunyaviridae. A high sequence divergence (55.9% identity at the nucleotide level) was observed between isolates of serogroup I (tomato spotted wilt virus) and isolates of serogroup III (Impatiens necrotic spot virus). The serogroup II isolates take an intermediate position. Their N genes have 75% identity with those of serogroup I isolates and 57% with those of serogroup III isolates. Whereas the isolates within serogroups I or III have almost identical sequences, the two isolates BR-03 and SA-05 of serogroup II diverged significantly from each other (82·1% sequence identity). The results obtained support the conclusion that, in addition to the species TSWV and INSV, the serogroup II isolates BR-03 and SA-05 have to be considered as distinct species within the genus Tospovirus for which the names tomato chlorotic spot virus and groundnut ringspot virus, respectively, are proposed.

Nucleotide sequence evidence for the occurrence of three distinct whitefly-transmitted geminiviruses in cassava.

Abstract: The complete nucleotide sequence of the DNA of Indian cassava mosaic virus (ICMV) and a key part of that of a group B isolate of African cassava mosaic virus from Malawi (ACMV-M) were determined and compared at the nucleotide and encoded amino acid levels with the published sequences of an ACMV group A isolate (ACMV-K) and other whitefly-transmitted geminiviruses (WTGs). The DNA of ICMV consists of two circular single-stranded molecules, DNA-A [2815 nucleotides (nt)] and DNA-B (2645 nt), which differ substantially in sequence from the genome components of ACMV-K (DNA-A 70%, DNA-B 47% sequence identity) and other WTGs. ICMV DNA-A contains eight open reading frames (ORFs) encoding proteins of > 100 amino acid residues, of which four ORFs (one genome sense, three complementary sense) are comparable to those of other WTGs. DNA-B contains one ORF in each sense, as in other WTGs. None of the putative viral proteins are more similar in amino acid sequence to the proteins of ACMV-K than to those of another WTG. The coat protein of ACMV-M is more like that of tomato yellow leaf curl virus from Sardinia (86% sequence identity) than those of ICMV or ACMV-K. The intergenic regions of ACMV-K, ACMV-M and ICMV DNAs differ in size, and largely in sequence, except for two 30 to 40 nt sequences which are also conserved in other WTGs and can form stem—loop structures. The intergenic region of ICMV DNA contains three copies of a 41 nt sequence, and that of ACMV-M DNA contains an imperfect repeat of a 34 nt sequence which resembles the repeated sequence in ICMV DNA. The differences between ACMV-K, ACMV-M and ICMV are considered great enough to justify their separation as isolates of three distinct WTGs: African cassava mosaic virus, East African cassava mosaic virus and Indian cassava mosaic virus.

Identification of a fifth neutralizable site on type O foot-and-mouth disease virus following characterization of single and quintuple monoclonal antibody escape mutants

Abstract: 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.

Tumour necrosis factor alpha stimulates the activity of the human cytomegalovirus major immediate early enhancer/promoter in immature monocytic cells.

Abstract: Both tumour necrosis factor α (TNF-α) and phorbol 12-myristate 13-acetate (PMA) stimulated human cytomegalovirus (HCMV) major immediate early (IE) enhancer/promoter activity in the HL-60 granulocyte/monocyte progenitor cell line when added to transfected cells. In U-937 monocytic cells, by contrast, TNF-α had no stimulatory effect and the addition of PMA produced only marginal stimulation. In the mature THP-1 monocytic cell line and in differentiated HL-60 cells, addition of TNF-α caused inhibition of the IE enhancer/promoter activity. The stimulating effect of PMA, as observed in the other cell lines, however, remained. Thus the effect of TNF-α on the major IE enhancer/promoter activity is determined by the degree of differentiation of the infected cells. Unlike TNF-α and PMA, the interleukins IL-1, IL-3, IL-6 as well as the cytokine GM-CSF were found to have no detectable influence on the activity of the IE enhancer/promoter activity which, likewise, was not affected by the presence of the modulator sequence. Since premonocytic cells are suggested to be sites of HCMV latency, the stimulation by TNF-α could be of potential pathophysiological significance.

An analysis of the complete sequence of a sugarcane bacilliform virus genome infectious to banana and rice.

Abstract: The genome of sugarcane bacilliform virus (ScBV), a badnavirus, consists of a circular dsDNA. The complete sequence of a cloned infective ScBV genome is reported here. The genome is 7568 bp in size and possesses a number of features suggesting that ScBV is a pararetrovirus. A tRNA(Met)-binding site that may serve as a primer for minus-strand synthesis is present. The plus-strand of the ScBV genome contains three open reading frames (ORFs) which are capable of encoding proteins with calculated M(r) values of 22K, 13K and 215K. The 215K protein has regions with similarity to the RNA-binding domains, aspartic proteases and replicases of retro-elements. In addition, the 215K protein also has a region with restricted similarity to the intercellular transport proteins of plant viruses. Comparisons with the other sequenced badnaviruses, Commelina yellow mottle (CoYMV) and rice tungro bacilliform (RTBV) viruses, indicate that the arrangement of the ORFs in these viruses is conserved. Located next to the putative RNA-binding domain is a cysteine-rich region that is unique to the badnaviruses. When the molecular relationships of a portion of the reverse transcriptases of plant pararetroviruses were determined, two badnaviruses, CoYMV and ScBV, form one distinct cluster, whereas three caulimoviruses, cauliflower mosaic virus, carnation etched ring virus and figwort mosaic virus, form a second cluster. The badnavirus RTBV and the caulimovirus soybean chlorotic mottle virus occupy intermediate positions between the clusters. When introduced by Agrobacterium-mediated inoculation, a construct containing 1.1 copies of the cloned ScBV genome is infectious to both rice and banana.

Antibody response to the M2 protein of influenza A virus expressed in insect cells

Abstract: A recombinant baculovirus expressing the M2 protein from influenza A/Ann Arbor/6/60 (H2N2) virus (AA60 virus) was constructed. The expressed M2 protein was recognized by a monoclonal antibody specific for the M2 protein and comigrated with the M2 protein from cells infected with AA60 virus on SDS-polyacrylamide gels. Immunofluorescence studies indicated that the expressed M2 protein was present on the surface of Spodoptera frugiperda (Sf9) cells infected with the recombinant baculovirus. Immunoassays using the expressed M2 protein were able to detect antibodies to the M2 protein in serum samples from humans and ferrets infected with influenza A viruses.

Subgenomic RNAs of Lelystad virus contain a conserved leader-body junction sequence.

Abstract: During the replication of Lelystad virus in alveolar lung macrophages, a 3′-coterminal nested set of six subgenomic RNAs (RNA2 to RNA7) is formed. These contain a common leader sequence derived from the 5′ non-coding region of the genomic RNA. In this study, the sequence of the junction sites, i.e. the sites where the leader sequence joins to the body of the subgenomic RNA, was determined for all six subgenomic RNAs. For each subgenomic RNA, six to nine cDNA clones were isolated by means of reverse transcription and PCR. The nucleotide sequence at the junction site was identical for all eight cDNA clones derived from subgenomic RNA4. However, heterogeneity was observed in the nucleotide sequence surrounding the junction sites of the cDNA clones derived from subgenomic RNAs 2, 3, 5, 6 and 7. This heterogeneity suggests that the fusion of the leader to the body of the subgenomic RNA may be imprecise. The junction sites of the six subgenomic RNAs had a conserved sequence motif of six nucleotides (UCAACC or a highly similar sequence). The distance between the junction site and the translation initiation codon of the downstream open reading frame varied from nine to 83 nucleotides.

Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid

Abstract: We have produced a plasmid expression vector for the coat protein of RNA bacteriophage MS2. The vector has been modified to introduce a unique KpnI restriction site within the coat protein gene at a site corresponding to the most radially distant feature of the bacteriophage capsid, namely the top of the N-terminal β-hairpin (between residues 15 and 16). Insertion of DNA oligonucleotides at this site allows the production of chimeric MS2 coat proteins having foreign peptide sequences expressed as the central part of the hairpin. We have produced chimeras with a number of different peptide sequences (up to 24 amino acids in length) chosen because of their known antigenic properties. The chimeric coat proteins self-assemble into largely RNA-free phage-like capsids in Escherichia coli and can be easily disassembled and reassembled in vitro. Such peptide-presenting particles may have a number of biotechnological applications, including use as a cost-effective, synthetic vaccine. We have tested the anti-genicity of one such construct in vivo in mice and have shown that these particles are immunogenic and that antibody titres against the inserted peptide epitope can be obtained.

Human polyomavirus JC promoter/enhancer rearrangement patterns from progressive multifocal leukoencephalopathy brain are unique derivatives of a single archetypal structure.

Abstract: 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

New observations on antigenic diversification of RNA viruses. Antigenic variation is not dependent on immune selection.

Abstract: Summary Recent results have revealed novel features in the process of antigenic diversification of FMDV. (i) Antigenic variation is not necessarily the result of immune selection. (ii) Single, critical amino acid replacements may either have a minor effect on antigenic specificity or cause a drastic antigenic change affecting many epitopes on an antigenic site. (iii) The effect of such a critical replacement may be suppressed by additional substitutions at neighbouring sites. (iv) Antigenic diversification does not necessarily involve net accumulation of amino acid substitutions over time. We review evidence that some of these features apply also to other riboviruses and retroviruses. A model is proposed to relate antigenic variation without immune selection to the quasispecies structure of RNA virus populations.

Identification of a new hepatitis B virus (HBV) genotype from Brazil that expresses HBV surface antigen subtype adw4

Abstract: 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.

The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation

Abstract: The 86K immediate early (IE) 2 protein of human cytomegalovirus trans-activates a number of homologous and heterologous promoters, including the cellular promoter for the 70K heat-shock protein (hsp 70), and the human immunodeficiency virus long terminal repeat. We have previously shown that IE2 trans-activates these two promoters in a TATA-dependent manner, and that IE2 is able to form a direct contact with TATA-box binding protein (TBP) in vitro. We now show that IE2 binds to the basic repeat region of TBP. In addition IE2 can contact a second general transcription factor, TFIIB. We have mapped the TBP- and TFIIB-binding regions within IE2 and show that these regions overlap, and also lie within parts of the protein previously identified as being required for the trans-activation and autoregulation functions of IE2.

The involvement of a spliceosome component in internal initiation of human rhinovirus RNA translation

Abstract: Human rhinoviruses (HRVs) and encephalomyocarditis virus (EMCV) belong to different genera of the picornavirus family, but the translation of the RNAs of both viruses is by the same mechanism, that is, internal ribosome entry. In rabbit reticulocyte lysates this translation initiation is efficient for mRNAs bearing the EMCV 5' untranslated region (5' UTR), but very inefficient for mRNAs bearing the HRV 5' UTR, unless factors from HeLa cells are added. The copurification of the HeLa cell translation stimulatory activity with proteins which can be specifically cross-linked to the HRV 5' UTR by u.v. irradiation has been examined. Both the EMCV and HRV 5' UTRs can be cross-linked to a 58/60K protein doublet present in HeLa cell extracts in higher amounts than in reticulocyte lysates, which is shown to be very similar, if not identical to the polypyrimidine tract binding protein (PTB) previously identified as a component of a multi-subunit complex necessary for pre-mRNA splicing. However, the activity in HeLa cell extracts that specifically stimulates translation initiation on mRNAs with the HRV 5' UTR does not copurify with the majority of the 58/60K protein present in these extracts, but copurifies with a minor fraction of these proteins and with a 97K protein which can be cross-linked to the HRV 5' UTR but not to the EMCV 5' UTR, and which is absent from reticulocyte lysates. It is proposed that the specific translation initiation stimulatory activity found in HeLa cells is due to a high M(r) complex containing the 97K polypeptide and PTB.

Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene

Abstract: One DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component-predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.

Synthesis and assembly of infectious bovine papillomavirus particles in vitro.

Abstract: Bovine papillomavirus type 1 (BPV-1) virions were produced in vitro using vaccinia virus (VV) recombinants expressing the BPV-1 L1 and L2 capsid proteins. Particles morphologically resembling papillomaviruses were observed in the nucleus of cells infected with a VV recombinant for the BPV-1 L1 protein, and greater numbers of similar particles were seen in the nuclei of cells infected with a VV double recombinant for L1 and L2. Virus-like particles (VLPs) assembled in cells infected with the VV double recombinant for BPV-1 L1 and L2, and not those assembled in cells infected with the VV recombinant for BPV-1 L1 alone, were able to package BPV-1 DNA. Transcription of the BPV-1 E1 viral open reading frame was observed after a mouse fibroblast cell line was exposed to VLPs produced using a BPV-1 L1/L2 VV recombinant in a cell line containing episomal BPV-1 DNA. E1 transcription was not observed when the VLPs were pre-incubated with antibodies to the capsid protein of BPV-1. This system should allow an in vitro approach to the definition of the BPV-1 cellular receptor.

Prophylactic and therapeutic vaccination against a mucosal papillomavirus

Abstract: Papillomaviruses are ubiquitous DNA viruses affecting humans and animals and causing a variety of tumours of mucosal and cutaneous epithelia. Some of these lesions, particularly those affecting mucosal epithelia, can progress to squamous cell carcinomas. Prevention or cure of viral infection would ultimately lead to a decrease in the incidence of papillomavirus-associated cancers. Using recombinant proteins, we have developed prophylactic and therapeutic vaccines against bovine papillomavirus type 4, a mucosal papillomavirus implicated in cancer of the alimentary canal in cattle; similar possibilities exist for the human mucosal papillomaviruses.

Swaledale sheep affected by natural scrapie differ significantly in PrP genotype frequencies from healthy sheep and those selected for reduced incidence of scrapie

Abstract: PrP glycoprotein gene polymorphisms were examined in Swaledale sheep affected by natural scrapie, in healthy sheep and in Swaledales selected for low susceptibility to scrapie. The three groups differed significantly in frequencies of PrP genotypes detected by the restriction enzymes EcoRI, HindIII and BspHI, the latter being indicative of a PrP protein amino acid difference at codon 136. These frequency differences were confirmed in a single-flock study and present good evidence that scrapie susceptibility and resistance are associated with PrP gene variants in Swaledale sheep.