- Volume 79, Issue 11, 1998
Volume 79, Issue 11, 1998
- Articles
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Recombinant Ebola virus nucleoprotein and glycoprotein (Gabon 94 strain) provide new tools for the detection of human infections
After cloning and sequencing the glycoprotein (GP) gene of one of the Gabonese strains of Ebola virus isolated during the 1994–1996 outbreak, it was shown that the circulating virus was of the Zaire subtype. This was confirmed in this study by cloning and sequencing the nucleoprotein (NP) gene of this strain. These two structural proteins were also expressed as recombinant proteins and used in ELISA tests. NP was expressed as a His-tagged fusion protein in Escherichia coliand was purified on resins charged with nickel ions. GP was expressed by means of recombinant baculoviruses in Spodoptera frugiperda cells. Both recombinant proteins reacted positively in ELISAs for the detection of IgG antibodies in convalescent human sera from Gabon and Zaire. The difference in the relative titres of anti-NP and -GP antibodies was variable, depending on the sera. In addition, the recombinant NP reacted with heterologous sera from Cĉte d’Ivoire and was used successfully to detect IgM antibodies by μ-capture ELISA in sera from Gabonese patients.
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Location of neutralizing epitopes on the G protein of bovine ephemeral fever rhabdovirus
More LessThe surface glycoprotein G is the major neutralizing and protective antigen of bovine ephemeral fever rhabdovirus (BEFV). Twelve neutralizing MAbs against BEFV strain BB7721 were used to select 33 neutralization escape mutants. The mutants had been classified previously into three major antigenic sites (G1-G3) based on their cross-neutralization patterns. The nucleotide sequence of the entire extracellular domain of the G protein gene was determined for all mutants. Each contained a single nucleotide change leading to a single amino acid substitution. The 16 mutants assigned to the linear antigenic site G1 mapped to aa 487-503 of the 623 aa G protein. Results of antibody binding to several overlapping octapeptides covering this region mapped the sequence of two common minimal B cell epitopes recognized by the five G1 MAbs to (488)EEDE(491) and (499)NPHE(502). Site G2 mutations mapped either at aa 169 or 187. The 12 mutants representing antigenic site G3 (G3a and G3b) mapped to aa 49, 57, 218, 229 and 265, indicating that this site is likely to combine complex discontinuous epitopes. Comparison of the deduced amino acid sequence from five BEFV field isolates and BB7721 identified aa 218 to be critical for the site G3a neutralization. Alignment of the glycoproteins of rabies virus, vesicular stomatitis Indiana virus, vesicular stomatitis New Jersey virus, infectious haematopoietic necrosis virus and BEFV revealed similarities in the location of the neutralizing epitopes and extensive conservation of cysteine residues, suggesting that basic elements of the folded structure of these glycoproteins are preserved.
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Recognition of measles virus-infected cells by CD8+ T cells depends on the H-2 molecule
More LessH-2d mice are resistant to measles virus-induced encephalitis (MVE) and develop Ld-restricted CD8 T cells which lyse target cells infected with measles virus or with a vaccinia virus recombinant expressing the nucleocapsid protein of measles virus (vvN). In contrast, H-2k mice are susceptible to MVE and generate CD8 T cells which lyse target cells infected with vvN, but not those infected with MV. We were able to demonstrate that this difference is not due to a defect in the antigen processing machinery, but that Kk molecules require 100-fold more peptide to sensitize target cells for lysis by CTL. vvN replicates well in target cells and therefore enhances the level of epitope peptide available for CTL recognition. In contrast, MV infection is abortive in mouse cells and low levels of epitope peptide are produced. As Ld requires 100-fold less peptide than Kk to sensitize target cells for lysis, the low level of epitope peptide is enough to induce lysis by CD8 T cells, whereas for recognition via Kk, increased synthesis of protein is required. We propose that the differences in peptide binding between the two H-2 molecules will have consequences for the kinetics of the generation of CD8 T cells as well as the absolute numbers of CD8 T cells generated.
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Intracellular IFN-gamma expression in natural killer cells precedes lung CD8+ T cell recruitment during respiratory syncytial virus infection
More LessNatural killer (NK) cells are recruited locally during the initial phases of virus infection and produce cytokines which may affect the subsequent emergence of specific T cells. In this study, cellular responses to primary respiratory syncytial virus (RSV) infection and after vaccination with individual viral proteins were investigated in BALB/c mice using the new NK cell antibody, DX5. Purified DX5 cells caused lysis of YAC-1 cell targets. DX5 cells did not express CD8, CD45R or MHC class II antigens. A small proportion of DX5 cells coexpressed CD4 (10·3%) and CD3 (10·6%). Of the DX5 /CD4 cells, the majority expressed the α/β T cell receptor and less than 1 % expressed the γ/δ T cell receptor. During infection with RSV, lung DX5 /CD3 NK cells peaked on day 4 of primary infection and were the most numerous subset producing IFN-γ, as determined by intracellular staining, at this time-point. Less than 1% of the DX5 cells secreting IFN-γ were CD4 . In the lungs of mice vaccinated with recombinant vaccinia virus expressing individual RSV proteins, increased NK cell cytotoxicity and IFN-γ production correlated with increased numbers of CD8 T cells. Mice with few NK cells subsequently had low CD8 T cells and developed lung eosinophilia. IFN-γ-producing NK cells therefore form a substantial component of the early cellular response to virus infection with important potential influences on the subsequent development of specific immunity.
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Isolation and characterization of Puumala hantavirus from Norway: evidence for a distinct phylogenetic sublineage
Puumala (PUU) hantavirus is the aetiological agent of nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome, which occurs in Fennoscandia, central Europe and Russia. In Norway, NE-like disease has been reported since 1946 and about 50 cases are diagnosed annually; however, the causative agent has not been characterized. In this study, a virus originating from bank voles (Clethrionomys glareolus) trapped near the town of Eidsvoll (Akershus county) was isolated and passaged in laboratory-bred bank voles. The bank vole strain was identified as a PUU virus by serological typing and by sequence analysis of the S and M gene segments. For comparison, complete or partial S sequences were determined for wild-type PUU strains from five locations in Sweden, two inhabited by the southern variant of bank vole present in Fennoscandia, and three by the northern variant. Phylogenetic analysis showed that Norwegian PUU strains are clustered together with Swedish strains from the first group forming a well- supported sublineage within the PUU genotype, distinct from other sublineages from northern Sweden, Finland, Russia and France. The results are consistent with the view of a complex evolutionary history of PUU strains in post-glacial Fennoscandia. Analyses of the current collection of nucleotide sequences suggest that PUU is the most variable genotype of the known hantaviruses.
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Completion of the Tula hantavirus genome sequence: properties of the L segment and heterogeneity found in the 3' termini of S and L genome RNAs
More LessIn this study the L segment and the 5′ and 3′ termini of the S, M and L segments of the prototype Tula hantavirus (TUL) were sequenced, thus completing the first determination of the genome sequence of a hantavirus that has not been linked to any human disease. The TUL L segment comprises 6541 nt with one ORF of 6459 nt in the antigenome sense. This ORF potentially encodes a 2153 aa protein with a predicted molecular mass of 247 kDa. The amino acid sequence includes all the motifs conserved in RNA-dependent RNA polymerases. The 5′ termini of all three genome RNAs (vRNAs) had the expected sequences conserved in hantaviruses. The 3′ termini of M vRNAs were also conserved. However, the 3′ termini of S and L vRNAs were heterogeneous as most of the sequenced 3′ termini had either deletions of 1 to 22 nt or an extra 1 to 3 nt. No increase in the level of heterogeneity was seen in vRNAs of virions collected 3, 6, 9 and 12 days postinfection, suggesting that the heterogeneity already exists at the early stages of infection. The S and L vRNAs from infected cells had more truncated 3′ termini than vRNAs from pelleted virus. Heterogeneity of the 3′ termini of genome RNAs could decrease the efficiency of antigenome and mRNA syntheses and contribute to the slow growth observed for TUL and other hantaviruses in cell culture.
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Genetic evolution of hepatitis G virus in chronically infected individual patients
Comparative sequence analysis of different isolates of hepatitis G virus (HGV) has demonstrated significant intersubject genetic heterogeneity, but few data on intrasubject genetic evolution have been reported. To further investigate the genetic diversification of the HGV genome, 36 plasma samples from eleven patients chronically infected with HGV serially obtained 2–4 years apart were analysed. We determined the viral nucleotide sequence of the 5′ non-coding (NC) and the NS3 regions by directly sequencing the RT-PCR amplified products obtained from the viral RNAs. Intrasubject sequence variation was found to be 1·3–2·4 × 10 3 base substitutions per genome site per year within the 5′ NC region and 1·3–9·4 × 10 3 base substitutions per genome site per year within the NS3 region. Depending on the genomic region analysed (i.e. 5′ NC or NS3 region), pairwise comparisons and phylogenetic reconstructions showed that intersubject genetic distances were 17·5- to 20·8-fold greater than intrasubject ones. Overall, the evolution rate of HGV in the regions analysed is not significantly different from that found in hepatitis C virus.
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Growth restriction of dengue virus type 2 by site-specific mutagenesis of virus-encoded glycoproteins
More LessThe three flavivirus glycoproteins prM, E and NS1 are formed by post-translational cleavage and are glycosylated by the addition of N-linked glycans. NS1 may form homodimers, whereas E may form homodimers, homotrimers or heterodimers (prM-E). Modification of these processes by mutagenesis of the proteins has the potential to generate viruses that are restricted in growth and are possible vaccine candidates. Using an SV40-based expression system, we previously analysed dimerization and secretion of the NS1 protein of dengue virus type 2 (DEN-2) with mutations in the conserved Cys residues, or within hydrophilic or hydrophobic regions, or at glycosylation sites. In this study, mutations which reduce cleavage at the DEN-2 prM/E signalase cleavage site are described. On the basis of earlier and current results with transient expression, six mutations which reduced NS1 dimerization and two mutations which inhibited prM/E cleavage were analysed individually for their effects on virus growth using a genomic length cDNA clone. Two viruses were obtained that showed reduced growth in cell culture and attenuation of neurovirulence when inoculated into 3-day-old mice. One of these viruses encoded NS1 that lacked the second glycosylation site, the other encoded a Ser → Ile change at the -3 position of the prM/E cleavage site. A third virus encoding a mutation in NS1 within a hydrophilic region grew as well as the parental virus. No virus was detected for the remaining five mutations.
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Molecular analysis of human parechovirus type 2 (formerly echovirus 23)
More LessPicornaviruses have been divided into five genera until recently, when a sixth genus, Parechovirus, was defined. Human parechovirus type 1 (HPeV1; formerly echovirus 22) was the first recognized member of this genus and preliminary sequence analysis of echovirus 23 [now renamed human parechovirus type 2 (HPeV2)] suggested that it is also a parechovirus. Here we describe the complete nucleotide and predicted amino acid sequences of HPeV2, which indicate a close relationship to HPeV1 throughout the genome. Sequence covariance in the 5′ untranslated region allows a prediction of the secondary structure, which indicates that these parechoviruses have a type 2 internal ribosome entry site, most closely related to that of cardio-viruses. Overall, HPeV2 has 87·9% amino acid identity with HPeV1, most divergence being seen in regions of the capsid proteins that probably define antigenic sites. The N-terminal sequence extension to VP3, seen only in parechoviruses, is highly basic in both viruses, but has a variable sequence, suggesting that it does not have a sequence-specific role. There is an RGD motif near the C terminus of VP1, in an analogous location to that in HPeV1 which is believed to be functionally significant. The results confirm that both viruses are parechoviruses and give insights into the molecular features of this genus.
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Co-expression of human eIF-4G and poliovirus 2Apro in Saccharomyces cerevisiae: effects on gene expression
More LessThe poliovirus 5′ untranslated region (5′ UTR) confers on mRNAs the capacity to be translated by internal initiation. The functionality of this RNA motif has been tested in yeast cells (Saccharomyces cerevisiae) using luciferase (luc) as a reporter gene. Although some luciferase is synthesized from luc mRNA containing the poliovirus 5′ UTR (Leader-luc mRNA), much more luciferase is synthesized in cells that express luc mRNA devoid of the poliovirus 5′ UTR. Since poliovirus 2Apro enhances the translation of Leader-luc mRNAs after eIF-4G cleavage in mammalian cells, yeast cells were produced that synthesize three heterologous proteins, luciferase, poliovirus 2Apro and human eIF-4G. Initially, S. cerevisiae cells constitutively expressing human eIF-4G were isolated. The human eIF-4G gene does not complement yeast cells defective in the initiation factor counterpart, p150, indicating that the human and yeast eIF-4G are not interchangeable. Expression of poliovirus 2Apro in an inducible manner does not affect p150, but led to the efficient cleavage of human eIF-4G in yeast cells. Induction of 2Apro was detrimental to luciferase synthesis either from luc mRNA or Leader-luc mRNA irrespective of the presence or absence of human eIF-4G. 2Apro blocked luciferase expression at the transcriptional level. Finally, the effects of 16 point mutations of poliovirus 2Apro on luciferase expression and human eIF-4G cleavage were analysed. Only those 2Apro variants that generate viable polioviruses actively cleave eIF-4G in yeast.
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Serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease
T. L. To, L. A. Ward, L. Yuan and L. J. SaifWe examined the antibody responses and protection to a human rotavirus (HRV) in gnotobiotic (Gn) pigs. Pigs were perorally (PO) inoculated with attenuated (group 1), virulent (group 2), or inactivated (group 3) Wa (P1A[8]G1) HRV. Afourth group was inoculated intramuscularly (IM) with inactivated Wa HRV in adjuvant. After PO challenge with virulent Wa HRV at post-inoculation day 21, most group 1,3 and 4 pigs shed virus and developed diarrhoea, whereas this occurred in only a few group 2 pigs. Antibodies to HRV (IgM, IgA or IgG) were detected in serum and intestinal contents of pigs of all groups after virus inoculation or challenge, and the antibody titres in intestinal contents, although lower, showed similar kinetics to the serum responses. There was no correlation between protection and neutralizing antibody titres of serum or intestinal contents, but a positive correlation existed between protection and serum IgA, intestinal IgA and intestinal IgG antibody titres. These findings suggest that serum IgA antibodies to HRV could act as an indicator of IgA antibodies in the intestine after rotavirus infection. The virulent HRV elicited protective immunity and higher levels of serum and intestinal IgA antibodies to HRV compared to attenuated and inactivated HRV. These findings suggest that more efficient mucosal delivery systems and/or adjuvants are needed to enhance the intestinal immune responses to attenuated or inactivated HRV if more successful vaccination is to be achieved in neonates.
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A new cysteine in rotavirus VP4 participates in the formation of an alternate disulfide bond
More LessMost animal rotaviruses bind to a cell surface molecule that contains sialic acid (SA). We have recently isolated variants from simian rotavirus RRV which show an SA-independent phenotype. The VP4 protein of these variants was shown to have three amino acid changes with respect to the wt protein, one of them being Tyr-267 → Cys. In this work, we have investigated whether the new cysteine could interfere with the disulfide bond (Cys-318/Cys-380) present in the VP5 * subunit of the wt protein. Cysteine residues 318 and 380 were mutagenized in gpr8 and RRV VP4 genes, and the wt and mutant genes were transcribed and translated in vitro. The protein products were analysed by electrophoresis under reducing and non-reducing conditions. This approach showed that, in the VP4 protein synthesized in vitro, Cys- 267 is capable of forming an alternate disulfide bond with Cys-318. This alternate bond also seems to occur in the VP4 protein present in the variant gpr8 virus particles.
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Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2
More LessWe have previously shown that a number of isoforms of the non-structural rotavirus protein NSP5 are found in virus-infected cells. These isoforms differ in their level of phosphorylation which, at least in part, appears to occur through autophosphorylation. NSP5 co-localizes with another non-structural protein, NSP2, in the viroplasms of infected cells where virus replication takes place. We now show that NSP5 can be chemically cross-linked in living cells with the viral polymerase VP1 and NSP2. Interaction of NSP5 with NSP2 was also demonstrated by co- immunoprecipitation of NSP2 and NSP5 from extracts of UV-treated rotavirus-infected cells. In addition, in transient transfection assays, NSP5 phosphorylation in vivo was enhanced by coexpression of NSP2. An NSP5 C-terminal domain deletion mutant, was completely unable to be phosphorylated either in the presence or absence of NSP2. However, a 33 aa N-terminal deletion mutant of NSP5 was shown to become hyperphosphoryl- ated in vivo and to be insensitive to NSP2 activation, suggesting a regulatory role for this domain in NSP5 phosphorylation and making it a candidate for the interaction with NSP2. These mutants also allow a preliminary mapping of NSP5 autophosphorylation activity.
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Novel retroviral sequences are expressed in the epididymis and uterus of Syrian hamsters
Combined androgen and oestrogen treatment of male or female Syrian hamsters results via an unknown mechanism in the formation of leiomyosarcomas in the reproductive tract. We have examined the possibility that retroviral gene expression may play a role in tumorigenesis. Evidence of virus-like particles in epididymis and seminal fluid is shown in electron micrographs. We identified expressed retroviral sequences by using RT-PCRto amplify a conserved retroviral reverse transcriptase coding region in RNA isolated from epididymis, testis, clarified seminal fluid and uterus. Phylogenetic analysis allowed us to classify the sequences into two distinct groups: (1) mammalian type-C viruses, having similarity to Moloney murine leu kaemia virus, feline leukaemia virus and gibbon ape leukaemia virus amongst others; (2) a mixed ABCD group containing, for example, Chinese hamster and murine intracisternal A-particle virus sequences, mouse mammary tumour virus and human and simian retroviral sequences. The presence of putative full-length retrovirus related to mammalian type-C viruses in the epididymis and uterus was confirmed by Northern blot analysis. However, steroid treatment did not alter retroviral RNA levels in the epididymis or in a uterine tumour relative to untreated uterus. In summary, Syrian hamster reproductive tissues were found to express unique retroviral sequences; however, their role, if any, in hormonal carcinogenesis remains unresolved.
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Evidence for a post-Columbian introduction of human T-cell lymphotropic virus [type I] [corrected] in Latin America
To investigate the origin and dissemination of human T-cell lymphotropic virus type I in Latin America, we performed phylogenetic analysis on the LTR and env sequences of 13 HTLV-I isolates from Peruvians of four different ethnic groups: blacks and some mulattos of African origin; Quechuas of Inca origin; Nikkei of Japanese descendance; and Mestizos, a mixed population of white and Indian origin. All Peruvian samples could be situated within the cosmopolitan subtype HTLV-Ia, yet one sample showed an indeterminate Western blot pattern, lacking reactivity towards the HTLV-I type specific MTA1 peptide. Within the LTR, we could confirm the previously reported subdivision into four subgroups - one big transcontinental clade A, a Japanese clade B, a West African/Caribbean clade C and a North African clade D - and we identified a new separate subgroup E of black Peruvian strains. The clustering of the Peruvian samples seemed to depend on the ethnic origin of the host. The largest heterogeneity was observed in the black Peruvian samples. The mitochondrial DNA type of one of these black Peruvian strains of subgroup E was identical to that of West African source populations of the slave trade. Both findings support the idea of multiple post-Columbian introductions of African HTLV-Ia strains into the black Latin American population. Additionally, a tight cluster of Nikkei and Japanese samples implied a separate and rather recent transmission of a Japanese lineage of HTLV-I into Peru. A well-supported cluster of Latin American strains (including Peruvian Quechuas and Colombian Amerindians) could be situated within the transcontinental group. Molecular clock analysis of the Latin American and Japanese clade resulted in an equal evolutionary rate for those strains. Along with the anthropologically documented peopling of the Americas, the analysis was more in favour of a recent (400 to 100 years ago) introduction of HTLV- Ia into the American continent rather than a Palaeolithic introduction.
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The neutralizing antibody response against a conserved region of human immunodeficiency virus type 1 gp41 (amino acid residues 731-752) is uniquely directed against a conformational epitope
Amino acids 731–752 (731PRGPDRPEGIEEEGGERD- RDRS752) of the transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1)are conserved in most virus isolates and are controversially reported to be implicated in virus neutralization. The humoral response in infected patients against this region is poor and humans immunized with gp160 show high levels of antibodies against the peptide but poor neutralization titres. Nonetheless, several groups have succeeded in obtaining neutralizing antibodies against this sequence using different antigen-presenting systems. In order to identify the sequence(s) against which the neutralizing response was generated, we used the flock house virus (FHV) antigen-presenting system to analyse neutralizing antisera from mice immunized with a cowpea mosaic virus (CPMV) chimera expressing the 731–752 sequence. Data show that the neutralizing response is uniquely directed against a conformational epitope mapping to the ERDRD portion of this sequence, although the major antibody response, which is non-linear, and is not neutralizing, is against an IEEE epitope. These results provide an explanation for the controversy regarding the immunogenicity of this region of gp41 and suggest that this conformational epitope, in the absence of the non-neutralizing epitope, should be considered for a subunit vaccine. In addition, this study highlights the usefulness of antigen- presenting systems that preserve epitope conformation in the investigation of immune responses.
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Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR down-modulation
More LessThe Nef protein of simian immunodeficiency virus (SIV) is dispensable for replication in established T- cell lines but essential for high level virus replication in the adult host, though the mechanism by which Nef contributes to this has remained unclear. We demonstrate here that SIV Nef binds to the ζ chain of the T-cell receptor (TCR). SIV Nef proteins that interact with TCR ζ in a yeast two-hybrid system also reduce T-cell surface expression levels of TCR αβ, CD3 and CD4. These findings are the first demonstration that Nef can bind directly to a component of the TCR-CD3 complex and modulate its surface expression.
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Feline CD8+ T cell non-cytolytic anti-feline immunodeficiency virus activity mediated by a soluble factor(s)
More LessFeline immunodeficiency virus (FIV) is more readily isolated from CD8 T cell-depleted peripheral blood mononuclear cells (PBMC) of FIV-infected cats than from unfractionated PBMC cultures. However, it is not known whether feline CD8 T cells down- regulate FIV expression by direct interaction with FIV-infected cells or via a soluble mediator. Furthermore, it is not known whether this anti-FIV activity involves a lytic or non-lytic mechanism. In the present study, we demonstrated that autologous and allogeneic CD8 T cells from asympto-matic FIV-infected cats inhibited the replication of FIV in CD8 T cell-depleted PBMC cultures in a dose-dependent manner. The inhibitory effect was mediated by a non-lytic mechanism, and was not dependent on direct cell-to-cell contact: an inhibitory effect was exerted by CD8 T cells across a semi-permeable membrane, and an inhibitory activity was also present in cell-free supernatants from CD8 T cells. These results suggest that this suppressive effect is mediated, at least in part, by soluble factors produced by CD8 T cells.
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Erk-independent partial activation of AP-1 sites by the hepatitis B virus HBx protein
More LessThe hepatitis B virus × protein (HBx) is suggested to regulate transcription by stimulation of intracellular signalling pathways. We have analysed the effects of HBx on activation of the MAP kinase (Erk) and JNK/SAPK signalling pathways and confirm a stimulation of the Erk/MAP kinase in quiescent cells. However, a substantial Erk-independent activation of AP-1, and phosphorylation of c-Jun (serine-63), but not Erk-2, was induced by HBx in dividing, serum-maintained cells. These data suggest that HBx promiscuously activates Erk and JNK responsive pathways and that its overall effect on signalling may be influenced by external mitogenic stimuli.
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Normal phosphorylation of duck hepatitis B virus L protein is dispensable for infectivity
More LessA fraction of the large surface protein (L) of duck hepatitis B virus (DHBV) is phosphorylated at serine or threonine residues (E. Grgacic & D. Anderson, Journal of Virology 68,7344–7350, 1994). We now report the identification of phosphorylation sites in DHBV L protein. Using site-directed mutagenesis, we have identified serine-118 (S118) as the major phosphorylation site, accepting approximately 64% of the total phosphate groups incorporated in L, and resulting in retarded migration of phos- phorylated L in SDS-PAGE. Proline-119 is indispensable for S118 phosphorylation. Mutation of other serine/threonine residues which are followed by prolines (T79, T89, S117 and T155) together with S118 further reduced phosphorylation to around 19% of wild-type. Non-equilibrium pH gel electrophoresis (NEPHGE) and SDS-PAGE of 33P- labelled L protein revealed two phosphorylated L species, while protein with the S118 to alanine mutation was detected as only one labelled species, consistent with multiple phosphorylations in wild- type L. Together, these results demonstrate that serine 118 is the major phosphorylation site for a proline-directed kinase, and that a proportion of L molecules are additionally phosphorylated at one of a number of secondary sites. DHBV mutants encoding L proteins with minimal phosphorylation (alanine mutants) or mimicking constitutive phosphorylation (aspartic acid mutants) remained infectious both in cell culture and in ducks, demonstrating that L phosphorylation may play only a minor role in DHBV replication.
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PEF-1, an epithelial cell transcription factor which activates the long control region of human papillomavirus type 16, is glycosylated with N-acetylglucosamine
More LessInfection by human papillomavirus type 16 (HPV- 16) has been linked to cervical cancer. The transcription of viral genes in HPV-16 is partially controlled by a number of cellular transcription factors. We have previously identified a novel cellular transcription factor, PEF-1, from its ability to interact with the long control region (LCR) of HPV- 16. This factor has a molecular mass of about 110 kDa and binds to a GC-rich sequence in the section of the LCR responsible for cell type-specific transcription from viral DNA. The factor Sp1 has similar properties and also interacts with the HPV- 16 LCR. We show that PEF-1 and Sp1 are distinct transcription factors: they recognize different DNA sequences, have different electrophoretic mobilities and different glycosylation patterns. Sp1 is O- glycosylated while PEF-1 appears to have a novel type of glycosylation, as shown by the interaction with pokeweed lectin and by the inhibition of this interaction by tunicamycin.
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Detection of sequences of TT virus, a novel DNA virus, in German patients
More LessRecently a novel DNA virus, designated TT virus (TTV), that possibly accounts for some of the cases of liver disease of unknown aetiology, was identified in Japanese patients. Using specific primer pairs for conserved regions, we detected TTV DNA by PCR in 16/84(19%) German patients awaiting orthotopic liver transplantation because of decompensated liver cirrhosis (of diverse causes); in 4/25 (16%) patients with non-A-G hepatitis; in 1/7 patients with autoimmune hepatitis; and in one intravenous drug user. Sequence analysis showed that in contrast to the findings in Japanese patients only about 37% of our TTV sequences belonged to genomic group 1 but about 58% belonged to group 2, including several sequences belonging to a further subgroup tentatively designated group 2c. Further studies to clarify whether the novel virus has hepatitis-inducing capacity or other clinical significance are needed.
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Translational control of viral and host protein synthesis during the course of herpes simplex virus type 1 infection: evidence that initiation of translation is the limiting step
More LessHerpes simplex virus type 1 (HSV-1) infection induces the selective shut-off of host protein synthesis, other than ribosomal proteins, and the successive synthesis of viral proteins. Because viral mRNAs persist in the cytoplasm after viral protein synthesis has been inhibited, we hypothesized that viral gene expression may be under translational control. Expression of genes encoding immediate early ICP27, early DBP and late US11 proteins, together with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was monitored over the course of infection at the level of mRNA and protein synthesis. After an efficient synthesis beginning with the appearance of successive viral mRNAs in the cytoplasm, synthesis of viral proteins was shut off similarly to the synthesis of GAPDH. This shut-off was not achieved by mRNA degradation but by progressive shifts of viral mRNAs from large polyribosomes to smaller ones, then to 40S ribosomal subunits. Transient expression of the UL41 gene alone, directing synthesis of virion-associated host shut-off (VHS) protein, induced efficient mRNA degradation, but did not impair recruitment of the remaining GAPDH and β-actin mRNAs into polyribosomes. These results indicate that HSV-1 induces a selective repression of initiation of mRNA translation which is probably the main cause of the shut-off of viral protein synthesis, and which contributes to the repression of host protein synthesis. VHS protein is not directly involved in this repression, at least in the absence of other viral proteins.
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Characterization of the herpes simplex virus type 2 (HSV-2) US2 gene product and a US2-deficient HSV-2 mutant
The herpes simplex virus type 2 (HSV-2) US2 gene product was identified by using a rabbit polyclonal antiserum raised against a recombinant 6 × His-US2 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 39 kDa protein in HSV-2 strain 186-infected cell lysates. The protein was not detectable in the presence of the virus DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies localized the US2 protein in the cytoplasm and as discrete granules at late times post-infection within and at the periphery of the nucleus, and nuclear fractionation studies showed that the protein was partially associated with the nuclear matrix of infected cells. The protein was easily detected in purified virions. Also, a US2 insertion mutant was constructed which contained an ICP6-lacZ insertion in the US2 gene. This mutant was as virulent as wild-type virus in mice when inoculated by the footpad route. The importance of the US2 protein of HSV-2 in the virus life-cycle may be apparent only in the natural human host.
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NF-kappaB activation is responsible for the synergistic effect of herpes simplex virus type 2 infection on interferon-gamma-induced nitric oxide production in macrophages
More LessNitric oxide (NO), produced in interferon (IFN)-γ- activated murine macrophages by the enzyme inducible nitric oxide synthase (iNOS), has been found to have antiviral properties. We have previously shown that herpes simplex virus type 2 (HSV-2) infection of macrophages synergistically enhances IFN-γ-induced NO production, and we now extend these findings by providing evidence that virus- induced tumour necrosis factor (TNF)-α mediates activation of the transcription factor nuclear factor (NF)- k B, which in turn is responsible for the synergistic effect. HSV-2 infection and IFN-γ stimulation of macrophages synergistically induced TNF-α secretion and nuclear translocation of NF- k B, which bound to a sequence corresponding to a k B site in the iNOS promoter. The effect of HSV-2 on NF- k B and NO production was eliminated when cells were treated with antibodies to TNF-α, and direct inhibition of NF- k B activation with pyrrolidinedithio- carbamate (PDTC) also blocked the effect of HSV-2 infection on NO production. The effect of the NF- k B activation inhibitor was not mediated through inhibition of the production of interferon regulatory factor (IRF)-1 or of TNF-α itself, and a possible alternative mechanism of activation of NF- k B through virus-induced activation of the kinase PKR was also ruled out. Thus, our data indicate that NF- k B activation, through virus-induced autocrine TNF-α secretion, is responsible for the synergistic effect of HSV-2 infection on IFN-γ-induced NO production, and that such activation might constitute a mechanism by which high-output NO production is targeted to infectious foci.
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Differential susceptibility to Marek's disease is associated with differences in number, but not phenotype or location, of pp38+ lymphocytes
More LessFlow cytometric and immunocytochemical techniques were used to quantify, identify and locate Marek’s disease herpesvirus (MDV)-infected lymphocytes in lymphoid organs of infected chickens, by expression of the virus antigen pp38. Two closely related lines of chicken, one susceptible to Marek’s disease (line 72) and another resistant (line 61), were infected at 2 weeks of age and compared at 10 sampling times between 0 and 50 days post-infection. In both lines 61 and 72, pp38 lymphocytes were detected at 4–6 days in the spleen, thymus and bursa. pp38 cells could not be detected from day 8 onwards. In both lines, pp38 lymphocytes were located in the peri-ellipsoidal area of the spleen, the medulla of the thymic lobes and the medulla of the bursal follicles. In both lines, pp38 cells were predominantly B lymphocytes, but CD4 and CD8 TCRαβ T lymphocytes were also detected in the thymus and spleen. For each organ, the mean number of pp38 lymphocytes was greater in line 72 than in line 61. pp38 lymphocytes were not detected in the peripheral blood at anytime. The data show that the differential susceptibility of lines 61 and 72 to the development of Marek’s disease lymphoma is not attributable to differences in phenotype or location of pp38 lymphocytes, or the time of expression of pp38. However, susceptibility is associated with greater numbers of pp38 lymphocytes.
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Murine cytomegalovirus induces expression and enzyme activity of cellular dihydrofolate reductase in quiescent cells
More LessMurine cytomegalovirus (MCMV) productively infects quiescent fibroblasts in which the levels of nucleoside triphosphate precursors and cell functions involved in DNA metabolism are minimal. It appears that MCMV has evolved molecular pathways in order to ensure the presence of nucleoside triphosphate precursors for the viral DNA polymerase. Here, we report that MCMV infection of quiescent NIH 3T3 cells markedly stimulates transcription, expression and activity of the cellular dihydrofolate reductase (DHFR), a key enzyme in the synthesis of DNA precursors. DHFR stimulation by MCMV is sensitive to UV irradiation and seems to depend on expression of the viral immediate-early protein pp89. Finally, it has been demonstrated that suppression of virus-induced DHFR activity by the specific inhibitor methotrexate prevents MCMV DNA replication. These observations indicate that induction of host cell DHFR activity by MCMV is required for viral DNA synthesis in quiescent fibroblasts.
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Diversity of naturally occurring Epstein-Barr virus revealed by nucleotide sequence polymorphism in hypervariable domains in the BamHI K and N subgenomic regions
The extent of nucleotide sequence microheterogeneity varies among subgenomic regions of Epstein-Barr virus (EBV). We examined, in EBV- carrying lymphoid cell lines, the extent of polymorphism in EBV DNA fragments amplified from the BamHI E, K, N and Z regions, and then investigated the diversity of the more hypervariable regions in tissues and body fluids. In cell lines, sequence dissimilarities in a genotype-specifying fragment of the EBNA-3C gene varied from < 1–4% within each genotype; dissimilarities in the first intron of the BZLF-1 gene were < 2% within each genotype. By contrast, dissimilarities in a C-terminal unique domain of the EBNA-1 gene, and in a fragment that encompasses and is upstream of the LMP-1 start codon, varied between 2 and 7% and were not genotype-specific. The sequence diversity in BamHI K and N regions was then examined in tissues and body fluids by single-strand conformation polymorphism (SSCP) analysis and cycle sequencing. Extensive inter-host diversity was observed, whether the host was co-infected by human immunodeficiency virus (HIV) or not. In the oral cavity of HIV-infected patients, inter-compartmental EBV diversity could be demonstrated, even between sites that were anatomically proximate. Studies of BamHI K clones derived from EBV in oral lesions revealed infection by multiple variants. Identification of hypermutable loci within the EBV genome such as those located in the BamHI K and N regions should permit fine discrimination of individual EBV variants.
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Resistance in plants transformed with the P1 or P3 gene of tobacco vein mottling potyvirus
More LessTobacco plants were transformed with genes encoding the tobacco vein mottling potyvirus (TVMV) P1 or P3 protein. When compared with vector- transformed or P1 transgenic lines, seedlings of P3 transgenic lines (except a low expressor line) developed poorly, suggesting a detrimental effect of P3 on the plant. All P1 and P3 transgenic lines were protected against the homologous TVMV strain and showed variable proportions of two resistance phenotypes: asymptomatic plants or symptomatic plants that recovered from infection. The resistance was effective against a high inoculum dose but had a narrow spectrum. The heterologous strain TVMV-S was able to overcome resistance in most P1 lines but did not break the resistance of most P3 lines. No line showed resistance to another potyvirus (potato virus Y) or to potato virus X. These features and the low levels of transgene expression in resistant plants suggest that protection in P1 and P3 lines is RNA-mediated. In contrast with most reports on virus-activated gene silencing, some P3 lines with the predominant ‘recovery’ phenotype showed silencing of the transgene that was activated at a certain developmental stage of the plant independently of virus infection.
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The capsid protein of tomato yellow leaf curl virus binds cooperatively to single-stranded DNA
More LessThe capsid protein (CP) of tomato yellow leaf curl virus (TYLCV) is the only known component of the virus coat. Here, we identify TYLCV CP as a singlestranded (ss) DNA binding protein. Purified TYLCV CP bound ssDNA in a highly cooperative and sequence-nonspecific fashion. TYLCV CP-ssDNA complexes were resistant to nucleolytic digestion and remained stable at relatively high salt concentrations. Because TYLCV CP is known to contain an active nuclear targeting signal, we propose that its association with the viral genomic ssDNA mediates TYLCV entry into the host cell nucleus during the infection process.
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Types of variation in DNA-A among isolates of East African cassava mosaic virus from Kenya, Malawi and Tanzania
More LessComplete nucleotide sequences of the DNA-A-like molecules of three East African cassava mosaic virus (EACMV) isolates from Kenya (-K, 2801 nt) and Malawi (-MH and -MK, both 2804 nt) were determined. These sequences were compared with that published for a Tanzanian isolate (-T, 2801 nt) and the partial sequence of athird Malawian isolate. Intergenic region sequences of all isolates, and deduced amino acid sequences of their AC1 (Rep) proteins, each formed a tightly related cluster that was distinct from the comparable components of other begomoviruses. Other complementary-sense genes (AC2, AC3, AC4) differed between EACMV isolates in a way consistent with the accumulation of point mutations. In contrast, virus-sense genes (CP, AV2) of isolates -MH and -MK differed (substantially for AV2) from those of other EACMV isolates but somewhat resembled those of tomato yellow leaf curl virus - Israel, suggesting they had been acquired by recombination with an unidentified begomovirus.
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Distinct gene arrangement in the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus genome
The genome organization of the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (BusuNPV) was largely elucidated and compared to those of other baculoviruses. A detailed physical map was constructed for the restriction enzymes BamHI, BglI, BglII, EcoRI, HindIII, KpnI, PstI, XbaI and XhoI. The 120·9 kbp viral genome was cloned as restriction fragments into a plasmid library from which about 43·5 kbp of dispersed sequence information was generated. Fifty-two putative open reading frames homologous to those of other baculoviruses were identified and their location in the genome of BusuNPV was determined. Although the gene content of BusuNPV is similar to that of Autographa californica multiple-nucleocapsid nucleopolyhedrovirus, Bombyx mori nucleopolyhedrovirus and Orgyia pseudotsugata multiple-nucleocapsid nucleopolyhedrovirus, the gene order is, however, significantly different from that observed in the other viruses, which have a high degree of collinearity. A new approach (GeneParity-Plot) was developed to represent the differences in gene order among baculoviruses when limited sequence information is available and to take advantage of the high degree of gene conservation. The data obtained show that BusuNPV is a distinct baculovirus species and the analyses suggest that gene distribution along baculovirus genomes may be used as a phylogenetic marker.
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Mapping and polyhedrin gene analysis of the Epiphyas postvittana nucleopolyhedrovirus genome
More LessThe light brown apple moth, Epiphyas postvittana, is a major insect pest of a variety of fruit crops grown in New Zealand and we are studying a nucleopolyhedrovirus, EppoNPV, isolated from this insect. Restriction endonuclease analysis of EppoNPV DNA shows that this is a single strain of virus with a genome size of approximately 119 kbp and a complete library of the EppoNPV genome has been cloned. A strategy of single-stranded sequencing of the termini of REN fragment clones was employed to map the virus genome. Sequence homologies to NPV gene sequences present in the GenBank database allowed a nearly complete restriction map of the EppoNPV genome to be constructed. The mapping was completed with Southern blotting and restriction analysis. Fifty-five open reading frames (ORFs) with similarity to genes from other NPVs have been identified and placement of these on the restriction map shows that EppoNPV has a nearly identical genome organization to Orgyia pseudotsugata (Op)MNPV. The polyhedrin gene of EppoNPV has been fully sequenced and an ORF of 738 bp encodes a predicted protein of 28·8 kDa. The conserved 12 bp consensus sequence typical of very late baculovirus gene promoters, AATAAGTAATTT, has been located upstream of the ATG initiation codon. An ORF located downstream of the polyhedrin gene shows homology to the 1629-capsid protein from OpMNPV. Phylogenetic comparison to polyhedrin gene sequences from 23 other NPVs shows EppoNPV to be a group I NPV closely related to OpMNPV.
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Analysis of the incubation periods, induction of obesity and histopathological changes in senescence-prone and senescence-resistant mice infected with various scrapie strains
More LessThe similarity in histopathological changes seen in scrapie-infected mice and in an uninfected senescence-accelerated mouse strain led to a study in which the mouse strain that is prone to senescence (SAMP8), a strain that is resistant to senescence (SAMR1) and a progenitor strain (AKR) of these two strains were infected with three different scrapie strains, ME7,139Aand 22L. For each scrapie strain, the incubation period was shortest in AKR mice and longest in SAMR1 mice. The induction of obesity was a function of scrapie strain and not mouse strain; ME7 caused obesity in all mouse strains, whereas the average weights of mice injected with 139A and 22L did not differ significantly from mice injected with homogenates of normal mouse brain. The pattern of vacuolation seen in the brain of each mouse strain was primarily dependent on the scrapie strain injected. There were, in general, similarities to the patterns induced in other inbred strains; e.g. ME7 caused extensive forebrain vacuolation, 22L caused prominent vacuolation in the cerebellum, and the 139A strain induced characteristic white matter vacuolation. Vacuolation was also seen inthe medulla and midbrain of SAMP8 mice injected with normal mouse brain, which is consistent with the occurrence of accelerated ageing changes in the brain of this strain. Further analysis of the differences among these mouse strains should provide information relating to the observed differences in scrapie incubation periods.
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Volumes and issues
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Volume 105 (2024)
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Volume 6 (1970)
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Volume 2 (1968)
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Volume 1 (1967)