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Volume 83,
Issue 7,
2002
Volume 83, Issue 7, 2002
- Animal: DNA Viruses
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Characterization of an Epstein–Barr virus-related gammaherpesvirus from common marmoset (Callithrix jacchus)
More LessA gammaherpesvirus related to Epstein–Barr virus (EBV; Human herpesvirus 4) infects otherwise healthy common marmosets (Callithrix jacchus). Long-term culture of common marmoset peripheral blood lymphocytes resulted in outgrowth of spontaneously immortalized lymphoblastoid cell lines, primarily of B cell lineage. Electron microscopy of cells and supernatants showed herpesvirus particles. There were high rates of serological cross-reactivity to other herpesviruses (68–86%), but with very low geometric mean antibody titres [1:12 to human herpesvirus 6 and 1:14 to Herpesvirus papio (Cercopithecine herpesvirus 12)]. Sequence analysis of the conserved herpesvirus DNA polymerase gene showed that the virus is a member of the lymphocryptovirus subgroup and is most closely related to a lymphocryptovirus from rhesus macaques and is closely related to EBV and Herpesvirus papio. High seroprevalence (79%, with geometric mean antibody titre of 1:110) among 28 common marmosets from two geographically distinct colonies indicated that the virus is likely present in many common marmosets in captivity. A New World primate harbouring a lymphocryptovirus suggests that this subgroup arose much earlier than previously thought.
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Identification of structural determinants of the first transmembrane domain of the small envelope protein of duck hepatitis B virus essential for particle morphogenesis
More LessThe envelope of duck hepatitis B virus (DHBV) consists of the small (S) and large (L) envelope proteins, which share a common C-terminal multispanning transmembrane region but differ by the long N-terminal pre-S domain of L, which is essential for interactions with both the receptor and nucleocapsid. To achieve these dual functions, L acquires mixed topologies through S-dependent post-translational translocation of its pre-S domain. This study has examined the role of S in this unusual mechanism of translocation by analysis of the α-helical transmembrane domains and their potential to engage in lateral interactions for envelope assembly. Through mutagenesis in constructs expressing the S and L envelope proteins independently, transmembrane domain 1 was identified as an essential structural determinant in S. Two polar residues in this helix were identified as contributing to L protein translocation through the assembly of S into particles, implying that the topological switch of L is part of the assembly and maturation process. The same domain in L was shown to be dispensable for L translocation and assembly, suggesting that transmembrane domain 1 of L and S have different functional roles and structural arrangements on the assembled particle. The conservation in all hepadnavirus envelope proteins of two polar residues at positions 24 and 27 of transmembrane domain 1, the former positively charged, points to this being a common determinant in particle morphogenesis for all hepadnaviruses.
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Experimental transfection of Macaca sylvanus with cloned human hepatitis B virus
Due to the absence of easily accessible animal models for the study of hepatitis B virus (HBV), the possibility of using Macaca sylvanus, a monkey originating from Morocco, North Africa, was investigated. Three monkeys were intrahepatically inoculated with a replication-competent head-to-tail HBV DNA plasmid dimer construct. The HBV surface antigen and HBV DNA were detected prior to alanine aminotransferase elevation in the serum of two of three HBV-inoculated monkeys at day 2 post-transfection and persisted for several weeks. This indicates that transfected animals developed markers of HBV infection. In addition, electron microscopy of the serum 3 weeks post-transfection showed the presence of virus particles whose shape and size were similar to complete 42 nm HBV Dane particles. Histological examination of liver tissues also revealed pathological changes not observed in uninfected controls, which strongly suggested acute hepatitis. HBV DNA was also detected by PCR in these monkey livers. Taken together, these results indicate that HBV can successfully replicate in this model and that M. sylvanus could be a potentially useful new primate model for the study of HBV replication.
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PTEN is a negative regulator of STAT3 activation in human papillomavirus-infected cells
More LessLaryngeal papillomas are caused by infection of the laryngeal epithelium by human papillomavirus type 6 or type 11 (HPV-6/-11). Previous studies in our laboratory have demonstrated an increase in PI3 kinase levels in papilloma tissue. However, activation of the downstream effector of PI3 kinase, protein kinase B (PKB/Akt), was reduced. This observation was explained by the elevated expression of the phosphatase and tensin homologue (PTEN), a recently characterized tumour suppressor, in papilloma tissue. Recent investigation of the possible functional roles of PTEN during papilloma development has now indicated that the level of tyrosine(705)-phosphorylated signal transducer and activator of transcription 3 [PTyr(705)STAT3] could be inversely correlated to that of PTEN as well. In vitro phosphatase assays suggested the presence of an increased level of a PTyr(705)STAT3 phosphatase in papilloma extract. Immunodepletion of PTEN from papilloma extracts resulted in a reduction of the PTyr(705)STAT3 phosphatase activity. Transfection of PTEN cDNA into HeLa cells attenuated STAT3 phosphorylation at Tyr(705) in a dose-dependent manner. This attenuation of STAT3 phosphorylation was independent of the STAT3 kinase. Interestingly, introduction of a lipid phosphatase mutant of PTEN (G129E) resulted in heightened PTyr(705)STAT3 phosphatase activity, relative to that obtained from wild-type PTEN transfection. These data indicate that PTEN negatively regulates STAT3 activation in HPV-infected papilloma cells. Induction of PTEN and reduction of activated STAT3 might be a result of a host defence mechanism or a virus-directed strategy to alter normal epithelial differentiation programming.
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Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA]2 motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication
More LessThe linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA]2, from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA]2 sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.
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- Plant
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Interaction between potyvirus helper component-proteinase and capsid protein in infected plants
Monoclonal antibodies were raised against helper component-proteinase (HcPro) purified from plants infected with the potyvirus Lettuce mosaic virus (LMV). These antibodies were used in a two-site triple antibody sandwich ELISA assay together with polyclonal antibodies directed against purified virions. An interaction between HcPro and the viral coat protein (CP) was demonstrated in extracts of LMV-infected leaves, as well as for two other potyviruses, Plum pox virus and Potato virus Y. The CP–HcPro interaction was not abolished in LMV derivatives with an HcPro GFP N-terminal fusion, or with a deletion from the CP of the amino acids involved in aphid transmission. Electron microscopy indicated that HcPro probably does not interact with the CP in the form of assembled virions or virus-like particles. Together, these results suggest that the interaction detected between CP and HcPro might be involved in a process of the potyvirus cycle different from aphid transmission.
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Substitution of a single amino acid in the 2b protein of Pea early-browning virus affects nematode transmission
More LessThe 2b protein of Pea early-browning virus (PEBV) is required for transmission of the virus by nematodes. Comparison of the 2b proteins of highly transmissible (TpA56) and poorly transmissible (SP5) isolates of PEBV identified two amino acid substitutions (G90S and G177R) that might be responsible for the poor transmission of isolate SP5. Hybrid viruses were created in which the TpA56 2b protein carried SP5-specific substitutions at residue 90 or 177, and in which the SP5 2b protein carried TpA56-specific substitutions at these positions. Transmission tests showed that the G177R substitution is sufficient to prevent nematode transmission of the virus. Examination of the 2b proteins from PEBV and other tobraviruses predicted the presence of a coiled-coil domain in the central region of the protein. This structural element is important for the association of interacting proteins and, thus, might mediate interaction of the 2b protein with the virus coat protein or with the vector nematode.
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RNA 2 of Citrus psorosis virus is of negative polarity and has a single open reading frame in its complementary strand
More LessCitrus psorosis virus (CPsV) causes a citrus disease occurring worldwide. Isolate CPV 4 has a genome with three single-stranded RNAs. The complete sequence of RNA 2 (1643 nucleotides) is reported here. Northern blot hybridization with strand-specific probes showed that most of the encapsidated RNA 2 is of negative polarity, although a small amount of the complementary strand may also be present in particles. The RNA 2 complementary strand contained a single open reading frame encoding a protein of 476 amino acids, which includes a motif resembling a nuclear localization signal. The sequence of this putative protein shows no significant similarity to any other in the databases. In the 3′-terminal untranslated region there is a putative polyadenylation signal. No subgenomic RNAs derived from RNA 2 were detected.
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Regulated nuclear targeting of cauliflower mosaic virus
More LessThe mature cauliflower mosaic virus (CaMV) capsid protein (CP), if expressed in the absence of other viral proteins, is transported into the plant cell nucleus by the action of a nuclear localization signal (NLS) close to the N terminus. In contrast, virus particles do not enter the nucleus, but dock at the nuclear membrane, a process inhibited by anti-NLS antibodies or by GTPγS, and apparently mediated by interaction of CP with host importin α. The very acidic N-terminal extension of the viral CP precursor inhibits nuclear targeting of the protein and hence the precursor is localized in the cytoplasm. We hypothesize that this provides a control mechanism which ensures that the CP precursor is used for virus assembly in the cytoplasm and that only mature virus particles reach the nuclear pore.
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Nucleotide sequence shows that Bean leafroll virus has a Luteovirus-like genome organization
More LessThe complete nucleotide sequence of the Bean leafroll virus (BLRV) genomic RNA and the termini of its smallest subgenomic RNAs were determined to better understand its mechanisms of gene expression and replication and its phylogenetic position within the Luteoviridae. The number and placement of open reading frames (ORFs) within the BLRV genome was Luteovirus-like. The nucleotide and predicted amino acid sequences of BLRV were most similar to those of Soybean dwarf virus (SbDV). Phylogenetic analyses employing the neighbour-joining method and sister-scanning analysis indicated that the BLRV nonstructural proteins were closely related to those of Barley yellow dwarf virus-PAV (BYDV-PAV), a Luteovirus. The region surrounding the frameshift at the junction between ORFs 1 and 2 also contained sequences very similar to those of BYDV-PAV and a Dianthovirus, Red clover necrotic mosaic virus. Similar analyses showed that the structural proteins were most similar to those of the Polerovirus genus. The 3′-noncoding regions downstream of ORF5 contained sequences similar to translational control elements identified in the BYDV-PAV genome. These data suggest that BLRV, like SbDV, is derived either through selection from a common ancestor with BYDV-PAV or that BLRV is the product of two recombination events between luteovirus-like and polerovirus-like ancestors where the 5′ 2900 nt and 3′ 700 nt of the BLRV genome are from a Luteovirus and the intervening sequences are derived from a Polerovirus.
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Sequence analysis of Potato leafroll virus isolates reveals genetic stability, major evolutionary events and differential selection pressure between overlapping reading frame products
More LessIn order to investigate the genetic diversity of Potato leafroll virus (PLRV), seven new complete genomic sequences of isolates collected worldwide were compared with the five sequences available in GenBank. Then, a restricted polymorphic region of the genome was chosen to further analyse new sequences. The sequences of PLRV open reading frames (ORFs) 3 and 4 were also compared with those of two other poleroviruses and the non-synonymous to synonymous substitution ratio distribution was analysed in overlapping and non-overlapping regions of the genome using maximum-likelihood models. Results confirmed that PLRV sequences from around the world are very closely related and showed that the region encoding protein P0 allowed the detection of three groups of isolates. When compared to other poleroviruses, PLRV was the most conserved in both ORFs 3 and 4. However, the results suggest that important events, such as deletion, mutation at a stop codon and intraspecific homologous recombination events, have occurred during the evolution of PLRV. Finally, it was shown that the translation products of ORFs 0 and 3 are significantly more conserved than those of the overlapping ORFs 1 and 4, respectively. All together, the results allow the proposal of new hypotheses to explain the apparent genetic stability of PLRV and its evolution.
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Volumes and issues
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Volume 106 (2025)
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