- Volume 82, Issue 7, 2001
Volume 82, Issue 7, 2001
- Animal: DNA Viruses
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Evidence for structural differences in the S domain of L in comparison with S protein of hepatitis B virus
More LessThe structures of the large (L), middle (M) and small (S) versions of the envelope proteins of hepatitis B virus remain poorly characterized due to the complex nature of their conformations. Several groups have proposed transmembrane topological models depicting the lumenally and cytosolically disposed regions of these proteins. Recently, post-translational topological changes in L have been described. However, no overall differences in the topology of the S domains of the L or M, to the S protein are predicted. In this report, we investigated a previously uncharacterized anti-S monoclonal antibody (MAb), 6B1, which recognizes a conformation-sensitive epitope in S. Unlike other anti-S MAbs tested, this MAb did not recognize its epitope in the S domain of L protein. Interestingly, however, the M protein was efficiently recognized. This unique characteristic of MAb 6B1 has allowed us to study the intracellular distribution of L and S proteins. In cells expressing both L and S, L re-localized from the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) to the membrane-associated distribution of S protein indicating that L and S interact with each other. This was confirmed by immunoprecipitation assays, which also showed that the interaction between L and S results in the secretion of L protein from cells. Overall, the ability of MAb 6B1 to selectively recognize S and M, but not L, strongly points to the existence of significant topological differences in the S domain of L. The availability of this important reagent should help further our understanding of the structure of HBV surface antigens.
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Coordinate effects of human immunodeficiency virus type 1 protein Tat and cellular protein Purα on DNA replication initiated at the JC virus origin
JC virus (JCV) causes progressive multifocal leukoencephalopathy, a demyelinating disease in brains of individuals with AIDS. Previous work has shown that the Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), can interact with cellular protein Purα to enhance both TAR-dependent HIV-1 transcription and JCV late gene transcription. Tat has been shown to activate JCV transcription through interaction with Purα, which binds to promoter sequence elements near the JCV origin of replication. DNA footprinting has shown that Purα and large T-antigen cooperatively interact at several binding sites in the origin and transcriptional control region. Overexpression of Purα inhibits replication initiated at the JCV origin by T-antigen. In transfected glial cells Tat reversed this inhibition and enhanced DNA replication. In an in vitro replication system maximal activation by Tat, more than sixfold the levels achieved with T-antigen alone, was achieved in the presence of Purα. Effects of mutant Tat proteins on both activation of replication and binding to Purα have revealed that Cys22 exerts a conformational effect that affects both activities. The origin of an archetypal strain of JCV was less susceptible to activation of replication by Tat relative to the rearranged Mad-1 strain. These results have revealed a previously undocumented role for Tat in DNA replication and have indicated a regulatory role for JCV origin auxiliary sequences in replication and activation by Tat.
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Analysis of canine parvovirus sequences from wolves and dogs isolated in Italy
The VP2 genes of Italian canine parvovirus (CPV) type 2 strains isolated from dogs and wolves were sequenced and a three-dimensional model of the VP2 capsid protein was constructed. Two mutations were detected in the VP2 sequences of the Italian strains: one at residue 297 and one at residue 265. Variant 297 is the predominant CPV isolate in Europe, whereas variant 265 has never been detected before. The mutation at residue 265 causes a disruption in a G strand of the β-barrel in the VP2 protein. Data on strains isolated from wolves demonstrated that the same strain of CPV can circulate among domestic and wild canids; therefore, this result leads us to exclude the possibility that a separate parvovirus pool exists in wild populations.
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Evaluation of the primary effect of brefeldin A treatment upon herpes simplex virus assembly
More LessAddition of the drug brefeldin A (BFA) to cells infected by herpes simplex virus (HSV) type 1 is known to result in a complex pattern of defects in particle assembly. BFA-treated, infected cells accumulate perinuclear enveloped virions and non-enveloped (‘naked’) cytoplasmic capsids, and it has been difficult to interpret these data in terms of the assembly pathway of HSV and the known effects of BFA on the secretory apparatus. Since BFA is a cytotoxic drug, and earlier studies commonly examined the effects of long-term BFA incubations on infected cells, it was hypothesized that the drug could have pleiotropic and indirect effects on HSV assembly. To test this, use was made of an HSV synchronized assembly assay, in which cells are infected with the virus mutant tsProt.A and maintained at 39 °C to induce reversible accumulation of a population of procapsids. By first adding BFA and then shifting these cells to 31 °C for 3 h to allow the accumulated procapsids to mature, it was possible to test the effect of short-term BFA treatment on only those HSV assembly events that are downstream of procapsid maturation. Under these conditions, it was found that procapsids matured and packaged the viral genome normally, but remained non-enveloped and failed to exit the nucleus. It is concluded that the primary effect of BFA on HSV replication is to inhibit budding at the inner nuclear membrane.
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The carboxyl terminus of the human cytomegalovirus UL37 immediate-early glycoprotein is conserved in primary strains and is important for transactivation
The human cytomegalovirus (HCMV) UL37 exon 3 (UL37x3) open reading frame (ORF) encodes the carboxyl termini of two immediate-early glycoproteins (gpUL37 and gpUL37M). UL37x3 homologous sequences are not required for mouse cytomegalovirus (MCMV) growth in vitro; yet, they are important for MCMV growth and pathogenesis in vivo. Similarly, UL37x3 sequences are dispensable for HCMV growth in culture, but their requirement for HCMV growth in vivo is not known. To determine this requirement, we directly sequenced the complete UL37x3 gene in multiple HCMV primary strains. A total of 63 of the 310 amino acids in the UL37x3 ORF differ non-conservatively in one or more HCMV primary strains. The HCMV UL37x3 genetic diversity is non-random: the N-glycosylation (46/186 aa) and basic (9/15 aa) domains have the highest proportion of non-conservative variant amino acids. Nonetheless, most (15/17 signals) of the N-glycosylation signals are retained in all HCMV primary strains. Moreover, new N-glycosylation signals are encoded by 5/20 primary strains. In sharp contrast, the UL37x3 transmembrane (TM) ORF completely lacks diversity in all 20 HCMV sequenced primary strains, and only 1 of 28 cytosolic tail residues differs non-conservatively. To test the functional significance of the conserved carboxyl terminus, gpUL37 mutants lacking the TM and/or cytosolic tail were tested for transactivating activity. The gpUL37 carboxyl-terminal mutants are partially defective in hsp70 promoter transactivation even though they trafficked similarly to the wild-type protein into the endoplasmic reticulum and to mitochondria. From these results, we conclude that N-glycosylated gpUL37, particularly its TM and cytosolic domains, is important for HCMV growth in humans.
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Epstein–Barr virus nuclear antigen 5 interacts with HAX-1, a possible component of the B-cell receptor signalling pathway
More LessUsing a yeast two-hybrid screen of a B-cell cDNA library with an Epstein–Barr nuclear antigen 5 (EBNA5) molecule containing seven repeats of the W1W2 domain as bait, we have isolated the EBNA5-interacting protein HAX-1. HAX-1 has previously been shown to associate with HS1, a protein specifically expressed in cells of the haematopoietic lineage, and is thought to be involved in signal transduction in B-cells. Immunofluorescence experiments showed that HAX-1 co-localized with the hsp60 protein that is associated with the mitochondria in the cell cytoplasm. Pull down experiments with a fusion protein between glutathione S-transferase and the seven copy repeat EBNA5 synthesized in bacteria and in yeast cells confirmed that HAX-1 can interact with EBNA5 in vitro. Conventionally, EBNA5 is regarded as a nuclear protein. However, we show here that the smallest EBNA5 species, composed of the unique Y domain and only one copy of the W1W2 repeat domain, like HAX-1, co-localizes with the mitochondrial hsp60 protein in the B-cell cytoplasm. Furthermore, immunoprecipitation experiments demonstrate that the single repeat EBNA5 associates with HAX-1 in transfected B-lymphoblastoid cells.
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- Insect
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Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication
More LessThe DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3′–5′ exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.
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Comparative pathogenesis of Helicoverpa zea S nucleopolyhedrovirus in noctuid larvae
More LessWe used a recombinant of Helicoverpa zea S nucleopolyhedrovirus containing the hsp70/lacZ reporter cassette (HzSNPV-hsp70/lacZ) to quantify mortality relationships and to elucidate early pathogenesis in two permissive hosts, Heliothis virescens and Helicoverpa zea, and one semi-permissive host, Trichoplusia ni. Fourth instar T. ni were highly resistant to fatal infection both by oral injection of occlusions and by intrahaemocoelic injection of budded virus, indicating the presence of both midgut and systemic mechanisms of resistance. In bioassays, newly moulted (40) H. zea were significantly more susceptible than 40 H. virescens to fatal infection, but mortality levels were the same for larval cohorts inoculated 16 h after the moult (416). Developmental resistance was stronger in H. zea and in both hosts, partially reversed by administration of the optical brightener M2R. In both species, developmental resistance was correlated with a reduced ability of HzSNPV to establish and/or maintain primary midgut infections. In time-course experiments using a dosage of 15 occlusions (∼LD90), lacZ expression marking the onset of primary and secondary infection was first observed in midgut columnar and tracheal cells at 4 and 12 h, respectively. Inoculation of 40 larvae resulted in approximately twofold more foci in H. zea larvae than in H. virescens, but H. zea larvae sloughed infected midgut cells at a faster rate. For both heliothines, interaction of occlusion-derived virus with primary cellular targets within the midgut epithelium was critical to the outcome of infection and a key process underlying acquisition of developmental resistance.
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- Plant
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Genomic organization of RNA2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro
More LessThe proteinase of Tomato ringspot virus (genus Nepovirus) is responsible for proteolytic cleavage of the RNA2-encoded polyprotein (P2) at two cleavage sites, allowing definition of the domains for the movement protein (MP) and coat protein. In this study, we have characterized a third cleavage site in the N-terminal region of P2 using an in vitro processing assay and partial cDNA clones. Results from site-directed mutagenesis of putative cleavage sites suggest that cleavage occurs at dipeptide Q301/G. Cleavage at this site is predicted to result in the release of two proteins from the N-terminal region of P2: a 34 kDa protein located at the N terminus of P2 (assuming translation initiation at the first AUG codon) and a 71 kDa protein located immediately upstream of the MP domain. In contrast, only one protein domain is present in the equivalent region of the P2 polyprotein of other characterized nepoviruses.
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Quasispecies nature of the genome of Grapevine fanleaf virus
More LessGenetic diversity was characterized in 14 isolates of Grapevine fanleaf virus (GFLV) recovered from grapevine (Vitis vinifera). Virions were collected by immunocapture, and a 1557 bp fragment containing part of the viral coat protein gene and part of the untranslated region to its 3′ side was amplified by RT–PCR. Sequence variation among isolates was characterized by restriction fragment length polymorphism (RFLP) analysis and by sequencing. The AvaII-generated RFLP patterns from the various isolates were highly variable. The isolates were passaged in Chenopodium quinoa. The RFLP patterns altered with passage through the alternate host, but the variation stabilized after a number of passages. Individual genomes were recovered by cloning. The subcloned sequences were found to vary from each other by as much as 13%, and the encoded amino acid sequences by as much as 9%. The data suggest that the GFLV genome consists of quasispecies populations.
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- Phage
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Peptide display on live MS2 phage: restrictions at the RNA genome level
More LessThe potential of the RNA phage MS2 to accommodate extra amino acids in its major coat protein has been examined. Accordingly, a pentapeptide was encoded in the genome as an N-terminal extension. In the MS2 crystal structure, this part of the coat protein forms a loop that extends from the outer surface of the icosahedral virion. At the RNA level, the insert forms a large loop at the top of an existing hairpin. This study shows that it is possible to maintain inserts in the coat protein of live phages. However, not all inserts were genetically stable. Some suffer deletions, while others underwent adaptation by base substitutions. Whether or not an insert is stable appears to be determined by the choice of the nucleic acid sequence used to encode the extra peptide. This effect was not caused by differential translation, because coat-protein synthesis was equal in wild-type and mutants. We conclude that the stability of the insert depends on the structure of the large RNA hairpin loop, as demonstrated by the fact that a single substitution can convert an unstable loop into a stable one.
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Volumes and issues
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