bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane-destabilizing activity of rotavirus NSP4 is mediated by a membrane-proximal amphipathic domain
Authors: Browne Edward P. , Bellamy A. Richard , Taylor John A.
Published: August 2015

Expression of the rotavirus non-structural glycoprotein NSP4 inE.colileads to a decrease in optical density of the culture and release of [3H]uridine into the medium, effects attributable to the ability of NSP4 to perturb the bacterial membrane. To identify a domain of NSP4 responsible, different regions of the polypeptide were expressed inE.coli. Membrane destabilization is associated with a region of the protein located within residues 48–91, which includes a potential cationic amphipathic helix. A second region of NSP4 that contains a coiled-coil oligomerization domain and a sequence reported to function as a viral enterotoxin enhances the membrane-destabilizing activity of residues 48–91, but has no direct effect on the membrane stability. These studies suggest that the membrane-destabilizing and enterotoxic properties of NSP4 may be mediated by different regions of the polypeptide and suggest a possible basis for the cytotoxicity of NSP4 in mammalian cells.
Hot-spot variations of Kaposi’s sarcoma-associated herpesvirus latent nuclear antigen and application in genotyping by PCR–RFLP
Authors: Zhang Yan-Jin , Deng Jian-Hong , Rabkin Charles , Gao Shou-Jiang
Published: August 2015

Kaposi’s sarcoma-associated herpesvirus (KSHV, human herpesvirus-8) is aetiologically associated with Kaposi’s sarcoma and several other lymphoproliferative disorders. The latent nuclear antigen (LNA) encoded by KSHV ORF73 has important functions in virus latent infection and shows molecular polymorphism. Sequence variations were identified in the internal repeat domain (IRD) of ORF73. DNA sequencing of ORF73 from one KSHV-infected cell line, PK-1, revealed that there were 558 bp (30·2%) deletions and 66 (3·6%) point mutations located mainly in repeat region 2, the glutamine-rich region of ORF73 IRD, compared with ORF73 of BC-1 KSHV. Similar sequence variations of ORF73 were also identified in two other isolates. None of the sequence variations caused any translational frame-shift in these four KSHV isolates examined, suggesting that LNA has a conservative function in virus latent infection. The frequent sequence variations in repeat region 2 of ORF73 IRD were also identified by PCR–RFLP genotyping in 26 KSHV isolates, suggesting that this region is a ‘hot-spot’ for genetic variations. Each Kaposi’s sarcoma lesion sample contained one virus genotype with a unique RFLP pattern, indicating that in vivo KSHV infection was established with single predominate genotypes, which was further supported by the presence of invariable genotypes in multifocal lesions from individual KS patients. Four KSHV subtypes were classified based on the RFLP patterns that represent the patterns of DNA sequence variations in the ORF73 IRD. PCR–RFLP genotyping is capable of identifying LNA genetic variations and differentiating individual KSHV isolates, and thus may be useful for KSHV molecular epidemiology studies.
In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase
Authors: Matsushita Yasuhiko , Hanazawa Kohtaro , Yoshioka Kuniaki , Oguchi Taichi , Kawakami Shigeki , Watanabe Yuichiro , Nishiguchi Masamichi , Nyunoya Hiroshi
Published: August 2015

The movement protein (MP) of tomato mosaic virus (ToMV) was produced inE.colias a soluble fusion protein with glutathioneS-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylatedin vitroby incubating with cell extracts ofNicotiana tabacumand tobacco suspension culture cells (BY-2) in the presence of [γ-32P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [γ-32P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.
N-Glycans on the short ectodomain of the primary envelope glycoprotein play a major role in the polyclonal activation of B cells by lactate dehydrogenase-elevating virus
Authors: Plagemann Peter G. W. , Jones Quentin A. , Cafruny William A.
Published: August 2015

The common biologically cloned isolates of lactate dehydrogenase-elevating virus (LDV-P and LDV-vx) invariably cause a polyclonal activation of B cells in immunocompetent mice. It is recognized by an at least 10-fold increase in plasma IgG2a levels and the de novo formation of immune complexes that most likely consist of autoantibodies and their antigens. The present study indicates that three closely spaced N-glycans on the short ectodomain of the primary envelope glycoprotein, VP-3P, of LDV-P/vx, play a major role in inducing the polyclonal proliferation of B cells. IFN-γ then seems to mediate the differentiation of the activated B cells to IgG2a-producing plasma cells. These conclusions are based on the finding that the IgG2a hypergammaglobulinaemia and immune complex formation were much lower in mice that were infected with LDV variants (LDV-C and LDV-v) whose VP-3P ectodomains lack two of the three N-glycans than in LDV-P/vx infected mice. In contrast, the VP-3P ectodomains of three neutralization escape variants of LDV-C/v whose VP-3P ectodomains possess three N-glycosylation sites caused a polyclonal activation of B cells comparable to that of LDV-P/vx.
The potential terminase subunit of human cytomegalovirus, pUL56, is translocated into the nucleus by its own nuclear localization signal and interacts with importin α
Authors: Giesen Kyra , Radsak Klaus , Bogner Elke
Published: August 2015

Human cytomegalovirus (HCMV) DNA-binding protein pUL56 is thought to be involved in the cleavage/packaging process of viral DNA and therefore needs to be transported into the nucleus. By using indirect immunofluorescence analysis, HCMV pUL56 (p130) was found to be localized predominantly in the nucleus of infected cells. Solitary expression of wild-type as well as epitope-tagged pUL56 also resulted in nuclear distribution after transfection, suggesting the presence of an endogenous nuclear localization signal (NLS). Deletion of a carboxy-terminal stretch of basic amino acids (aa 816–827) prevented nuclear translocation, indicating that the sequence RRVRATRKRPRR of HCMV pUL56 mediates nuclear targetting. The signal character of the NLS sequence was demonstrated by successful transfer of the NLS to a reporter protein chimera. Furthermore, sequential substitutions of pairs of amino acids by alanine in the context of the reporter protein as well as substitutions within the full-length pUL56 sequence indicated that residues at positions 7 and 8 of the NLS (R and K at positions 822 and 823 of pUL56) were essential for nuclear translocation. In order to identify the transport machinery involved, the potential of pUL56 to bind importin α (hSRP1α) was examined. Clear evidence of a direct interaction of a carboxy-terminal portion as well as the NLS of pUL56 with hSRP1α was provided byin vitrobinding assays. In view of these findings, it is suggested that nuclear translocation of HCMV pUL56 is mediated by the importin-dependent pathway.
Codon usage in nucleopolyhedroviruses
Authors: Levin David B. , Whittome Beatrixe
Published: August 2015

Phylogenetic analyses based on baculovirus polyhedrin nucleotide and amino acid sequences revealed two major nucleopolyhedrovirus (NPV) clades, designated Group I and Group II. Subsequent phylogenetic analyses have revealed three Group II subclades, designated A, B and C. Variations in amino acid frequencies determine the extent of dissimilarity for divergent but structurally and functionally conserved genes and therefore significantly influence the analysis of phylogenetic relationships. Hence, it is important to consider variations in amino acid codon usage. The Genome Hypothesis postulates that genes in any given genome use the same coding pattern with respect to synonymous codons and that genes in phylogenetically related species generally show the same pattern of codon usage. We have examined codon usage in six genes from six NPVs and found that: (1) there is significant variation in codon use by genes within the same virus genome; (2) there is significant variation in the codon usage of homologous genes encoded by different NPVs; (3) there is no correlation between the level of gene expression and codon bias in NPVs; (4) there is no correlation between gene length and codon bias in NPVs; and (5) that while codon use bias appears to be conserved between viruses that are closely related phylogenetically, the patterns of codon usage also appear to be a direct function of the GC-content of the virus-encoded genes.
T-helper and humoral responses to Puumala hantavirus nucleocapsid protein: identification of T-helper epitopes in a mouse model
Authors: de Carvalho Nicacio Cristina , Sällberg Matti , Hultgren Catharina , Lundkvist Åke
Published: August 2015

Puumala hantavirus (PUUV) is a rodent-borne agent causing nephropathia epidemica in humans, a milder form of haemorrhagic fever with renal syndrome occurring in Fennoscandia, central Europe and western Russia. In this study we characterized the immunogenicity of an E. coli-expressed nucleocapsid (N) protein of PUUV (strain Kazan-E6) in inbred mice (BALB/c, CBA and C57/BL6). The recombinant N (rN) protein raised PUUV-specific antibodies in all three tested murine haplotypes, and all IgG subclasses were detected. Epitope mapping using peptides spanning the N protein revealed that the B-cell recognition sites were mainly located at the amino-terminal part of the protein. Proliferative T-helper (Th) lymphocyte responses were detected in all haplotypes after a single immunization with rN. Several Th-recognition sites, spanning amino acids 6–27, 96–117, 211–232 and 256–277, were identified using overlapping peptides. Peptides representing the identified sites could also prime Th-lymphocytes to proliferate in response to recall with rN protein, thereby confirming the authenticity of the identified sites. The rN-primed Th-lymphocytes produced predominantly interleukin (IL)-2 and gamma interferon, together with lower levels of IL-4 and IL-6, indicating a mixed Th1/Th2 response.
VP5 and the N terminus of VP2 are not responsible for the different pathotype of serotype I and II infectious bursal disease virus
Authors: Schröder Anja , van Loon Adriaan A. W. M. , Goovaerts Danny , Teifke Jens Peter , Mundt Egbert
Published: August 2015

Two serotypes have been identified in infectious bursal disease virus (IBDV), a member of the family Birnaviridae. A reverse genetics system was used for generation of chimeras in genome segment A of the two serotypes, in which the complete viral VP5 gene and 3′ noncoding region (NCR), or parts thereof, were exchanged. The engineered viruses were characterized in vitro and in vivo in comparison to serotype I and II IBDV. Our results show that IBDV chimeras exhibit a different phenotype in cell culture compared to the wild-type viruses. In in vitro-cultivated bursal-derived cells, chimeric viruses infected B lymphocytes, as does serotype I IBDV. Surprisingly, serotype II virus was also able to infect in vitro-cultivated bursal cells, but these were neither B lymphocytes nor macrophages. After infection of susceptible chickens all chimeras replicated in the bursa of Fabricius (BF), and three chimeric viruses caused mild depletion of bursal cells. In contrast, after infection of chickens with a chimeric IBDV containing exchanged VP5 as well as 3′-NCR, no depletion was detectable. The serotype II strain did not replicate in the BF nor did it cause depletion of bursal cells. Thus, the origin of VP5 does not explain the different pathotype of IBDV serotype I and II.
Oestrogen and progesterone increase the levels of apoptosis induced by the human papillomavirus type 16 E2 and E7 proteins
Authors: Webster Kenneth , Taylor Anna , Gaston Kevin
Published: August 2015

Human papillomavirus (HPV) type 16 infects the genital tract and is generally acknowledged to be a causative agent of cervical cancer. HPV infection alone is not sufficient to induce cervical cancer and other factors such as steroid hormones are thought to play a role in the establishment and/or progression of this disease. The HPV-16 E2 protein is required for virus replication and modulates viral gene expression whereas the HPV-16 E7 protein is required for cell transformation. We and others have shown that both the E2 and E7 proteins can induce apoptotic cell death in HPV-transformed and non-HPV transformed cell lines. Here we show that the steroid hormones oestrogen and progesterone can both increase the levels of E2- and E7-induced apoptosis. The oestrogen metabolite 16α-hydroxyoestrone also increases E2- and E7-induced cell death and the dietary component indole-3-carbinol, which reduces the formation of 16α-hydroxyoestrone from oestrogen, blocks the effects of oestrogen. Thus the metabolism of oestrogen to 16α-hydroxyoestrone appears to be required for the effects of this hormone on E2- and E7-induced cell death. We also show that the oestrogen receptor antagonist 3-hydroxytamoxifen blocks the effects of oestrogen on E2- and E7-induced cell death, whereas the anti-progesterone RU486 blocks the effects of both progesterone and oestrogen. We discuss these results in terms of the origin and progression of cervical cancer.
The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome
Authors: Chen Xinwen , IJkel Wilfred F. J. , Tarchini Renato , Sun Xiulian , Sandbrink Hans , Wang Hualin , Peters Sander , Zuidema Douwe , Lankhorst René Klein , Vlak Just M. , Hu Zhihong
Published: August 2015

The nucleotide sequence of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) DNA genome was determined and analysed. The circular genome encompasses 131403 bp, has a G+C content of 39·1 mol% and contains five homologous regions with a unique pattern of repeats. Computer-assisted analysis revealed 135 putative ORFs of 150 nt or larger; 100 ORFs have homologues in Autographa californica multicapsid NPV (AcMNPV) and a further 15 ORFs have homologues in other baculoviruses such as Lymantria dispar MNPV (LdMNPV), Spodoptera exigua MNPV (SeMNPV) and Xestia c-nigrum granulovirus (XcGV). Twenty ORFs are unique to HaSNPV without homologues in GenBank. Among the six previously sequenced baculoviruses, AcMNPV, Bombyx mori NPV (BmNPV), Orgyia pseudotsugata MNPV (OpMNPV), SeMNPV, LdMNPV and XcGV, 65 ORFs are conserved and hence are considered as core baculovirus genes. The mean overall amino acid identity of HaSNPV ORFs was the highest with SeMNPV and LdMNPV homologues. Other than three ‘baculovirus repeat ORFs’ (bro) and two ‘inhibitor of apoptosis’ (iap) genes, no duplicated ORFs were found. A putative ORF showing similarity to poly(ADP-ribose) glycohydrolases (parg) was newly identified. The HaSNPV genome lacks a homologue of the major budded virus (BV) glycoprotein gene, gp64, of AcMNPV, BmNPV and OpMNPV. Instead, a homologue of SeMNPV ORF8, encoding the major BV envelope protein, has been identified. GeneParityPlot analysis suggests that HaSNPV, SeMNPV and LdMNPV (group II) have structural genomic features in common and are distinct from the group I NPVs and from the granuloviruses. Cluster alignment between group I and group II baculoviruses suggests that they have a common ancestor.
The open reading frame 1-encoded (‘36K’) protein of Carnation Italian ringspot virus localizes to mitochondria
Authors: Rubino L. , Weber-Lotfi F. , Dietrich A. , Stussi-Garaud C. , Russo M.
Published: August 2015

The localization of the 36 kDa (‘36K’) protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K–GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.
Genome characterization of Botrytis virus F, a flexuous rod-shaped mycovirus resembling plant ‘potex-like’ viruses
Authors: Howitt Robyn L. J. , Beever Ross E. , Pearson Michael N. , Forster Richard L. S.
Published: August 2015

This study reports the first sequence of a flexuous rod-shaped mycovirus and also the first molecular characterization of a virus that infects the plant-pathogenic fungus Botrytis cinerea. The mycovirus Botrytis virus F (BVF) contains an ssRNA genome of 6827 nucleotides and a poly(A) tract at or very near the 3′ terminus. Computer analysis of the genomic cDNA sequence of BVF revealed two potential open reading frames (ORFs) encoding proteins of 212 kDa (ORF1) and 32 kDa (ORF2). ORF1 showed significant sequence identity to the RNA-dependent RNA polymerase (RdRp)-containing proteins of plant ‘tymo-’ and ‘potex-like’ viruses. However, the ORF1 protein contained an opal putative readthrough codon between the helicase and RdRp regions, a feature not seen in this position in ‘tymo-’ and ‘potex-like’ replicases sequenced to date. ORF2 shared amino acid similarity with coat proteins of plant ‘potex-like’ viruses. Three untranslated regions were present in the genome, comprising a region of 63 nucleotides preceding the initiation codon of ORF1, a 93 nucleotide stretch between ORFs 1 and 2 and a 3′-terminal region of 70 nucleotides preceding the poly(A) tract. The nucleotide sequence of a putative defective RNA (D-RNA) of 829 nucleotides was also determined. The D-RNA contained one potential ORF comprising the N-terminal region of the replicase fused in-frame to the C-terminal region of the coat protein. It is proposed that the mycovirus BVF belongs to a new, as yet unassigned genus in the plant ‘potex-like’ virus group.
Rapid identification of a tobacco mosaic virus epitope by using a coat protein gene-fragment–pVIII fusion library
Authors: Holzem Achim , Nähring Jörg M. , Fischer Rainer
Published: August 2015

This study describes the identification of the epitope recognized by the tobacco mosaic virus (TMV) coat protein (CP)-specific monoclonal antibody 29 (MAb29) by displaying a CP gene-fragment library on pVIII of filamentous phage M13. More than 80% of the clones isolated after one round of panning bound specifically to MAb29. DNA sequencing of ten randomly chosen MAb29-specific clones and subsequent sequence comparison revealed a common seven amino acid epitope (ELIRGTG) representing amino acids 131–137 of the TMV CP. The reactivity of MAb29 in competition ELISA towards glutathione S-transferase fused to this epitope was stronger than that towards full-length wild-type TMV CP, confirming the epitope sequence determined by gene-fragment phage display. This demonstrated that gene-fragment libraries displayed on the phage surface as fusion proteins with the filamentous bacteriophage gene VIII are useful tools for rapid identification of linear epitopes recognized by MAbs.
Deletion or substitution of the aphthovirus 3′ NCR abrogates infectivity and virus replication
Authors: Sáiz M. , Gómez S. , Martínez-Salas E. , Sobrino F.
Published: August 2015

The 3′ noncoding region (NCR) of the genomic picornaviral RNA is believed to contain majorcis-acting signals required for negative-strand RNA synthesis. The 3′ NCR of foot-and-mouth disease virus (FMDV) was studied in the context of a full-length infectious clone in which the genetic element was deleted or exchanged for the equivalent region of a distantly related swine picornavirus, swine vesicular disease virus (SVDV). Deletion of the 3′ NCR, while maintaining the intact poly(A) tail as well as its replacement for the SVDV counterpart, abrogated virus replication in susceptible cells as determined by infectivity and Northern blot assays. Nevertheless, the presence of the SVDV sequence allowed the synthesis of low amounts of chimeric viral RNA at extended times post-transfection as compared to RNAs harbouring the 3′ NCR deletion. The failure to recover viable viruses or revertants after several passages on susceptible cells suggests that the presence of specific sequences contained within the FMDV 3′ NCR is essential to complete a full replication cycle and that FMDV and SVDV 3′ NCRs are not functionally interchangeable.
Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1–E2 interaction
Authors: Woytek Kelly J. , Rangasamy Dhandapani , Bazaldua-Hernandez Cynthia , West Mike , Wilson Van G.
Published: August 2015

The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using bothin vivoandin vitroassays to evaluate the regions of the two proteins necessary for the E1–E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1–311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315–605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N–E2TAD interaction is not essential for replication function and is probably involved in some other E1–E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.
Roles of Epstein–Barr virus glycoproteins gp350 and gp25 in the infection of human epithelial cells
Authors: Maruo Seiji , Yang Lixin , Takada Kenzo
Published: August 2015

Epstein–Barr virus (EBV) is associated with various epithelial malignancies such as nasopharyngeal carcinoma and gastric carcinoma, and causes oral hairy leukoplakia, a productive EBV infection of the differentiated epithelium of the tongue. However, it is not clear by what mechanism EBV infects epithelial cells. We generated a recombinant EBV that expresses enhanced green fluorescent protein in order to monitor EBV entrance into epithelial cells quickly and quantitatively. Using this monitoring system, we examined the roles of gp350 and gp25 in EBV infection of epithelial cells by utilizing soluble forms of the gp350 and gp25 proteins. EBV infection of three of four examined epithelial cell lines, 293, NU-GC-3 and Lovo, was almost completely blocked by pretreatment of cells with a soluble form of gp350 (designated gp350Ig), and this blockage was dependent on the CD21-binding region of gp350. On the other hand, infection of the other epithelial cell line, AGS, was not inhibited at all by pretreatment with gp350Ig. Moreover, we found that a soluble form of gp25 (designated gp25Ig) preferentially bound to epithelial cells rather than B cells, and pretreatment of cells with gp25Ig substantially blocked EBV infection of some epithelial cells. These results indicate the existence of two distinct pathways in EBV infection of epithelial cells, a gp350-dependent pathway and a gp350-independent pathway, and that gp25 can play a role in the infection of some epithelial cells.
Human cytomegalovirus binding to heparan sulfate proteoglycans on the cell surface and/or entry stimulates the expression of human leukocyte antigen class I
Authors: Song Byung H. , Lee Gyu C. , Moon Myung S. , Cho Yeon H. , Lee Chan H.
Published: August 2015

Human cytomegalovirus (HCMV) is known to down-regulate the expression of human leukocyte antigen (HLA) class I, the process of which involves a subset of virus genes. Infection of human foreskin fibroblast (HFF) cells with UV-inactivated HCMV (UV-HCMV), however, resulted in an increase in HLA class I presentation on the cell surface in the absence of HCMV gene expression. Heparin, which inhibits the interaction of virus particles with cell surface heparan sulfate proteoglycans (HSPGs), blocked the effect of UV-HCMV on HLA class I expression. Pretreatment of cells with heparinase I decreased in a dose-dependent manner the effect of UV-HCMV on HLA class I expression enhancement. Sodium chlorate, which is known to inhibit the sulfation of HSPGs, gave a similar result. Pretreatment of UV-HCMV with trypsin or monoclonal antibody reactive with the envelope glycoprotein gB reduced the increase in HLA class I expression on the HFF cell surface by UV-HCMV. RT–PCR analysis demonstrated that the increase in HLA class I presentation on the HFF cell surface was due to an increase in HLA class I transcription. Thus, binding of HCMV particles to cell surface HSPGs appears to be required for the stimulation of HLA class I expression. It is also possible that virus entry, in addition to binding to HSPGs, may be involved in the stimulation of HLA class I expression, since the UV-HCMV entered the cells and all treatments to block virus binding to HSPGs would necessarily prevent virus entry.
Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis–splenomegaly syndrome in the United States
Authors: Haqshenas G. , Shivaprasad H. L. , Woolcock P. R. , Read D. H. , Meng X. J.
Published: August 2015

Hepatitis–splenomegaly (HS) syndrome is an emerging disease in chickens in North America; the cause of this disease is unknown. In this study, the genetic identification and characterization of a novel virus related to human hepatitis E virus (HEV) isolated from bile samples of chickens with HS syndrome is reported. Based upon the similar genomic organization and significant sequence identity of this virus with HEV, the virus has been tentatively named avian HEV in order to distinguish it from human and swine HEV. Electron microscopy revealed that avian HEV is a non-enveloped virus particle of 30–35 nm in diameter. The sequence of the 3′ half of the viral genome (∼4 kb) was determined. Sequence analyses revealed that this genomic region contains the complete 3′ non-coding region, the complete genes from open reading frames (ORFs) 2 and 3, the complete RNA-dependent RNA polymerase (RdRp) gene and a partial helicase gene from ORF 1. The helicase gene is the most conserved gene between avian HEV and other HEV strains, displaying 58–61% aa and 57–60% nt sequence identities. The RdRp gene of avian HEV shares 47–50% aa and 52–53% nt sequence identities and the putative capsid gene (ORF 2) of avian HEV shares 48–49% aa and 48–51% nt sequence identities with the corresponding regions of other known HEV strains. Phylogenetic analysis indicates that avian HEV is genetically related to, but distinct from, other known HEV strains. This discovery has important implications for HEV animal models, nomenclature and natural history.
Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus
Authors: Tsuchiya Emi , Sugawara Kanetsu , Hongo Seiji , Matsuzaki Yoko , Muraki Yasushi , Li Zhu-Nan , Nakamura Kiyoto
Published: August 2015

The antigenic structure of influenza A/H2N2 virus haemagglutinin (HA) was analysed using 19 monoclonal antibodies (MAbs) against the HA of A/Kayano/57. The antibodies were classified into three groups: group I had both haemagglutination inhibition and neutralization activities, group II had neutralization activity but no haemagglutination inhibition activity and group III had neither activity. Analysis of escape mutants selected by each of the group I and II antibodies identified six distinct antigenic sites: four (I-A to I-D) were recognized by group I MAbs and two (II-A and II-B) were recognized by group II MAbs. Sequence analysis of the HA genes of the escape mutants demonstrated that sites I-A, I-B and I-C form a contiguous antigenic area that contains the regions corresponding to antigenic sites A, B and D on the H3 molecule and that sites I-D and II-B are the equivalents of sites E and C, respectively, suggesting that the antigenic structure of the H2 molecule is largely similar to that of the H3 molecule. However, the H2 molecule differed from the H3 molecule in having a highly conserved antigenic site (II-A) in the stem domain. It was also found that most of the escape mutants selected by antibodies to sites I-A, I-B and I-C acquired a new glycosylation site at position 160, 187 or 131, respectively, which indicates that A/H2N2 viruses have the potential to gain at least one additional oligosaccharide on the tip of the HA, although this has never occurred during 11 years of its circulation in humans.
Inhibition of release of lentivirus particles with incorporated human influenza virus haemagglutinin by binding to sialic acid-containing cellular receptors
Authors: Bosch Valerie , Kramer Beatrice , Pfeiffer Tanya , Stärck Lilian , Steinhauer David A.
Published: August 2015

Mutants of the haemagglutinin (HA) gene of human influenza virus A/Aichi/2/68 (H3N2) encoding HA proteins that are proteolytically cleaved intracellularly, defective in binding to cellular receptors or defective for acylation within the cytoplasmic C terminus have been generated. Here, the properties of these mutated HA molecules are described and their incorporation into the lipid membrane of released human immunodeficiency virus (HIV)-like particles is analysed. It is demonstrated that, when produced from cells coexpressing any of the binding-competent Aichi-HA molecules, release of HIV-like particles into the extracellular medium is reduced and the particles that are released fail to incorporate Aichi-HA. These blocks in release and incorporation, respectively, can both be overcome. The release of normal amounts of particles with incorporated HA can be achieved either by mutation of the receptor-binding site on the Aichi-HA molecule or by removal of sialic acid from surface proteins with neuraminidase. In contrast, as a result of blockage of the sialic acid-binding site by sialidated oligosaccharides on the HA itself, the HA of influenza virus A/FPV/Rostock/34 (H7N1) is efficiently incorporated into HIV-like particles. These results, namely that particle release can be inhibited by interactions between the incorporated glycoprotein and the cell surface and/or that interactions with other cellular components can be inhibitory to incorporation into retrovirus envelopes, probably reflect general principles that may hold for many viral and cellular glycoproteins.
Molecular characterization of a complex, recombinant human immunodeficiency virus type 1 (HIV-1) isolate (A/G/J/K/?): evidence to support the existence of a novel HIV-1 subtype
Authors: Paraskevis D. , Magiorkinis E. , Magiorkinis G. , Anastassopoulou C. , Lazanas M. , Chrysos G. , Vandamme A. M. , Hatzakis A.
Published: August 2015

Recombination is one of several factors that contribute to the great genetic diversity of human immunodeficiency virus type 1 (HIV-1). In the current study, analysis of the full-length genome of a novel complex mosaic HIV-1 isolate (99GR303) from a Greek sailor who was possibly infected in Sierra Leone, Africa is presented. The 99GR303 isolate was found to comprise genomic regions belonging to subtypes A, G, J and K as well as of regions of a subtype that remains unclassified. For a partial region of env as well as vpr, no apparent similarity to the known HIV-1 subtypes or to any of the circulating recombinant forms was found. In fact, in the partial env gene, including the C2-V3 region, the 99GR303 isolate formed a new clade, suggesting the existence of an additional HIV-1 subtype. Thus, novel recombinants embody partial genomic regions which may have originated either from subtypes that existed in the past and became extinct or from contemporary subtypes that are extremely rare.
Molecular characterization of the Rep protein of the blackgram isolate of Indian mungbean yellow mosaic virus
Authors: Pant V. , Gupta D. , Choudhury N. Roy , Malathi V. G. , Varma A. , Mukherjee S. K.
Published: August 2015

The complete nucleotide sequence of the blackgram isolate of mungbean yellow mosaic virus, IMYMV-Bg, which infects legumes in India, was determined and compared at the amino acid level with those of other whitefly-transmitted geminiviruses. The genome organization of IMYMV-Bg was similar to that of the begomoviruses. A unique feature of the genome organization was the sequence divergence of the common region (CR) between DNA-A and DNA-B. In order to understand the mechanism of viral DNA replication, the replication initiator protein, Rep, of IMYMV-Bg was overexpressed in E. coli. The recombinant and refolded Rep bound to CR-sequences of IMYMV-Bg in a specific manner. In this study, evidence is presented for ATP-upregulated cleavage function and ATP-mediated conformational change of Rep. It is hypothesized that, although ATP is not required for cleavage, ATP-mediated conformational changes may result in better access of Rep to the DNA-cleavage site. Evidence is also presented for a site-specific topoisomerase function of Rep, which has not been demonstrated before. The Rep protein can be classified as a type-I topoisomerase because of its nicking activity and sensitivity towards camptothecin, a topoisomerase type-I inhibitor.
Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively
Authors: Nurkiyanova Kulpash M. , Ryabov Eugene V. , Kalinina Natalia O. , Fan Yongchang , Andreev Igor , Fitzgerald Alexander G. , Palukaitis Peter , Taliansky Michael
Published: August 2015

Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP–RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed.
Localization of viral proteins in cells infected with bovine viral diarrhoea virus
Authors: Grummer B. , Beer M. , Liebler-Tenorio E. , Greiser-Wilke I.
Published: August 2015

Bovine viral diarrhoea virus (BVDV) is a member of the genusPestiviruswithin the familyFlaviviridae. In this report, protein localization studies were performed to assess the mechanism for the release of mature virus particles from infected cells. Since BVDV is an enveloped virus, budding from either intra- or extracellular membranes is feasible. A prerequisite for the latter mechanism is the integration of viral glycoproteins into the host cell membrane. Using monoclonal antibodies (MAbs) directed against the viral envelope glycoproteins E2 and ERNS, no specific signals were detected on the surface of BVDV-infected cells by indirect fluorescence, confocal microscopy or fluorescence-activated cell sorter analyses. Furthermore, biotin-labelled cell surface proteins of virus-infected and non-infected cells were not detected by immunoprecipitation using MAbs directed against ERNSand E2 or the non-structural protein NS2-3. None of these proteins was detected on the cell surface. In addition, to analyse the intracellular localization of the two viral glycoproteins ERNSand E2 and the non-structural proteins NS2-3 and NS3, subcellular fractionation of virus-infected cells followed by radioimmunoprecipitation with the MAbs were performed. These results led to the conclusion that the BVDV envelope glycoproteins ERNSand E2 as well as the non-structural proteins NS2-3 and NS3 were almost quantitatively associated with intracellular membranes. These findings indicate that BVDV is released by budding into the cisternae of the endoplasmic reticulum and that there seems to be no correlation between the location and function of the analysed proteins.
Viable porcine arteriviruses with deletions proximal to the 3′ end of the genome
Authors: Verheije M. H. , Kroese M. V. , Rottier P. J. M. , Meulenberg J. J. M.
Published: August 2015

In order to obtain attenuated live vaccine candidates of porcine reproductive and respiratory syndrome virus (PRRSV), a series of deletions was introduced at the 3′ end of the viral genome using an infectious cDNA clone of the Lelystad virus isolate. RNA transcripts from the full-length cDNA clones were transfected into BHK-21 cells. The culture supernatant of these cells was subsequently used to infect porcine alveolar macrophages to detect the production of progeny virus. It is shown that C-terminal truncation of the nucleocapsid (N) protein, encoded by ORF7, was tolerated for up to six amino acids without blocking the production of infectious virus. Mutants containing larger deletions produced neither virus nor virus-like particles containing viral RNA. Deletion analysis of the 3′ UTR immediately downstream of ORF7 showed that infectious virus was still produced after removal of seven nucleotides behind the stop codon of ORF7. Deletion of 32 nucleotides in this region abolished RNA replication and, consequently, no infectious virus was formed. Serial passage on porcine alveolar macrophages demonstrated that the viable deletion mutants were genetically stable at the site of mutation. In addition, the deletions did not affect the growth properties of the recombinant viruses in vitro, while their antigenic profiles were similar to that of wild-type virus. Immunoprecipitation experiments with the six-residue N protein-deletion mutant confirmed that the truncated protein was indeed smaller than the wild-type N protein. The deletion mutants produced in this study are interesting candidate vaccines to prevent PRRS disease in pigs.
Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele
Authors: D’Aloja Paola , Santarcangelo Anna Claudia , Arold Stefan , Baur Andreas , Federico Maurizio
Published: August 2015

The primary human immunodeficiency virus type 1 (HIV-1) Nef mutant F12-HIVNef is characterized by three rare amino acid substitutions, G140E, V153L and E177G. It was reported previously that the expression of F12-HIVNef in the context of the highly productive NL4-3 HIV-1 strain blocks virus replication at the level of virus assembly and/or release by a mechanism depending on the presence of the CD4 intracytoplasmic tail. Here, it is reported that NL4-3 HIV-1 strains expressing F12-HIVnef alleles that were back-mutated in each amino acid substitution readily replicated in CD4+ cells. Attempting to correlate possible functional alterations with antiviral effects, both F12-HIVNef and its back mutants were tested in terms of well-characterized markers of Nef expression. Both F12-HIVNef and its G177E back mutant did not down-regulate CD4 as the consequence of a greatly reduced rate of CD4 internalization. On the other hand, F12-HIVNef as well as the E140G and L153V back mutants failed to activate the p62 Nef-associated kinase (p62NAK). Thus, only F12-HIVNef was defective in both accelerated rates of CD4 internalization and p62NAK activation, whereas at least one Nef function was restored in all of the back mutants. Infection of cells expressing Nef-resistant CD4 molecules with HIV-1 strains encoding F12-HIVNef back mutants showed that both the lack of accelerated CD4 endocytosis and an, as yet, still unidentified function are required for the F12-HIVNef inhibitory phenotype. These results provide a detailed functional analysis of the F12-HIVnef allele and support the idea that both CD4 accelerated internalization and p62NAK activation are part of the essential steps in the virus replication cycle.
Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae
Authors: Phenix Kerry V. , Weston Jonathan H. , Ypelaar Ingrid , Lavazza Antonio , Smyth Joan A. , Todd Daniel , Wilcox Graham E. , Raidal Shane R.
Published: August 2015

The circular, single-stranded DNA genome of a novel circovirus of canaries, tentatively named canary circovirus (CaCV), was cloned and sequenced. Sequence analysis indicated that the genome was 1952 nucleotides (nt) in size and had the potential to encode three viral proteins, including the putative capsid and replication-associated (Rep) proteins. The CaCV genome shared greatest sequence similarity (58·3% nt identity) with the newly characterized columbid circovirus (CoCV) and was more distantly related to the two porcine circovirus strains, PCV1 and PCV2, beak and feather disease virus (BFDV) and a recently isolated goose circovirus (GCV) isolate (46·8–50·9% nt identity). In common with other members of the Circovirus genus, several nt structures and amino acid motifs thought to be implicated in virus replication were identified on the putative viral strand. Phylogenetic analysis of both the capsid and Rep protein-coding regions provided further evidence that CaCV is more closely related to CoCV and BFDV and more distantly related to GCV, PCV1 and PCV2.
Genome organization of the densovirus from Bombyx mori (BmDNV-1) and enzyme activity of its capsid
Authors: Li Y. , Zádori Z. , Bando H. , Dubuc R. , Fédière G. , Szelei J. , Tijssen P.
Published: August 2015

Bombyx mori densovirus (BmDNV-1), on the basis of the previously reported genome sequence, constitutes by itself a separate genus (Iteravirus) within the Densovirinae subfamily of parvoviruses. Inconsistencies in the genome organization, however, necessitated its reassessment. The genome sequence of new clones was determined and resulted in a completely different genome organization. The corrected sequence also contained conserved sequence motifs found in other parvoviruses. Some amino acids in the highly conserved domain in the unique region of VP1 were shared by critical amino acids in the catalytic site and Ca2+-binding loop of secreted phospholipase A2, such as from snake and bee venoms. Expression of this domain and determination of enzyme activity demonstrated that capsids have a phospholipase A2 activity thus far unknown to occur in viruses. This viral phospholipase A2, which is required shortly after entry into the cell, showed a substrate preference for phosphatidylethanolamine and phosphatidylcholine over phosphatidylinositol.
Comparative reactions of recombinant papaya ringspot viruses with chimeric coat protein (CP) genes and wild-type viruses on CP-transgenic papaya
Authors: Chiang Chu-Hui , Wang Ju-Jung , Jan Fuh-Jyh , Yeh Shyi-Dong , Gonsalves Dennis
Published: August 2015

Transgenic papaya cultivars SunUp and Rainbow express the coat protein (CP) gene of the mild mutant of papaya ringspot virus (PRSV) HA. Both cultivars are resistant to PRSV HA and other Hawaii isolates through homology-dependent resistance via post-transcriptional gene silencing. However, Rainbow, which is hemizygous for the CP gene, is susceptible to PRSV isolates from outside Hawaii, while the CP-homozygous SunUp is resistant to most isolates but susceptible to the YK isolate from Taiwan. To investigate the role of CP sequence similarity in overcoming the resistance of Rainbow, PRSV HA recombinants with various CP segments of the YK isolate were constructed and evaluated on Rainbow, SunUp and non-transgenic papaya. Non-transgenic papaya were severely infected by all recombinants, but Rainbow plants developed a variety of symptoms. On Rainbow, a recombinant with the entire CP gene of YK caused severe symptoms, while recombinants with only partial YK CP sequences produced a range of milder symptoms. Interestingly, a recombinant with a YK segment from the 5′ region of the CP gene caused very mild, transient symptoms, whereas recombinants with YK segments from the middle and 3′ parts of the CP gene caused prominent and lasting symptoms. SunUp was resistant to all but two recombinants, which contained the entire CP gene or the central and 3′-end regions of the CP gene and the 3′ non-coding region of YK, and the resulting symptoms were mild. It is concluded that the position of the heterologous sequences in the recombinants influences their pathogenicity on Rainbow.