bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic heterogeneity of human endogenous retrovirus particles produced by teratocarcinoma cell lines
Authors: Bieda Katrin , Hoffmann Andreas , Boller Klaus
Published: August 2015

Human endogenous retrovirus (HERV) sequences represent about 0·5% of the human genome. The only HERV known to express virus particles is human teratocarcinoma-derived virus (HTDV), which is now termed HTDV/HERV-K. Between 25 and 50 different copies of HERV-K are present in the human genome, three of which contain full-length genes for viral structural proteins. To determine whether genes of different HERV-K proviruses can be expressed, the morphologies and protein expression patterns of HTDV/HERV-K produced by various human teratocarcinoma cell lines were compared. Three different types of retrovirus-like particles were observed, showing differences in the presence of viral surface proteins and the existence of free mature virions. These distinct morphological features between virion types were in accordance with the results of immunoblotting analyses that revealed differences in the cleavage of a viral Gag protein precursor and the presence of a putative Env protein. These data suggest that different HERV-K proviruses are transcribed in human teratocarcinoma cell lines.
Characterization of H5N2 influenza viruses from Italian poultry
Authors: Donatelli Isabella , Campitelli Laura , Di Trani Livia , Puzelli Simona , Selli Lucia , Fioretti Alessandro , Alexander Dennis J. , Tollis Maria , Krauss Scott , Webster Robert G.
Published: August 2015

From October 1997 to January 1998, highly pathogenic H5N2 avian influenza viruses caused eight outbreaks of avian influenza in northern Italy. A nonpathogenic H5N9 influenza virus was also isolated during the outbreaks as a result of virological and epidemiological surveillance to control the spread of avian influenza to neighbouring regions. Antigenic analysis showed that the Italian H5N2 isolates were antigenically similar to, although distinguishable from, A/HK/156/97, a human influenza H5N1 virus isolated in Hong Kong in 1997. Phylogenetic analysis of the haemagglutinin (HA) genes showed that the highly pathogenic Italian viruses clustered with the Hong Kong strains, whereas the nonpathogenic H5N9 virus, despite its epidemiological association with the highly pathogenic Italian isolates, was most closely related to the highly pathogenic A/Turkey/England/91 (H5N1) strain. Like the HA phylogenetic tree, the nonstructural (NS) phylogenetic tree showed that the H5N2 Italian virus genes are clearly separate from those of the H5N9 strain. In contrast, results of the phylogenetic analysis of nucleoprotein (NP) genes indicated a closer genetic relationship between the two Italian virus groups, a finding suggesting a common progenitor. Comparison of the HA, NS and NP genes of the Italian H5 strains with those of the H5N1 viruses simultaneously circulating in Hong Kong revealed that the two groups of viruses do not share a recent common ancestor. No virological and serological evidence of bird-to-human transmission of the Italian H5N2 influenza viruses was found.
The size of encapsidated single-stranded DNA determines the multiplicity of African cassava mosaic virus particles
Authors: Frischmuth Thomas , Ringel Margit , Kocher Cornelia
Published: August 2015

Transgenic Nicotiana benthamiana plants harbouring a defective interfering (DI) DNA of African cassava mosaic virus (ACMV) and control plants were inoculated with ACMV. Virus particles were purified from infected plants, separated in sucrose gradients and fractions were analysed by Southern blotting. Transgenic plant-derived virus particles taken from the top fractions of sucrose gradients contained DI DNA, middle fractions contained a mixture of genomic and DI DNA and bottom fractions contained a mixture of multimeric, genomic and DI DNA. Virus particles from selected top, middle and bottom fractions were analysed by electron microscopy. In fractions containing only DI DNA, isometric particles of 18–20 nm were detected. In fractions containing DI DNA as well as genomic size DNA, isometric and geminate particles were found. Fractions containing multimeric size DNA were found to comprise particles consisting of three subunits adjacent to geminate particles. From these data, it is concluded that the size of encapsidated DNA determines the multiplicity of ACMV particles.
Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system
Authors: López Lissett , Urzainqui Ana , Domínguez Elvira , García Juan Antonio
Published: August 2015

Potyvirus CI RNA helicase is a protein involved in RNA genome replication and virus movement. The protein aggregates in the cytoplasm of infected cells to form typical cylindrical inclusions. A yeast two-hybrid system was used to analyse interactions of the CI RNA helicase from plum pox potyvirus (PPV) with itself and with other viral proteins. No interactions could be detected of full-length CI protein with itself or with PPV P3/6K1, NIa, NIb or CP proteins. However, positive self-interactions were detected for N-terminal fragments of the CI protein, allowing the mapping of a CI–CI binding domain to the N-terminal 177 aa of the protein. Further deletion analysis suggested that several regions of this domain contribute to the interaction. Moreover, pull-down experiments demonstrate that, at leastin vitro, full-length PPV CI protein is able to self-interact in the absence of other virus or plant factors.
Vascular cell adhesion molecule-1 induced by human T-cell leukaemia virus type 1 Tax protein in T-cells stimulates proliferation of human T-lymphocytes
Authors: Valentin Hélène , Hamaia Samir , König Stéphane , Gazzolo Louis
Published: August 2015

Human T-cell leukaemia/lymphotropic virus type 1 (HTLV-1), aetiologically linked to lymphoproliferative as well as inflammatory diseases, infects and activates CD4+helper T-cells and thus alters immunoregulatory pathways. The viral regulatory Tax protein has been shown previously to induce the expression of vascular cell adhesion molecule-1 (VCAM-1) by T-cells. To determine the functional role of this adhesion molecule, Jurkat T-cells stably expressing either Tax or both Tax and Rex (another viral regulatory protein) were used in binding and coculture assays performed with either control Jurkat cells or primary human T-lymphocytes. Evidence was provided that VCAM-1 acting in synergy with leucocyte function-associated antigen-3 promotes T-cell–T-cell interactions and increases T-cell proliferation. Interestingly, Rex was found to modulate these events. These data establish that VCAM-1 induced by Tax on T-cells thus contributes to the immunopathological process triggered by HTLV-1 infection.
Porcine endogenous retroviruses: in vitro host range and attempts to establish small animal models
Authors: Specke Volker , Tacke Stefan J. , Boller Klaus , Schwendemann Jochen , Denner Joachim
Published: August 2015

Using transgenic pigs as the source of cells or organs for xenotransplantation is associated with the risk of porcine endogenous retrovirus (PERV) transmission. Multiple proviruses are integrated into the genome of all pigs, and virus particles, some of which are able to infect human cells, are released from normal pig cells. In order to evaluate the potential risk posed by the transmission of PERVs, in vitro infection studies were performed as a basis for small animal as well as non-human primate models. In vitro infectivity was demonstrated for permanent cell lines and primary cells from a wide range of species. Productive infection was shown using reverse transcriptase (RT) assays and RT–PCR for mink, feline and human kidney cell lines, primary rhesus peripheral blood mononuclear cells (PBMCs), and baboon spleen cells and PBMCs as well as for different human lymphoid and monocyte cell lines and PBMCs. In an attempt to establish a small animal model, naive guinea pigs, non-immunosuppressed rats, rats immunosuppressed by cyclosporin-A and immunosuppressed rats treated with cobra venom factor were inoculated with PERVs produced from porcine kidney PK-15 cells, infected human 293 kidney cells and mitogen-stimulated porcine PBMCs. Animals were also inoculated with PERV-producing PK-15 and 293 cells. No antibodies against PERV and no provirus integration were observed in any of the treated animals. This suggests that productive infection of these animals did not occur in this experimental setting.
Human telomerase reverse transcriptase-immortalized MRC-5 and HCA2 human fibroblasts are fully permissive for human cytomegalovirus
Authors: McSharry B. P. , Jones C. J. , Skinner J. W. , Kipling D. , Wilkinson G. W. G.
Published: August 2015

MRC-5 cells are a well-characterized human diploid fibroblast cell line approved for vaccine production and favoured for the routine propagation of human cytomegalovirus (HCMV). Ectopic expression of telomerase in fibroblasts is capable of overcoming replicative senescence induced by telomere shortening. Following delivery of the hTERT gene to MRC-5 cells using a retrovirus vector three clones were generated that (i) expressed functional telomerase activity, (ii) exhibited telomere extension and (iii) were sustained for >100 population doublings. Immortalized MRC-5-hTERT and also HCA2-hTERT human fibroblasts were both fully permissive for HCMV as determined by plaque assay, studies of virus growth kinetics and measurement of virus yields. Furthermore, telomerase-immortalized HCA2 cells proved capable of supporting the stable maintenance of an EBV-based episomal vector with efficient transgene expression when driven by the HCMV immediate early promoter. An indicator cell line suitable for the efficient detection of HCMV infection was also generated using an episome containing a reporter gene (lacZ) under the control of the HCMV β-2.7 early promoter. Telomerase immortalization of human fibroblasts will thus facilitate the growth and detection of HCMV and also the generation of helper cell lines for the propagation of HCMV deletion mutants. Immortalization of fibroblasts by telomerase does not affect cell morphology or growth characteristics. The MRC-5-hTERT clones may therefore be suitable for additional applications in virology, cell biology, vaccine production and biotechnology.
A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness
Authors: Sugauchi Fuminaka , Mizokami Masashi , Orito Etsuro , Ohno Tomoyoshi , Kato Hideaki , Suzuki Seiji , Kimura Yoshihide , Ueda Ryuzo , Butterworth L. A. , Cooksley W. G. E.
Published: August 2015

There have been no reports of DNA sequences of hepatitis B virus (HBV) strains from Australian Aborigines, although the hepatitis B surface antigen (HBsAg) was discovered among them. To investigate the characteristics of DNA sequences of HBV strains from Australian Aborigines, the complete nucleotide sequences of HBV strains were determined and subjected to molecular evolutionary analysis. Serum samples positive for HBsAg were collected from five Australian Aborigines. Phylogenetic analysis of the five complete nucleotide sequences compared with DNA sequences of 54 global HBV isolates from international databases revealed that three of the five were classified into genotype D and were most closely related in terms of evolutionary distance to a strain isolated from a healthy blood donor in Papua New Guinea. Two of the five were classified into a novel variant genotype C, which has not been reported previously, and were closely related to a strain isolated from Polynesians, particularly in the X and Core genes. These two strains of variant genotype C differed from known genotype C strains by 5·9–7·4% over the complete nucleotide sequence and 4·0–5·6% in the small-S gene, and had residues Arg122, Thr127 and Lys160, characteristic of serotype ayw3, which have not been reported previously in genotype C. In conclusion, this is the first report of the characteristics of complete nucleotide sequences of HBV from Australian Aborigines. These results contribute to the investigation of the worldwide spread of HBV, the relationship between serotype and genotype and the ancient common origin of Australian Aborigines.
The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences
Authors: Donnelly Michelle L. L. , Hughes Lorraine E. , Luke Garry , Mendoza Heidi , ten Dam Edwin , Gani David , Ryan Martin D.
Published: August 2015

The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins ‘cleaving’ at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to β-glucuronidase (GUS) – forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the invitrotranslation products (∼90%) were in the form of the ‘cleavage’ products GUS and [GFP2A]. Alternative models have been proposed to account for the ‘cleavage’ activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring ‘2A-like’ sequences were used to program invitrotranslation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that ‘cleavage’ occurred in constructs in which all the candidate nucleophilic residues were substituted – with the exception of aspartate-12. This residue is not, however, conserved amongst all functional ‘2A-like’ sequences. ‘2A-like’ sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences inTrypanosomaspp. and a eubacterial α-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial α-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of thePicornaviridae. Taken together, these data provide additional evidence that neither FMDV 2A nor ‘2A-like’ sequences are autoproteolytic elements.
Comparison of capsid sequences from human and animal astroviruses
Authors: Jonassen Christine M. , Jonassen Tom Ø. , Saif Yehia M. , Snodgrass David R. , Ushijima Hiroshi , Shimizu Mitsugu , Grinde Bjørn
Published: August 2015

We have sequenced the genomic 3′-end, including the structural gene, of human astrovirus (HAstV) serotype 7 and morphologically related viruses infecting pig (PAstV), sheep (OAstV) and turkey (TAstV-1). These sequences were compared with corresponding astrovirus sequences available in the nucleic acid databases, including sequences of the seven other HAstV serotypes, two other avian astroviruses (TAstV-2 and avian nephritis virus) and astrovirus from cat (FAstV). A 35 nt stem–loop motif near the 3′-end of the genome, previously described as being highly conserved, was present in all of the astroviruses except TAstV-2. In the N-terminal half of the capsid precursor protein, there were several short conserved peptide motifs. Otherwise the capsid proteins of astroviruses infecting different hosts were highly divergent. Calculation of genetic distances revealed that the distance between FAstV and HAstV is comparable to the largest distances between different HAstV serotypes. Higher similarities between the HAstV, FAstV and PAstV capsid sequences suggest interspecies transmissions involving humans, cats and pigs relatively recently in the evolutionary history of astroviruses.
Differential processing and presentation of the H-2Db-restricted epitope from two different strains of influenza virus nucleoprotein
Authors: Potter Paul , Tourdot Sophie , Blanchard Tom , Smith Geoffrey L. , Gould Keith G.
Published: August 2015

The influenza virus strains A/NT/60/68 and A/PR/8/34 both have an immunodominant Db-restricted epitope in their nucleoprotein (NP) at amino acid residues 366–374, with two amino acid differences between the epitopes. Cross-reactive cytotoxic T lymphocytes (CTLs) were generated by priming mice with the influenza virus A/NT/60/68 NP and restimulatingin vitrowith influenza virus A/PR/8/34. CTLs that gave high levels of specific lysis recognized target cells infected with either strain of influenza virus with similar efficiency. Surprisingly, when target cells were infected with recombinant vaccinia viruses (VV) expressing the two different NPs, presentation of the Db-restricted epitope from the A/NT/60/68 NP was extremely poor, whereas presentation of the equivalent epitope from the A/PR/8/34 NP was as efficient as in influenza virus-infected cells. This difference was observed in spite of the fact that the two NP sequences show 94% identity at the amino acid sequence level. Experiments with additional cross-reactive CTL cell lines which recognized target cells less efficiently revealed a similar difference in presentation between the two NP epitopes in influenza virus-infected cells and showed a difference in the efficiency of presentation of the Db-restricted epitope from the two NP molecules independent of VV infection. The results show that two equivalent epitopes in highly similar proteins are processed with very different efficiency, even though they are both immunodominant epitopes. They also suggest that the previously described inhibition of antigen presentation by VV is a general, non-specific effect, which is more apparent for epitopes that are processed and presented less efficiently.
Nucleotides at the extremities of the viral RNA of influenza C virus are involved in type-specific interactions with the polymerase complex
Authors: Crescenzo-Chaigne Bernadette , van der Werf Sylvie
Published: August 2015

Influenza A and C viruses share common sequences in the terminal noncoding regions of the viral RNA segments. Differences at the 5′- and 3′-ends exist, however, that could contribute to the specificity with which the transcription/replication signals are recognized by the cognate polymerase complexes. Previously, by making use of a transient expression system for the transcription and replication of a reporter RNA template bearing either type A or type C extremities, it was shown that a type C RNA template is transcribed and replicated with equal efficiency by either the type A or the type C polymerase complex, whereas a type A RNA template is less efficiently transcribed and replicated by the type C polymerase complex than by the type A complex. To explore the contribution of the nucleotides at the extremities of the RNAs to this type-specificity, the effect of mutations introduced either alone or in combination at nucleotide 5 at the 3′-end and at nucleotides 3′, 6′ or 8′ at the 5′-end of type A or C RNA templates were studied in the presence of either the type A or the type C polymerase complex. The results indicate that the nature of nucleotides 5 and 6′ contribute to type-specificity. Moreover, these results underline the importance of the base pairing between nucleotide 3′ and 8′ at the 5′-end of the RNA. Thus, it could be suggested that the nature of the nucleotides as well as the stability of the secondary structure at the extremities of the viral RNA are important determinants of type-specificity.
A bovine macrophage cell line supports bovine herpesvirus-4 persistent infection
Authors: Donofrio Gaetano , van Santen Vicky L.
Published: August 2015

Although bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, has been demonstrated in many tissues during persistent BHV-4 infection, a likely site of virus persistence is in cells of the monocyte/macrophage lineage. To establish an in vitro model of persistent infection potentially useful for examining the molecular mechanisms of BHV-4 persistence/latency, we infected the bovine macrophage cell line BOMAC. Following extensive cell death, surviving cells were found to be persistently infected, maintaining the viral genome over many passages and producing low levels of infectious virus. Although selection was unnecessary for the maintenance of the viral genome, cells persistently infected with recombinant BHV-4 containing a neomycin-resistance gene could be selected with geneticin, thus confirming that persistent BHV-4 infection was compatible with cell survival and replication. Furthermore, persistent BHV-4 infection caused no decrease in the growth rate of BOMAC cells. Sodium butyrate, which reactivates latent gammaherpesviruses in vitro, or dexamethasone, which reactivates latent BHV-4 in vivo, increased viral DNA by 10- to 15-fold in persistently infected BOMAC cells. This suggests that reactivation of latent BHV-4 by dexamethasone in vivo might involve direct action of dexamethasone on latently infected cells.
Reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations
Authors: Nielsen Henriette S. , Oleksiewicz Martin B. , Forsberg Roald , Stadejek Tomasz , Bøtner Anette , Storgaard Torben
Published: August 2015

A live attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine virus has been shown to revert to virulence under field conditions. In order to identify genetic virulence determinants, ORF1 from the attenuated vaccine virus and three Danish vaccine-derived field isolates was sequenced and compared with the parental strain of the vaccine virus (VR2332). This revealed five mutations that had occurred independently in all three vaccine-derived field isolates, indicating strong parallel selective pressure on these positions in the vaccine virus when used in swine herds. Two of these parallel mutations were direct reversions to the parental VR2332 sequence and were situated in a papain-like cysteine protease domain and in the helicase domain. The remaining parallel mutations might be seen as second-site compensatory mutations for one or more of the mutations that accumulated in the vaccine virus sequence during cell-culture adaptation. Evaluation of the remaining mutations in the ORF1 sequence revealed stronger selective pressure for amino acid conservation during spread in pigs than during vaccine production. Furthermore, it was found that the selective pressure did not change during the time period studied. The implications of these findings for PRRS vaccine attenuation and reversion are discussed.
Infectious cDNA clone of the hepatitis C virus genotype 1 prototype sequence
Authors: Lanford Robert E. , Lee Helen , Chavez Deborah , Guerra Bernadette , Brasky Kathleen M.
Published: August 2015

A full-length cDNA clone of the hepatitis C virus (HCV) genotype 1 prototype (subtype 1a) sequence was constructed. Synthetic RNA produced from the initial cDNA clone was not infectious following intrahepatic inoculation of a chimpanzee. A consensus clone was prepared by comparison with multiple full-length HCV sequences of genotypes 1, 2 and 3. A total of 11 non-consensus amino acid residues were altered by mutagenesis. Synthetic RNA from the repaired clone initiated a typical, acute-resolving HCV infection following intrahepatic inoculation of a chimpanzee. In addition, at least one of three chimeric cDNA clones constructed between the HCV-1 and H77 genotype 1a strains of HCV was infectious in a chimpanzee. This is the first example of an infectious chimeric HCV clone. An infectious cDNA clone of HCV-1 will be of particular value, since it is the prototype HCV sequence and many commonly used reagents are based on this sequence.
Distribution and characterization of tick-borne encephalitis viruses from Siberia and far-eastern Asia
Authors: Hayasaka Daisuke , Ivanov Leonid , Leonova Galina N. , Goto Akiko , Yoshii Kentaro , Mizutani Tetsuya , Kariwa Hiroaki , Takashima Ikuo
Published: August 2015

In this study, tick-borne encephalitis (TBE) viruses from Siberia and far-eastern Asia were characterized in order to determine virus subtype distribution. TBE viruses were isolated from ticks (Ixodes persulcatus) collected in the far-eastern (Khabarovsk and Vladivostok) and Siberian (Irkutsk) regions of Russia in 1999. Phylogenetic analysis showed that isolates formed distinct clusters of far-eastern and Siberian subtypes. There was also a minor difference in antigenicity between the Irkutsk isolates and other TBE virus strains, as demonstrated by the reactivity of monoclonal antibodies. Amino acid alignments of the E gene showed that the Irkutsk isolates had a single amino acid change at position 234 (Q or H); this amino acid position is considered to be a ‘signature’ of Siberian subtype TBE viruses. Strains isolated in Irkutsk also exhibited equivalent or somewhat higher virulence in mice compared with far-eastern TBE virus isolates. All viruses isolated in this study (i.e. far-east Asian and Siberian isolates) have 3′ non-coding regions (NCRs) of almost the same length, which contrasts with the various sizes of 3′NCRs of other TBE viruses strains reported previously. The data presented in this study show that the 3′NCR is uniform among TBE viruses isolated from Siberia and far-eastern Asia and that the 3′NCR is essential for TBE virus growth in tick and/or rodent host cells.
Genetic characterization of the coxsackievirus B2 3′ untranslated region
Authors: Polacek Charlotta , Lindberg A. Michael
Published: August 2015

The secondary structure of the 3′ untranslated region (3′UTR) of picornaviruses is thought to be important for the initiation of negative-strand RNA synthesis. In this study, genetic and biological analyses of the 3′ terminus of coxsackievirus B2 (CVB2), which differs from other enteroviruses due to the presence of five additional nucleotides prior to the poly(A) tail, is reported. The importance of this extension was investigated using a 3′UTR mutant lacking the five nucleotides prior to the poly(A) tail and containing two point mutations. The predicted secondary structure within the 3′UTR of this mutant was less energetically favourable compared with that of the wild-type (wt) genotype. This mutant clone was transfected into green monkey kidney cells in four parallel experiments and propagated for multiple passages, enabling the virus to establish a stable revertant genotype. Genetic analysis of the virus progeny from these different passages revealed two major types of revertant. Both types showed wt-like growth properties and more stable and wt-like predicted secondary structures than the parent mutant clone. The first type of revertant neutralized the introduced point mutation with a compensatory second-site mutation, whereas the second type of revertant partly compensated for the deletion of the five proximal nucleotides by the insertion of nucleotides that matched the wt sequence. Therefore, the extended 3′ end of CVB2 may be considered to be a stabilizing sequence for RNA secondary structure and an important feature for the virus.
A haemagglutinin (HA1)-specific FAb neutralizes influenza A virus by inhibiting fusion activity
Authors: Edwards M. J. , Dimmock N. J.
Published: August 2015

H9-D3-4R2 (referred to as H9), a murine monoclonal HA1-specific IgG3, recognizes an epitope within antigenic site Cb of influenza virus A/PR/8/34 (H1N1). At 50% neutralization, inhibition of virus-mediated fusion was responsible for the majority of neutralization but, at higher antibody concentrations, the attachment of virus to target cells was also inhibited and may have contributed to neutralization. H9 FAb was also neutralizing, although the concentration needed was two orders of magnitude greater than for the IgG. Functional affinity of the IgG and affinity of the FAb were almost identical, and it is not clear why the neutralization efficiency of the FAb was so low. Unlike its IgG, H9 FAb had no detectable effect on virus attachment but inhibited virus fusion activity. It thus appears that monovalent binding by this antibody is sufficient to inhibit fusion activity and that this was directly responsible for neutralization of infectivity.
Characterization and functional analysis of Serp3: a novel myxoma virus-encoded serpin involved in virulence
Authors: Guerin Jean-Luc , Gelfi Jacqueline , Camus Christelle , Delverdier Maxence , Whisstock James C. , Amardeihl Marie-France , Py Robert , Bertagnoli Stéphane , Messud-Petit Frédérique
Published: August 2015

Myxoma virus (MV), a member of the familyPoxviridae, is the causative agent of myxomatosis, a fatal disease of the European rabbit. The MV genome is a linear, double-stranded DNA molecule that encodes several factors important for evasion of the host immune system. Sequencing the right-end region of the MV genome identified an 801 bp open reading frame (ORF) encoding a polypeptide that belongs to the serpin superfamily. To date, two MV-encoded serpins have been characterized: SERP-1 binds to several targets and is an anti-inflammatory molecule, whereas Serp2 is essential for virus virulence and has both anti-inflammatory and anti-apoptotic effects. Thus, Serp3 is the third MV-encoded serpin. DNA sequence analysis of Serp3 indicated a similarity to poxvirus late promoters, which was confirmed by mRNA expression analysis. Serp3 has an atypical serpin motif and has significant sequence deletions as compared to most cellular and viral serpins. However, molecular modelling studies suggested that Serp3 can retain the overall serpin fold. Insertional inactivation of theserp3ORF led to a significant attenuation of virulencein vivo(as measured by the increase in survival of infected rabbits) and limited dissemination of the virus to secondary sites of infection. In rabbits infected with a Serp3 deletion mutant (MV-Serp3−), the main histopathological feature is the absence of secondary myxomas. Both wild-type MV and MV-Serp3−replicate at comparable levelsin vivo. Serp3 may represent a significant virulence factor of MV and probably acts in synergy with other viral proteins.
Herpes simplex virus type 2 UL34 protein requires UL31 protein for its relocation to the internal nuclear membrane in transfected cells
Authors: Yamauchi Y. , Shiba C. , Goshima F. , Nawa A. , Murata T. , Nishiyama Y.
Published: August 2015

Herpes simplex virus type 2 UL34 protein is expressed late in infection and is required for envelopment of nucleocapsids at the nuclear membrane and possibly at the endoplasmic reticulum (ER). It is a type II membrane protein with a C-terminal anchor that localizes mainly to the nuclear membrane in infected cells. However, in single transient expression, UL34 protein localizes predominantly to the ER. Relocation of UL34 protein from the ER to the internal nuclear membrane and the nucleus was observed in the presence of UL31 protein, a phosphoprotein known to interact physically with UL34. It is suggested here that interaction with UL31 protein is important for the nuclear targetting of UL34 protein and also that the trans-membrane region of UL34 protein is responsible for its localization at the internal nuclear membrane. The results also suggest possible sites for the interaction.
Insertion/deletion frequencies match those of point mutations in the hypervariable regions of the simian immunodeficiency virus surface envelope gene
Authors: Cheynier Rémi , Kils-Hütten Laurens , Meyerhans Andreas , Wain-Hobson Simon
Published: August 2015

A method for encoding insertions and deletions (indels) has been developed and adapted to the SplitsTree program. Following phylogenetic reconstruction, the relative frequencies of indels were estimated for a large number ofin vivosequence sets corresponding to theenvV1 hypervariable region of the simian immunodeficiency virus SIVmac251. The method allowed recovery of many point mutations hitherto lost due to gap stripping. Deletions were as frequent as transversions and were 4- to 8-fold more frequent than insertions, invariably duplications. The high proportion of deletions among mutation events suggests that lentivirus vectors may readily delete parts of their cargo.
Coexpression of hepatitis C virus envelope proteins E1 and E2 in cis improves the stability of membrane insertion of E2
Authors: Cocquerel Laurence , Meunier Jean-Christophe , Op de Beeck Anne , Bonte Dorine , Wychowski Czeslaw , Dubuisson Jean
Published: August 2015

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins, E1 and E2. These proteins contain a large N-terminal ectodomain, and are anchored into membranes by their C-terminal transmembrane domain (TMD). The TMDs of HCV envelope proteins are multifunctional. In addition to their role as membrane anchors, they possess a signal sequence function in their C-terminal half, and play a major role in subcellular localization and assembly of these envelope proteins. In this work, the expression of full-length E2 led to secretion of a proportion of this protein, which is likely to be due to inefficient membrane insertion of a fraction of E2 expressed alone. However, when E1 and E2 were coexpressed from the same polyprotein, E2 was not secreted and remained tightly associated with membranes, suggesting that an early interaction between the TMDs of HCV envelope proteins improves the stability of membrane insertion of E2. These results reinforce the hypothesis that the TMDs of E1 and E2 are major factors in the assembly of the HCV envelope glycoprotein complex.
Antigen-presenting cells from calves persistently infected with bovine viral diarrhoea virus, a member of the Flaviviridae, are not compromised in their ability to present viral antigen
Authors: Glew E. Jane , Howard Chris J.
Published: August 2015

The aim of this study was to assess whether the infection of antigen-presenting cells (APC)in vivo, evident in calves persistently infected (PI) with bovine viral diarrhoea virus (BVDV), compromised their ability to stimulate virus-specific T cell responses. Major histocompatibility complex (MHC) molecule-identical cattle were identified from the inbred family at the Institute for Animal Health. One was PI and immunotolerant to BVDV. Virus was not isolated from the remaining calves, which were classified as BVDV-immune or BVDV-naïve depending on the presence or absence of BVDV-specific antibodies in sera. Two-colour flow-cytometric analysis of PBMC from the PI calf showed that 40% of CD14+monocytes were infectedin vivo. Monocytes from the PI calf (PI monocytes) were used as naturally infectedex vivoAPC with CD4+or CD8+T cells isolated from the BVDV-naïve or BVDV-immune animals. PI monocytes stimulated proliferative responses with CD4+and CD8+T cells from BVDV-immune animals, but not from BVDV-naïve calves. This provided evidence for the presence of virus-specific CD4+and CD8+memory T cells after acute infection and indicated thatex vivomonocytes from PI, immunotolerant calves stimulated both MHC class I- and MHC class II-restricted T cell responses to BVDV. Additionally, naturally infectedex vivomonocytes culturedin vitrofor 3 days stimulated effective T cell responses to the virus with which they were infected.
Acute hepatitis caused by a novel strain of hepatitis E virus most closely related to United States strains
Authors: Kabrane-Lazizi Yamina , Zhang Mingdong , Purcell Robert H. , Miller Kirk D. , Davey Richard T. , Emerson Suzanne U.
Published: August 2015

A unique hepatitis E virus (HEV) strain was identified as the aetiological agent of acute hepatitis in a United States (US) patient who had recently returned from vacation in Thailand, a country in which HEV is endemic. Sequence comparison showed that this HEV strain was most similar, but not identical, to the swine and human HEV strains recovered in the US. Phylogenetic analysis revealed that this new HEV isolate was closer to genotype 3 strains than to the genotype 1 strains common in Asia. The fact that this HEV was closely related to strains recovered in countries where HEV is not endemic and was highly divergent from Asian HEV strains raises the questions of where the patient’s infection was acquired and of whether strains are geographically as localized as once thought.
Peptide display on live MS2 phage: restrictions at the RNA genome level
Authors: van Meerten Dico , Olsthoorn Rene C. L. , van Duin Jan , Verhaert Raymond M. D.
Published: August 2015

The 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.
Distribution of ecotropic retrovirus receptor protein in rat brains detected by immunohistochemistry
Authors: Takase-Yoden Sayaka , Watanabe Rihito
Published: August 2015

Friend murine leukaemia virus (FrMLV) FrC6 clone A8 causes spongiform degeneration in the central nervous system (CNS) of newborn but not 3-week-old rats. To assess whether expression of the ecotropic MLV receptor (CAT-1) in the CNS correlates with the pathogenicity of the A8 virus, we generated an anti-CAT-1 antibody raised against a synthetic peptide that corresponds to the carboxyl-terminal amino acid sequence of CAT-1. In the CNS of newborn and 3 to 4-week-old rats, a strong immunoreactivity against the antibody was detected in most of the endothelial cells. However, almost no expression of CAT-1 was detected in the CNS of 21-week-old rats. In newborn rats, many parenchymal cells in the brain as well as the vascular wall expressed CAT-1 antigen. These findings suggest that retrovirus receptor-bearing glial cells contribute to the neuropathogenesis of MLV, including clone A8, which induces spongiosis in rats only when inoculated into newborns.
Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2
Authors: Owsianka Ania , Clayton Reginald F. , Loomis-Price Lawrence D. , McKeating Jane A. , Patel Arvind H.
Published: August 2015

Structure–function analysis of the hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, has been difficult due to the unavailability of HCV virions. Truncated soluble forms of E2 have been used as models to study virus interaction with the putative HCV receptor CD81, but they may not fully mimic E2 structures on the virion. Here, we compared the CD81-binding characteristics of truncated E2 (E2660) and full-length (FL) E1E2 complex expressed in mammalian cells, and of HCV virus-like particles (VLPs) generated in insect cells. All three glycoprotein forms interacted with human CD81 in an in vitro binding assay, allowing us to test a panel of well-characterized anti-E2 monoclonal antibodies (MAbs) for their ability to inhibit the glycoprotein–CD81 interaction. MAbs specific for E2 amino acid (aa) regions 396–407, 412–423 and 528–535 blocked binding to CD81 of all antigens tested. However, MAbs specific for regions 432–443, 436–443 and 436–447 inhibited the interaction of VLPs, but not of E2660 or the FL E1E2 complex with CD81, indicating the existence of structural differences amongst the E2 forms. These findings underscore the need to carefully select an appropriate ligand for structure–function analysis.
Cys9, Cys104 and Cys207 of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells
Authors: Gharakhanian Editte , Fasching Clare L. , Orlando Salvatore J. , Perez Ana R.
Published: August 2015

Structural studies have implicated Cys9, Cys104and Cys207of simian virus 40 (SV40) Vp1 in disulfide bond formation. Recently, we have shown the three cysteines to be essential for disulfide linkage of Vp1 complexesin vitro. Here, the role of the three cysteines was explored during the course of SV40 infection. Single-, double- and triple-mutant Vp1 at Cys9, Cys104and Cys207continued to localize to the nuclei of transfected CV-1 cells and to bind DNA, but showed a range of abilities to form plaques. Only mutants containing the Cys9→Ser change showed defects in plaque formation. Single mutants at Cys9formed small plaques; mutants at Cys9. Cys104, Cys9. Cys207and Cys9. Cys104. Cys207formed no plaques. All three isolated revertants contained back-mutations at the Vp1 Cys9codon. These results further confirm the involvement of the three Vp1 cysteines in protein–protein interactions during virus assembly. Cys9is critical for production of wild-type infectious virions, whereas Cys104and Cys207play secondary roles.
Human herpesvirus-8 (Kaposi’s sarcoma-associated herpesvirus) ORF50 interacts synergistically with the tat gene product in transactivating the human immunodeficiency virus type 1 LTR
Authors: Caselli Elisabetta , Menegazzi Paola , Bracci Arianna , Galvan Monica , Cassai Enzo , Di Luca Dario
Published: August 2015

Human herpesvirus-8 (HHV-8) is a lymphotropic virus associated with several AIDS-related neoplasms. Two ORFs play a critical role in the regulation of virus replication: ORF50, encoding an immediate-early transcriptional activator, and ORF57, encoding a post-transcriptional regulator. We analysed their effects on the activation of the human immunodeficiency virus type 1 (HIV-1) LTR. ORF50 interacted synergically with tat, inducing a 10-fold enhancement of HIV-1 LTR transactivation. This effect occurred both in BCBL-1 cells, latently infected with HHV-8, and in HL3T1 cells, an epithelial cell line non-permissive to HHV-8 infection. Also, ORF57 enhanced tat-induced transactivation of HIV-1 LTR, but only in BCBL-1 cells, suggesting that its action was likely mediated by the induction of other viral functions. Finally, when both ORFs were expressed, the enhancement of transactivation induced by ORF50 was partially inhibited. The findings suggest that ORF57 can modulate ORF50 activity and that ORF50 may render biologically active small amounts of tat.
Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus
Authors: Erokhina Tatyana N. , Vitushkina Maria V. , Zinovkin Roman A. , Lesemann Dietrich E. , Jelkmann Wilhelm , Koonin Eugene V. , Agranovsky Alexey A.
Published: August 2015

Monoclonal antibodies (MAbs) specific to the methyltransferase (MT) and helicase (HEL) domains of the closterovirus Beet yellows virus (BYV) were used for immunogold labelling of ultrathin sections of virus-infected Tetragonia expansa plants. MAbs 4A2 and 4A5 from the MT panel, and 1C4 from the HEL panel, specifically labelled distinct closterovirus-induced membranous structures, the ‘BYV-type vesicles’, thus suggesting that the closterovirus MT-like and HEL-like proteins co-localize in these structures. Probing of the MT and HEL MAbs with synthetic octapeptides spanning the sequences of the recombinant MT and HEL fragments that had been used as immunogens showed that 4A5 and 4A2 recognized a single epitope, SRLLENET (aa 686–692 in the BYV 1a protein), and 1C4 reacted with the DDPF epitope (aa 2493–2496). These epitopes apparently reside on the exposed parts of the membrane-associated molecules of the closterovirus MT-like and HEL-like proteins. Two other epitopes determined for the MT MAbs that were nonreactive in the immunogold labelling, namely TMVTPGEL (aa 750–757; MAbs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806–813; MAb 2A4), are possibly buried in the MT domain fold or shielded by membranes or other proteins involved in the viral replicative complex.
Characterization of a novel genital human papillomavirus by overlapping PCR: candHPV86 identified in cervicovaginal cells of a woman with cervical neoplasia
Authors: Terai Masanori , Burk Robert D.
Published: August 2015

A novel human papillomavirus (HPV), candHPV86, was cloned and characterized from cervicovaginal cells obtained from a 37-year-old Hispanic woman with cervical intraepithelial neoplasia grade 1 (CIN1) using an overlapping PCR technique. Primers were designed by phylogenetic alignment of closely related HPV genomes using the L1 fragment sequence amplified by GP5+/6+. The 7983 bp complete nucleotide sequence of the HPV genome was determined by sequence walking. A basic local alignment sequence tool (BLAST) homology search using the L1 open reading frame demonstrated that this HPV was most closely related to HPVHAN2294 (GenBank, AJ400628; 86% homology) and HPV84 (84% homology). candHPV86 was placed in the HPV genome homology group A3 by phylogenetic analyses. The overlapping PCR technique is applicable for characterizing the complete spectrum and variation of HPVs in a population.
Phylogeny of the Simbu serogroup of the genus Bunyavirus
Authors: Saeed Mohammad F. , Li Li , Wang Heiman , Weaver Scott C. , Barrett Alan D. T.
Published: August 2015

The Simbu serogroup of the genus Bunyavirus, family Bunyaviridae contains 25 viruses. Previous serological studies provided important information regarding some but not all of the relationships among Simbu serogroup viruses. This report describes the nucleotide sequence determination of the nucleocapsid (N) gene of the small genomic segment of 14 Simbu serogroup viruses and partial nucleotide sequence determination of the G2 glycoprotein-coding region (encoded by the medium RNA segment) of 19 viruses. The overall phylogeny of the Simbu serogroup inferred from analyses of the N gene was similar to that inferred from analyses of the G2 protein-coding region. Both analyses revealed that the Simbu serogroup viruses have evolved into at least five major phylogenetic lineages. In general, these phylogenetic lineages were consistent with the previous serological data, but provided a more detailed understanding of the relatedness amongst many viruses. In comparison to previous phylogenetic studies on the California and Bunyamwera serogroups of the Bunyavirus genus, the Simbu serogroup displays much larger genetic variation in the N gene (up to 40% amino acid sequence divergence).
p53-dependent transcriptional repression of p21waf1 by hepatitis C virus NS3
Authors: Kwun Hyun Jin , Jung Eun Young , Ahn Ji Young , Lee Mi Nam , Jang Kyung Lib
Published: August 2015

Hepatitis C virus (HCV) NS3 protein is known to affect normal cellular functions, such as cell proliferation and cell death, and to be involved, either directly or indirectly, in HCV hepatocarcinogenesis. In this study, we demonstrated that NS3 protein could specifically repress the promoter activity of p21 in a dose-dependent manner. The effect was not cell type-specific and was synergistic when combined with HCV core protein. Repression of the p21 promoter by NS3 was almost completely lost when p53 binding sites present on the p21 promoter were removed. Furthermore, p53 binding sites were sufficient to confer a strong NS3 responsiveness to an heterologous promoter, suggesting that NS3 represses the transcription of p21 by modulating the activity of p53. Although the NS3 protein domain required for the majority of p21 repression was located on the protease domain, the proteinase activity itself does not seem to be necessary for repression. Both transcription and protein stability of p53 were unaffected by NS3, suggesting that NS3 might repress transcription of p21 by inhibiting the regulatory activity of p53 via protein–protein interaction(s). Finally, the growth rate of NS3-expressing cell lines was at least twice as fast as that of the parent NIH 3T3 cells, indicating that the repression of p21 is actually reflected by the stimulation of cell growth.