bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatitis B virus maturation is affected by the incorporation of core proteins having a C-terminal substitution of arginine or lysine stretches
Authors: Hui Eric Ka-Wai , Chen Kun-Lin , Lo Szecheng J.
Published: August 2015

Assembly of replication-competent hepadnavirus nucleocapsids requires interaction of core protein, polymerase and encapsidation signal (ϵ) with viral pregenomic RNA. The N-terminal portion (aa 1–149) of the core protein is able to self-assemble into nucleocapsids, whereas the C-terminal portion (aa 150–183) is known to interact with pregenomic RNA. In this study, two hepatitis B virus (HBV) core mutants (C144Arg and C144Lys) in which the C-terminal SPRRR (Ser-Pro-Arg-Arg-Arg) motif was replaced by a stretch of arginine or lysine residues were generated to test their role in pregenome encapsidation and virus maturation. Mutant or wild-type core-expression plasmids were co-transfected with a core-negative plasmid into human hepatoma HuH-7 cells to comparetrans-complementation efficiency for virus replication. Both low- and high-density capsids were present in the cytoplasm and culture medium of HuH-7 cells in all transfections. Nucleocapsids formed by C144Arg and C144Lys, however, lost the endogenous polymerase activity to repair HBV DNA. Furthermore, in co-transfection of pHBVC144Arg or pHBVC144Lys with a plasmid which produces replication-competent nucleocapsids, the HBV DNA repairing signal was reduced 40- to 80-fold. This is probably due to formation of mosaic particles of wild-type and mutant cores. Results indicated that the SPRRR motif at the core protein C terminus is important for HBV DNA replication and maturation. Additionally, triple-plasmid transfection experiments showed that nucleocapsids containing various amounts of C144Arg and wild-type core proteins exhibited a bias in selecting a shorter pregenome for encapsidation and DNA replication. It is therefore suggested that unknown factors are also involved in HBV pregenome packaging.
A model for vaccinia virus pathogenesis and immunity based on intradermal injection of mouse ear pinnae
Authors: Tscharke David C. , Smith Geoffrey L.
Published: August 2015

Vaccinia virus (VV) proteins that interfere with the host response to infection are of interest because they provide insight into virus–host relationships and may affect the safety and immunogenicity of recombinant VV (rVV) vaccines. Such vaccines need assessment in animal models and with this aim a model of VV infection based on intradermal injection of BALB/c ear pinnae was developed and characterized. In this model, the outcome of infection is affected by the dose of virus inoculated but virus spread is minimal and the mice suffer no signs of systemic illness. Cellular and humoral immune responses to these infections were measured readily and were independent of virus dose over a 100-fold range. Thus the model seems suitable for the analysis of the safety and immunogenicity of VV mutants lacking specific immunomodulatory proteins or bearing foreign antigens.
Role of the beet western yellows virus readthrough protein in virus movement in Nicotiana clevelandii
Authors: Mutterer J. D. , Stussi-Garaud C. , Michler P. , Richards K. E. , Jonard G. , Ziegler-Graff V.
Published: August 2015

Luteoviruses such as beet western yellows polerovirus (BWYV) are confined to and multiply within the phloem compartment of their hosts. The readthrough domain (RTD) of the minor BWYV capsid protein P74 is required for efficient virus accumulation in Nicotiana clevelandii. Experiments were carried out to determine if the low virus titres observed following agro-inoculation of whole plants with certain RTD mutants are due to a defect in virus multiplication in the nucleate cells of the phloem compartment or to inefficient virus movement to new infection sites. Immuno-localization of wild-type and an RTD-null mutant virus in thin sections of petioles and in phloem cells of leaf lamina, as well as electron microscopy observations, were all consistent with the conclusion that the RTD is not essential for efficient virus multiplication in the nucleate phloem cells but intervenes in virus movement to increase the rate at which new infection foci are established and expand.
Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome
Authors: Sinzger C. , Schmidt K. , Knapp J. , Kahl M. , Beck R. , Waldman J. , Hebart H. , Einsele H. , Jahn G.
Published: August 2015

Following extensive propagation in fibroblasts, human cytomegalovirus (HCMV) loses tropism for a number of otherwise natural host cells, in particular, endothelial cells. In this study, the hypothesis was tested that loss of endothelial tropism is associated with the appearance of genomic variants. Initial quantitative focus expansion assays on endothelial monolayers demonstrated that, while the laboratory strains AD169 and Towne failed to form detectable foci, 29 out of 30 recent clinical HCMV isolates had the potential to expand in endothelial cell culture. By long-term adaptation in fibroblast cultures, nonendotheliotropic strains could be selected from clinical HCMV isolates, while long-term endothelial-adapted strains of the same isolates retained both fibroblast tropism and endothelial tropism. Such differentially adapted isolate pairs always displayed genomic differences in restriction fragment length analyses. Coinfection of endothelial cells by two nonendotheliotropic HCMV strains yielded an endotheliotropic recombinant HCMV variant combining portions of the genomes of both parental viruses. When DNA purified from various isolates was transfected into fibroblasts, progeny virus retained the specific tropism of parental virus from which the DNA was isolated. These findings demonstrate that endothelial tropism is an inherent property of most clinical HCMV isolates and is determined by the viral genome. Although the specific determinants of HCMV cell tropism are still unknown, this study provides the first evidence for a genetic contribution.
Effect of virulence on immunogenicity of single and double vaccinia virus recombinants expressing differently immunogenic antigens: antibody-response inhibition induced by immunization with a mixture of recombinants differing in virulence
Authors: Kutinová Luda , Ludvíková Viera , Marešová Lucie , Němečková šárka , Brouček Jaroslav , Hainz Petr , Vonka Vladimír
Published: August 2015

It has been shown recently that the residual virulence of vaccinia virus (VV) is an important factor that influences the outcome of immunization with VV recombinants. This study focused on the correlation of the residual virulence of several VV recombinants with antibody responses against the strongly immunogenic extrinsic glycoprotein E of varicella-zoster virus and the weakly immunogenic extrinsic protein preS2–S of hepatitis B virus and against VV proteins, with mice used as a model organism. Furthermore, the effects of mixing different recombinants on the antibody response were studied. The results obtained indicated that: (i) the antibody response depended on the residual virulence of the recombinants, more so in the case of the weakly immunogenic protein; (ii) the residual virulence, the growth rate of the VV recombinants in extraneural tissues and the immunogenicity were associated features; (iii) immunization with mixtures of two differently virulent recombinants or with unequal amounts of two similarly virulent recombinants sometimes led to the suppression of antibody response. The appearance of this suppression was dependent on three factors: the residual virulence of the recombinants, the immunogenicity of the extrinsic proteins and the ratio of the recombinants in the mixtures. Thus, the data obtained demonstrate that there are various limitations to the use of replicating VV recombinants for immunization purposes.
Human immunodeficiency virus infection in vitro activates naturally integrated human papillomavirus type 18 and induces synthesis of the L1 capsid protein
Authors: Dolei Antonina , Curreli Sabrina , Marongiu Patrizia , Pierangeli Alessandra , Gomes Eduarda , Bucci Mauro , Serra Caterina , Degener Anna Marta
Published: August 2015

Human papillomavirus (HPV) infections are prevalent in human immunodeficiency virus (HIV)-positive individuals. To highlight the effect of HIV on HPV expression, HPV-18-positive HIV-permissive HeLa-T4 cells were either infected with HIV-1 or treated with Tat or with the cytokines IL-1α, IL-1β, IL-6 and TNF-α . The presence of HPV-18 E1 (early) and L1 (late) transcripts was then determined by dot-blot or Northern blot hybridization with E1 and L1 or with genomic HPV-18 DNA probes, respectively. Protein extracts from parallel cultures were challenged by Western blotting with an antiserum raised against an L1–β-galactosidase hybrid protein. Results indicated that HeLa-T4 cells constitutively express E1 and L1 transcripts. When cells were infected with HIV, the amounts of E1 and L1 RNAs increased with time, followed by thede novoappearance of L1 protein. E1 and L1 transcripts were also increased, in a dose-dependent manner, by treatment of uninfected cultures with Tat or with IL-6, but were not affected by IL-1α, IL-1β and TNF- α. Neither Tat nor IL-6 could induce L1 translation. These findings raise the hypothesis that the increase of HPV shedding and of HPV-associated diseases in HIV-infected individuals could be due in part to a direct or cytokine-mediated action of HIV, in addition to the HIV-induced immunodeficiency.
Pseudotyping human immunodeficiency virus type 1 by vesicular stomatitis virus G protein does not reduce the cell-dependent requirement of Vif for optimal infectivity: functional difference between Vif and Nef
Authors: Akari Hirofumi , Uchiyama Tsuneo , Fukumori Tomoharu , Iida Shinya , Koyama A. Hajime , Adachi Akio
Published: August 2015

The functions of Vif and Nef in human immunodeficiency virus type 1 (HIV-1) infection have some similarities: Vif- and Nef-dependent enhancement of HIV-1 replication is cell type-specific, and defective mutations in these genes result in restricted proviral DNA synthesis in infected cells. It has recently been shown that pseudotyping HIV-1 by the envelope glycoprotein of vesicular stomatitis virus (VSV-G) targets HIV-1 entry to an endocytic pathway and suppresses the requirement of Nef for virus infectivity. In this study, we examined whether VSV-G pseudotyping suppresses the requirement of Vif for HIV-1 infectivity. It was found that pseudotyping HIV-1 by VSV-G did not compensate for the Vif function. Together with the findings that Vif does not influence virus binding/entry and virion incorporation of Env, it is concluded that Vif enhances HIV-1 infectivity at the post-entry step(s) independently of the Env function by a different mechanism to that of Nef.
Characterization of Sendai virus M protein mutants that can partially interfere with virus particle production
Authors: Mottet Geneviève , Müller Virginie , Roux Laurent
Published: August 2015

Substitution of Val113in Sendai virus (SeV) M protein generates non-functional polypeptides, characterized by their exclusion from virus particles and by their ability to interfere with virus particle production. These phenotypic traits correlate with a single-band PAGE migration profile, in contrast to wild-type M (Mwt), which separates into two species, one of which is a phosphorylated form. The single-band migration is likely to result from a conformational change, as evidenced by the lack of maturation of a native epitope and by a particular tryptic digestion profile, and not from the phosphorylation of all M molecules, an assumption consistent with the PAGE migration feature. One of the M mutants (HA–M30, an M protein carrying Thr112Met and Val113Glu substitutions tagged with an influenza virus haemagglutinin epitope) was characterized further in the context of SeV infection, i.e. under conditions of co-expression with Mwt. HA–M30is shown (i) to bind mainly to membrane fractions, (ii) not to co-precipitate Mwt, as HA–Mwtdoes, (iii) to interfere with the binding of nucleocapsids to membranes and (iv) to accumulate in perinuclear regions, in contrast to HA-Mwt, which is also found at the cell periphery. Such mutants constitute potential tools for the identification of critical steps in paramyxovirus assembly and budding.
Inducible nitric-oxide synthase plays a minimal role in lymphocytic choriomeningitis virus-induced, T cell-mediated protective immunity and immunopathology
Authors: Bartholdy C. , Nansen A. , Christensen J. Erbo , Marker O. , Thomsen A. Randrup
Published: August 2015

By using mice with a targetted disruption in the gene encoding inducible nitric-oxide synthase (iNOS), we have studied the role of nitric oxide (NO) in lymphocytic choriomeningitis virus (LCMV)-induced, T cell-mediated protective immunity and immunopathology. The afferent phase of the T cell-mediated immune response was found to be unaltered in iNOS-deficient mice compared with wild-type C57BL/6 mice, and LCMV- induced general immunosuppression was equally pronounced in both strains.In vivoanalysis revealed identical kinetics of virus clearance, as well as unaltered clinical severity of systemic LCMV infection in both strains. Concerning the outcome of intracerebral infection, no significant differences were found between iNOS-deficient and wild-type mice in the number or composition of mononuclear cells found in the cerebrospinal fluid on day 6 post-infection. Likewise, NO did not influence the up-regulation of proinflammatory cytokine/chemokine genes significantly, nor did it influence the development of fatal meningitis. However, a reduced virus-specific delayed-type hypersensitivity reaction was observed in iNOS-deficient mice compared with both IFN-γ-deficient and wild-type mice. This might suggest a role of NO in regulating vascular reactivity in the context of T cell-mediated inflammation. In conclusion, these findings indicate a minimal role for iNOS/NO in the host response to LCMV. Except for a reduced local oedema in the knockout mice, iNOS/NO seems to be redundant in controlling both the afferent and efferent phases of the T cell-mediated immune response to LCMV infection.
Sequence of the non-structural protein gene encoded by RNA1 of striped jack nervous necrosis virus
Authors: Nagai Takahiro , Nishizawa Toyohiko
Published: August 2015

Striped jack nervous necrosis virus (SJNNV), the causative agent of viral nervous necrosis in marine fish, is a member of the family Nodaviridae whose genome consists of two positive-sense RNA molecules encapsidated in a single virion. In this study, the nucleotide sequence of SJNNV RNA1 was determined. The SJNNV RNA1 was 3081 bases long and contained a single ORF encoding 983 aa of approximately 110 kDa. The sequence identities between RNA1 of SJNNV and RNA1 of insect nodaviruses were 28% at the nucleotide and amino acid levels, although the conserved motifs for the RNA-dependent RNA polymerase were located at almost the same positions in the amino acid sequences. The present study, together with our previous work on SJNNV RNA2, suggests that a new genus, Piscinodavirus, should be created in the family Nodaviridae.
Pathogenesis of the oral route of infection of mice with scrapie and bovine spongiform encephalopathy agents
Authors: Maignien Thomas , Lasmé zas Corinne Ida , Beringue Vincent , Dormont Dominique , Deslys Jean-Philippe
Published: August 2015

Transmissible spongiform encephalopathies can be transmitted via the oral route. The understanding of this mode of contamination has become a major issue since it is responsible for the appearance of bovine spongiform encephalopathy (BSE) and is probably implicated in new variant Creutzfeldt–Jakob disease. In this study, we addressed the questions of the propagation pathway and the strain specificity of the pathogenesis of oral contamination of mice with the C506M3 scrapie strain and the 6PB1 BSE strain. PrPres was used as a marker of infectivity and was searched for sequentially in 22 organs during the whole incubation period and clinical stage. PrPres was first detectable in the Peyer’s patches and mesenteric lymph nodes at 45 days post-inoculation. It became detectable 1 to 3 months later in the other tissues of the lymphoreticular system (LRS) such as the spleen and the lymph nodes not related to the digestive tract. These data indicate that after an oral route of entry, the infectious agent is propagated from the Peyer’s patches to the mesenteric lymph nodes by the lymphatic route, then enters the bloodstream and is distributed to the secondary replication site, the LRS. The major difference between the two agents is that PrPres could be detected in the digestive tract (from the stomach to the colon) with the scrapie agent only. This observation may have implications for the horizontal transmission of scrapie in endemically affected sheep flocks.
Functional characterization of a piscine retroviral promoter
Authors: Zhang Z. , Kim E. , Martineau D.
Published: August 2015

Walleye dermal sarcoma virus (WDSV) is a retrovirus aetiologically associated with a multifocal skin tumour of walleye. Tumours synchronously develop on 27% of fish and regress seasonally; their severity is influenced by water temperature. To functionally characterize the LTR of WDSV, the LTR was fused to theluciferasereporter gene. WDSV LTR was found to be transcriptionally active in both fish and mammalian cells. WDSV LTR deletion mutants were constructed to identify specific regions that were functionally important in modulating viral gene expression and in temperature responsiveness. The 5′ end 60 bp, which contain a putative ecdysone-response element also present in another fish retrovirus, positively modulated transcription from the WDSV LTR at 25 °C, but not at lower temperatures. A 13 bp region (nt −288 to −275) comprising a putative activator protein-1 element was necessary for maintaining WDSV LTR activity at all temperatures. In marked contrast to the short direct repeats found in mammalian retroviral LTRs, five 5 bp direct repeats (nt −336 and −272) were found to negatively regulate transcription from the WDSV LTR. A region spanning nt −440 to −218 stimulated the activity of a heterologous mammalian promoter in an orientation-dependent manner, modestly in fish cells (1·3- to 2-fold), but markedly (3·7- to 5·1-fold) in mammalian cells. Our results strongly suggest that the putative promoter elements present in the WDSV LTR function differentially in a temperature-specific manner and that complex interactions between these elements modulate WDSV LTR activity in response to temperature changes.
Characterization of aggregates of hepatitis C virus glycoproteins
Authors: Choukhi Amélie , Pillez André , Drobecq Hervé , Sergheraert Christian , Wychowski Czeslaw , Dubuisson Jean
Published: August 2015

Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which assemble in oligomeric structures. Studies of HCV glycoprotein assembly using heterologous expression systems have shown that these glycoproteins can follow two pathways: a productive pathway leading to the formation of a non-covalent heterodimer; and a non-productive pathway leading to the formation of large disulfide-linked aggregates. The non-covalent HCV glycoprotein complex is probably the functional complex which plays an active role in the entry process in host cells. The aggregates are believed to be waste products; however, one can imagine that, in infected cells, they could provide HCV glycoproteins with additional functions. To further understand the potential role played by HCV glycoprotein aggregates in HCV infection, a MAb (H14) was produced which specifically recognizes these aggregates but not the non-covalent E1E2 heterodimer. The H14 epitope was shown to be present on both HCV glycoproteins and was sensitive to deglycosylation. An additional characterization of HCV glycoprotein aggregates, with the help of MAb H14, indicates that they share an epitope with a cellular protein called Mac-2 binding protein. The presence of such an epitope on HCV glycoprotein aggregates could potentially lead to the production of autoantibodies recognizing Mac-2 binding protein in HCV-infected patients.
Phylogenetic and virulence analysis of tick-borne encephalitis viruses from Japan and far-eastern Russia
Authors: Hayasaka Daisuke , Suzuki Yoshiyuki , Kariwa Hiroaki , Ivanov Leonid , Volkov Vladimir , Demenev Vladimir , Mizutani Tetsuya , Gojobori Takashi , Takashima Ikuo
Published: August 2015

We have previously reported that tick-borne encephalitis (TBE) is endemic in a specific area of Hokkaido, Japan. In Oshima, the southern part of Hokkaido, TBE virus was isolated from sentinel dogs, ticks and rodents in 1995 and 1996. To identify when these TBE viruses emerged in Hokkaido, the times of divergence of TBE virus strains isolated in Oshima and far-eastern Russia were estimated. TBE virus was isolated in Khabarovsk in 1998 and the nucleotide sequences of viral envelope protein genes of isolates from Oshima and Khabarovsk were compared. From the synonymous substitution rate of these virus strains, the lineage divergence time of these TBE virus strains was predicted phylogenetically to be about 260–430 years ago. Furthermore, the virulence of TBE virus isolates from Oshima and Khabarovsk were compared in a mouse model. The results showed that the isolates possessed very similar virulence in mice. This report provides evidence that the Oshima strains of TBE virus in Hokkaido emerged from far-eastern Russia a few hundred years ago and this explains why these strains possess virulence similar to the TBE viruses isolated in Russia.
La protein is required for efficient translation driven by encephalomyocarditis virus internal ribosomal entry site
Authors: Kim Yoon Ki , Jang Sung Key
Published: August 2015

Translation of internal ribosomal entry site (IRES)-dependent mRNAs is mediated by RNA-binding proteins as well as canonical translation factors. In order to elucidate the roles of RNA-binding proteins in IRES-dependent translation, the role of polypyrimidine tract-binding protein (PTB) and La protein in encephalomyocarditis virus (EMCV) IRES-dependent translation was investigated. PTB was required for efficient EMCV IRES-driven translation but, intriguingly, an excess of PTB suppressed it. Such a translational suppression by surplus PTB was relieved by addition of La protein. A possible role for La protein in IRES-dependent translation is discussed.
Mutations in the conserved carboxy-terminal hydrophobic region of glycoprotein gB affect infectivity of herpes simplex virus
Authors: Wanas Essam , Efler Sue , Ghosh Kakoli , Ghosh Hara P.
Published: August 2015

Glycoprotein gB is the most highly conserved glycoprotein in the herpesvirus family and plays a critical role in virus entry and fusion. Glycoprotein gB of herpes simplex virus type 1 contains a hydrophobic stretch of 69 aa near the carboxy terminus that is essential for its biological activity. To determine the role(s) of specific amino acids in the carboxy-terminal hydrophobic region, a number of amino acids were mutagenized that are highly conserved in this region within the gB homologues of the family Herpesviridae. Three conserved residues in the membrane anchor domain, namely A786, A790 and A791, as well as amino acids G743, G746, G766, G770 and P774, that are non-variant in Herpesviridae, were mutagenized. The ability of the mutant proteins to rescue the infectivity of the gB-null virus, K082, in trans was measured by a complementation assay. All of the mutant proteins formed dimers and were incorporated in virion particles produced in the complementation assay. Mutants G746N, G766N, F770S and P774L showed negligible complementation of K082, whereas mutant G743R showed a reduced activity. Virion particles containing these four mutant glycoproteins also showed a markedly reduced rate of entry compared to the wild-type. The results suggest that non-variant residues in the carboxy-terminal hydrophobic region of the gB protein may be important in virus infectivity.
Human papillomavirus type 16 E6 variants in cervical carcinoma: relationship to host genetic factors and clinical parameters
Authors: Brady Claire S. , Duggan-Keen Margaret F. , Davidson Judith A. , Varley Jenny M. , Stern Peter L.
Published: August 2015

Infection with human papillomavirus type 16 (HPV-16) confers a high risk for the development of cervical neoplasia. Variants of this virus may interact differentially with host genetic factors, possibly altering the disease course. Thus, HPV-16 E6 variants may differ in their ability to degrade p53 whereas the polymorphic p53 alleles may provide more or less susceptible substrates for the viral oncogene product. Also, E6 variants may differ in immunogenicity by generating different peptides for presentation by polymorphic HLA molecules to specific T cells. This study examines HPV-16 E6 sequence variation in cervical carcinomas from the UK and its relationship to polymorphism of HLA and p53 and to clinical parameters. Sequence analysis of the HPV-16 E6 ORF from 77 tumour biopsies detected the viral prototype sequence in 38% of cases. The most common variation detected was a T to G transition at base pair 350, resulting in an amino acid change from a leucine to a valine. Overall, the frequencies of 350T and 350G sequences were similar (49·4% and 50·6% respectively). Other mutations of lower frequencies were detected together with and independently of 350G. HPV-16 E6 sequence variation at base pair 350 did not correlate with HLA genotype or clinical outcome. There was no difference in the distribution of p53 proline and arginine alleles between HPV-16-positive cervical carcinoma patients and local controls, and no influence on clinical outcome; however, there was a trend for an increased frequency of p53 arginine homozygotes among the 350T carcinoma patients.
Failure of viral oncoproteins to target the p53-homologue p51A
Authors: Roth Judith , Dobbelstein Matthias
Published: August 2015

The p51/p63/KET proteins were identified based on their strong homology to the tumour suppressor p53 and a related set of proteins termed p73. All these protein species were shown to activate transcription from at least some p53-responsive promoters. To evaluate a possible role of the transcriptionally active splicing variant p51A/p63γ in tumour suppression, we determined whether viral oncoproteins that inactivate p53 might also target p51A. Neither the large T-antigen of simian vacuolating virus 40 (SV40) nor the E6 protein from human papillomavirus type 18 were found to inhibit p51A-mediated transcription, whereas they strongly suppress the activity of p53. Further, SV40 T-antigen directly interacts with p53 but not detectably with p51A. Finally, a cytoplasmic mutant (K128A) of SV40 T-antigen relocalizes p53 from the nucleus to the cytoplasm, but p51A remains in the nucleus when coexpressed with cytoplasmic T-antigen. These results strongly suggest that the inhibitory effect of these viral oncoproteins is specific for p53 and does not measurably affect p51A. Thus, unlike p53, p51A does not appear to be a necessary target in virus-induced cell transformation and may not exert a role comparable to p53 in tumour suppression.
Atypical nucleoprotein complexes mediate CRE-dependent regulation of the early promoter of minute virus of mice
Authors: Perros Manoussos , Fuks François , Kherrouche Zoulika , Rommelaere Jean
Published: August 2015

The P4 promoter of the parvovirus minute virus of mice (MVMp) directs transcription of the genes encoding non-structural proteins. We have previously shown that functional upstream CRE elements contribute to both therasoncogene-dependent activation of promoter P4 and its down-modulation by known activators of cyclic AMP-dependent protein kinase A (PKA). In the present work, the nucleoprotein complexes formed with the P4 CRE elements were characterized with regard to their polypeptide constituents and the nucleotides taking part in the interaction. Atypical interactions, both at the protein–protein and protein–DNA level, were observed, which may be a reflection of the divergence of the parvoviral CREs from the usual consensus. The CRE-mediated regulation of promoter P4 by PKA and Ras is discussed in light of these findings.
Nonstructural proteins of Tobacco rattle virus which have a role in nematode-transmission: expression pattern and interaction with viral coat protein
Authors: Visser Peter B. , Bol John F.
Published: August 2015

RNA 2 ofTobacco rattle virusisolate PpK20 encodes the viral coat protein (CP) and two nonstructural proteins of 40 kDa (‘40K protein’) and 32·8 kDa (‘32·8K’). The 40K protein is required for transmission of the virus by the vector nematodeParatrichodorus pachydermuswhereas the 32·8K protein may be involved in transmission by other vector nematode species. An antiserum was raised against the 40K protein expressed inE.coliand used to study the expression and subcellular localization of this protein in infectedNicotiana benthamianaplants. The time-course of the expression of the 40K protein in leaves and roots was similar to that of CP and both proteins were similarly distributed over the 1000 gpellet, 30000 gpellet and 30000 gsupernatant fractions of leaf and root homogenates. Using the yeast two-hybrid system, a strong interaction between CP subunits was observed and weaker interactions between CP and the 32·8K protein and between CP and the 40K protein were detected. A deletion of the C-terminal 19 amino acids of CP interfered with the CP–40K interaction but not with CP–32·8K or CP–CP interactions, whereas a C-terminal deletion of 79 amino acids interfered with CP–40K and CP–32·8K interactions but not with the CP–CP interaction. As the C terminus of CP is known to be involved in nematode-transmission of tobraviruses, the data support the hypothesis that interactions between CP and RNA 2-encoded nonstructural proteins play a role in the transmission process.
Evidence for the role of His-142 of protein 1C in the acid-induced disassembly of foot-and-mouth disease virus capsids
Authors: Ellard Fiona M. , Drew Jeff , Blakemore Wendy E. , Stuart David I. , King Andrew M. Q.
Published: August 2015

Foot-and-mouth disease virus (FMDV) capsids are inherently labile under mildly acidic conditions, dissociating to pentamers at pH values in the region of 6·5, with the release of protein 1A and the viral RNA. This acid-induced disassembly is thought to be required for the entry of the virus genome into the host cell. Previous work has highlighted a histidine–α-helix charge-dipole interaction at the twofold axes of symmetry between pentamers and has suggested that this interaction plays a role in acid-induced disassembly. The validity of this theory has now been tested by converting the implicated residue, His-142 of protein 1C, to Arg, Phe and Asp. The effects of such changes were studied by using a previously described vaccinia virus expression system, in which synthesis and processing of FMDV capsid proteins results in the self-assembly of capsids. In agreement with the histidine–α-helix charge-dipole theory, assembly in the arginine mutant was found to be greatly reduced, while capsids of the aspartic acid mutant were considerably more stable under acidic conditions than the wild-type. Aberrant but acid-stable complexes were obtained in the phenylalanine mutant.
Human neutralizing human immunodeficiency virustype 2-specific Fab molecules generated by phage display
Authors: Björling Ewa , von Garrelts Eva , Mörner Andreas , Ehnlund Mariethe , Persson Mats A. A.
Published: August 2015

A panel of human immunodeficiency virus type 2 (HIV-2)-neutralizing, recombinant Fab fragments was generated by using the phage display technique. The combinatorial library was derived from an asymptomatic, HIV-2-seropositive individual and constructed on the surface of filamentous phage by using the pComb3 phagemid vector and then screened against native HIV-2 envelope glycoprotein (gp125). Ten of 30 Fab fragments generated displayed strong reactivity in an ELISA and were therefore selected for further study. Six of these possessed neutralizing capacity, with titres varying from 20 to 80 against the homologous HIV-2 strain, and one also had a weak neutralizing capacity against a heterologous HIV-2 isolate, K135. Sequencing of the heavy chain CDR3 regions showed that the gp125-specific Fabs represented individual clones. These reagents may be useful for studies on the conformational structures of the HIV-2 envelope antigens and their immunogenicity, which may help in vaccine design. Furthermore, the cloned Fab genes may be transformed into whole IgG for eukaryotic expression, and as such used for therapeutic and immunoprophylactic studies in HIV-2-infected macaques and, possibly, for human immunoprophylaxis against HIV-2.
Mapping of domains on the human parainfluenza virus type 2 nucleocapsid protein (NP) required for NP–phosphoprotein or NP–NP interaction
Authors: Nishio Machiko , Tsurudome Masato , Ito Morihiro , Kawano Mitsuo , Kusagawa Shigeru , Komada Hiroshi , Ito Yasuhiko
Published: August 2015

The epitopes recognized by 41 monoclonal antibodies directed against the nucleocapsid protein (NP) of human parainfluenza virus type 2 (hPIV-2) were mapped on the primary structure of the hPIV-2 NP protein by testing their reactivities with deletion mutants. By Western immunoblotting using these monoclonal antibodies, the analysis of deletion mutants of the hPIV-2 NP protein was performed to identify the region essential for NP–NP interaction and phosphoprotein (P)-binding sites on the NP protein. The results indicate that the N-terminal 294 aa of the NP protein are all required for NP–NP self-assembly, and that two C-terminal parts of the NP protein are essential for NP–P binding: one region, aa 295–402, is required for binding to the C-terminal part of the P protein and another region, aa 403–494, to the N-terminal part of the P protein.
Tissue culture infectivity of different strains of infectious bursal disease virus is determined by distinct amino acids in VP2
Authors: Mundt Egbert
Published: August 2015

Two types of strains of serotype I of infectious bursal disease virus (IBDV) have been described, on the basis of their ability (IBDV-TC) or inability (IBDV-BU) to infect chicken embryonic cells in culture. However, both types infect B lymphocytes in the bursa of Fabricius of young chickens. To determine the molecular basis for tissue culture infectivity, virus recombinants with chimeric segments A were constructed from IBDV-TC and IBDV-BU by reverse genetics. The region responsible for the different phenotypes was located in VP2. Site-directed mutagenesis identified single amino acids that are responsible for the restriction in infectivity. However, the appropriate amino acid exchanges are strain-specific.
Overlapping YY1- and aberrant SP1-binding sites proximal to the early promoter of human papillomavirus type 16
Authors: Dong Xiao-Ping , Pfister Herbert
Published: August 2015

Transcription of oncogenes E6 and E7 of human papillomavirus type 16 (HPV-16) from the P97 promoter is regulated by viral and cellular proteins. The transcription factor YY1 represses transcription through binding to cognate sequences in the long control region (LCR). In HPV-16 DNA from cervical carcinomas, mutations of YY1-binding sites have been identified that increase P97 activity 3–6-fold. A second, SP1-binding site has now been identified in the HPV-16 LCR (nt 7842–7847), which overlaps the YY1-binding site at positions 7840–7848. A point mutation within this YY1 site in viral DNA from a cervical cancer, previously shown to prevent YY1 binding, was shown to increase SP1 binding and P97 activity 4·7-fold. An engineered mutant eliminating SP1 binding showed only 1- to 1·6-fold increased P97 activity. It is concluded that competition between SP1 and YY1 for DNA binding plays a major role in YY1 repression mediated by the binding site at positions 7840–7848.
Herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture
Authors: Hukkanen Veijo , Mikola Hannamari , Nykänen Marja , Syrjänen Stina
Published: August 2015

This study describes the outcome of herpes simplex virus type 1 (HSV-1) infection in an organotypic raft culture of spontaneously immortalized HaCat keratinocytes and human fibroblasts, as related to the virus load and epithelial stratification and differentiation. In this model, a confluent monolayer of HaCat keratinocytes was formed 60 h after seeding. Inoculation of HSV-1 before induction of differentiation by lifting of the culture to the air–liquid interface always resulted in a productive infection, but the virus yield was highest when the inoculation took place 72 h after seeding. Even at 0·1 p.f.u. per culture, the HaCat cultures became HSV positive. Infection of the full-thickness epithelium at 5 p.f.u. per culture resulted in a productive infection of the whole epithelium. The HaCat cells were about 10 times more sensitive to HSV-1 infection than the Vero cells in which the virus stocks were titrated. The raft cultures infected 30 min after lifting were negative by HSV-1 culture, and no HSV-1 antigen was detected by immunocytochemistry. PCR showed the presence of HSV-1 DNA and in situ hybridization showed reactivity with a latency-associated RNA probe, indicating the presence of a non-productive infection. Two different patterns of virus spread in epithelia were found: (i) lateral spread through the superficial layers of the epithelium and (ii) a demarcated infection throughout the whole thickness of the epithelium at the margins of the culture.
Nucleolar localization of the UL3 protein of herpes simplex virus type 2
Authors: Yamada Hiroshi , Jiang Yue-Mei , Zhu Hong-Yan , Inagaki-Ohara Kyoko , Nishiyama Yukihiro
Published: August 2015

A rabbit polyclonal antiserum was raised against a recombinant 6×His–UL3 fusion protein expressed inEscherichia coliand used to examine the intracellular localization of the UL3 protein of herpes simplex virus type 2 (HSV-2). The antiserum reacted specifically with 31 and 34 kDa proteins in HSV-2 186-infected Vero cells and with 31 and 35 kDa proteins in UL3-expressing COS-7 cells. The UL3 protein localized both in the cytoplasm and in five to ten bright fluorescent granules in the nucleus close to the nuclear membrane at 4 h post-infection (p.i.). These structures became bigger at 5 h p.i. and showed doughnut-like forms at 6 h p.i. In transfected Vero cells, the UL3 protein localized exclusively in the nucleoplasm and specifically in the nucleolus. Five deletion mutants of the UL3 protein were constructed for transfection assays and the results showed that the region containing amino acids 100–164 was important for nucleolar localization. Moreover, green fluorescent protein (GFP)-targetting experiments showed that the region containing amino acids 100–164 was able to transport non-nucleolar GFP to the nucleolus as a fusion protein.
Epstein–Barr virus lacking latent membrane protein 2 immortalizes B cells with efficiency indistinguishable from that of wild-type virus
Authors: Speck Peter , Kline Kimberly A. , Cheresh Paul , Longnecker Richard
Published: August 2015

Epstein–Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807–2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an E. coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFP+LMP2− recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2− virus to immortalize primary human B cells when compared to that of wild-type virus.
Transcriptional activation by the human herpesvirus-8-encoded interferon regulatory factor
Authors: Roan Florence , Zimring James C. , Goodbourn Stephen , Offermann Margaret K.
Published: August 2015

Human herpesvirus-8 (HHV-8), a gammaherpesvirus that is thought to be the viral aetiologic agent of Kaposi’s sarcoma and primary effusion lymphoma, encodes a homologue to cellular interferon regulatory factors (IRFs). The HHV-8 IRF homologue (vIRF; ORF K9) has previously been shown to inhibit gene induction by interferons and IRF-1 and to transform NIH3T3 cells or Rat-1 cells. Additionally, expression of antisense to vIRF in BCBL-1 cells results in the repression of certain HHV-8 genes, suggesting that vIRF may also positively regulate gene expression. We demonstrate that vIRF activates transcription when directed to DNA by the GAL4 DNA-binding domain. GAL-vIRF truncation constructs that individually are incapable of activating transcription can cooperate in transactivation when coexpressed in HeLa cells, suggesting that multiple regions of vIRF are involved in transactivation. These studies broaden the potential mechanisms of action of vIRF to include transcriptional activation as well as transcriptional repression.
A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses
Authors: Pooggin Mikhail M. , Fütterer Johannes , Skryabin Konstantin G. , Hohn Thomas
Published: August 2015

In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5′-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem–loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5′-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation. The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism. For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF.
Integrin αvβ3 (vitronectin receptor) is a candidate receptor for the virulent echovirus 9 strain Barty
Authors: Nelsen-Salz Birgit , Eggers Hans J. , Zimmermann Holger
Published: August 2015

The enterovirus echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the αvβ3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3- and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this echovirus infection is controlled by the availability of αvβ3 integrin.
Lower concentration of La protein required for internal ribosome entry on hepatitis C virus RNA than on poliovirus RNA
Authors: Isoyama Takeshi , Kamoshita Nobuhiko , Yasui Kotaro , Iwai Atsushi , Shiroki Kazuko , Toyoda Haruka , Yamada Akio , Takasaki Yoshinari , Nomoto Akio
Published: August 2015

Translation initiation of poliovirus and hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal RNA sequence, called the internal ribosomal entry site (IRES). Both IRES bind to the La protein and are thought to require the protein for their translation initiation activity, although they are greatly different in both the primary and predicted secondary structures. To compare the La protein requirement for these IRES, we took advantage of I-RNA from the yeastSaccharomyces cerevisiae, which has been reported to bind to La protein and block poliovirus IRES-mediated translation initiation. In a cell-free translation system prepared from HeLa cells, yeast I-RNA inhibited translation initiation on poliovirus RNA as expected, but did not significantly inhibit translation initiation on HCV RNA. However, the translation initiation directed by either IRES was apparently inhibited by I-RNA in rabbit reticulocyte lysates, in which La protein is limiting. I-RNA-mediated inhibition of HCV IRES-dependent translation in rabbit reticulocyte lysates was reversed by exogenous addition of purified recombinant La protein of smaller amounts than necessary to reverse poliovirus IRES-dependent translation. These results suggest that HCV IRES requires lower concentrations of La protein for its function than does poliovirus IRES. Immunofluorescence studies showed that HCV infection appeared not to affect the subcellular localization of La protein, which exists mainly in the nucleus, although La protein redistributed to the cytoplasm after poliovirus infection. The data are compatible with the low requirement of La protein for HCV IRES activity.