bluuebunny/crossref_metadata_2025 huggingface dataset:
Circuit-Specific Coinfection of Neurons in the Rat Central Nervous System with Two Pseudorabies Virus Recombinants
Authors: Kim Jin-Sang , Enquist Lynn W. , Card J. Patrick
Published: December 2019

Neurotropic alphaherpesviruses have become popular tools for transynaptic analysis of neural circuitry. It has also been demonstrated that coinfection with two viruses expressing unique reporters can be used to define more complicated circuitry. However, the coinfection studies reported to date have employed nonisogenic strains that differ in their invasive properties. In the present investigation we used two antigenically distinct recombinants of the swine pathogen pseudorabies virus (PRV) in single and double infections of the rat central nervous system. Both viruses are derivatives of PRV-Bartha, a strain with reduced virulence that is widely used for circuit analysis. PRV-BaBlu expresses β-galactosidase, and PRV-D expresses the PRV membrane protein gI, the gene for which is deleted in PRV-BaBlu. Antibodies to β-galactosidase identify neurons infected with PRV-BaBlu, and antibodies monospecific for PRV gI identify neurons infected with PRV-D. The ability of these strains to establish coinfections in neurons was evaluated in visual and autonomic circuitry in which the parental virus has previously been characterized. The following conclusions can be drawn from these experiments. First, PRV-D is significantly more neuroinvasive than PRV-Bartha or PRV-BaBlu in the same circuitry. Second, PRV-D is more virulent than either PRV-Bartha or PRV-BaBlu, and PRV-BaBlu is less virulent than PRV-Bartha. Third, in every model examined, PRV-D and PRV-BaBlu coinfect some neurons, but single infections predominate. Fourth, prior infection with one virus renders neurons less permissive to infection by another virus. Fifth, prior infection by PRV-D is more effective than PRV-BaBlu in reducing invasion and spread of the second virus. Collectively, the data define important variables that must be considered in coinfection experiments and suggest that the most successful application of this approach would be accomplished by using isogenic strains of virus with equivalent virulence.
Scrapie Pathogenesis in Subclinically Infected B-Cell-Deficient Mice
Authors: Frigg Rico , Klein Michael A. , Hegyi Ivan , Zinkernagel Rolf M. , Aguzzi Adriano
Published: December 2019

Prion infections can present without clinical manifestations. B-cell deficiency may be a model for subclinical transmissible spongiform encephalopathy, since it protects mice from disease upon intraperitoneal administration of scrapie prions; however, a proportion of B-cell-deficient mice accumulate protease-resistant prion protein in their brains. Here, we have characterized this subclinical disease. In addition, we have studied the possibility that a neurotoxic factor secreted by B cells may contribute to pathogenesis.
Preferential Associations of Alleles of Three Distinct Genes Argue for the Existence of Two Prototype Variants of Human Herpesvirus 7
Authors: Franti Michael , Aubin Jean-Thierry , Gautheret-Dejean Agnes , Malet Isabelle , Cahour Annie , Huraux Jean-Marie , Agut Henri
Published: December 2019

We had previously described six distinct alleles of the glycoprotein B (gB) gene of human herpesvirus 7 (HHV-7). The genetic changes corresponding to these alleles did not affect gB gene transcription or translation in in vitro assays. The study of distinct HHV-7-positive human samples showed preferential associations of some gB alleles with some alleles of two other genes, distantly located on the HHV-7 genome, coding for the phosphoprotein p100 (p100) and the major capsid protein (MCP). Two allele combinations, corresponding to 44 and 31% of the samples studied, respectively, were interpreted as the genetic signatures of two major prototype HHV-7 variants.
The Y-S-L-I Tyrosine-Based Motif in the Cytoplasmic Domain of the Human T-Cell Leukemia Virus Type 1 Envelope Is Essential for Cell-to-Cell Transmission
Authors: Delamarre Lélia , Pique Claudine , Rosenberg Arielle R. , Blot Vincent , Grange Marie-Pierre , Le Blanc Isabelle , Dokhélar Marie-Christine
Published: December 2019

The human T-cell leukemia virus type 1 (HTLV-1) transmembrane glycoprotein has a 24-amino-acid cytoplasmic domain whose function in the viral life cycle is poorly understood. We introduced premature-stop mutations and 18 single-amino-acid substitutions into this domain and studied their effects on cell-to-cell transmission of the virus. The results show that the cytoplasmic domain is absolutely required for cell-to-cell transmission of HTLV-1, through amino acids which cluster in a Y-S-L-I tyrosine-based motif. The transmission defect in two motif mutants did not result from a defect in glycoprotein incorporation or fusion. It appears that the Y-S-L-I tyrosine-based motif of the HTLV-1 glycoprotein cytoplasmic domain has multiple functions, including involvement in virus transmission at a postfusion step.
Human Cytomegalovirus UL144 Open Reading Frame: Sequence Hypervariability in Low-Passage Clinical Isolates
Authors: Lurain Nell S. , Kapell Kathi S. , Huang Diana D. , Short Jeffery A. , Paintsil Jeanette , Winkfield Ernest , Benedict Chris A. , Ware Carl F. , Bremer James W.
Published: December 2019

Human cytomegalovirus (HCMV) infects a number of organs and cell types in vivo, leading to the hypothesis that HCMV disease and tissue tropism may be related to specific sequence variants. A potential component of HCMV variant strains is the UL144 open reading frame (ORF), which encodes a homologue of the herpesvirus entry mediator, HveA, a member of the tumor necrosis factor receptor superfamily. Sequence analysis of the UL144 ORF in 45 low-passage clinical isolates demonstrated significant strain-specific variability. In individual isolates, nucleotide substitutions occur at up to 21% of the 531 positions, resulting in approximately the same percentage of substitutions in the predicted 176-amino-acid sequence. Phylogenetic analysis indicated that the nucleotide and amino acid sequences diverge into three major groups. For genotypic comparison, the known hypervariable region encompassing the proteolytic cleavage site of the glycoprotein B (gB) gene was also sequenced. All of the isolates could be typed according to the four known gB groups; however, the gB and UL144 sequence groups appeared to be phylogenetically unlinked. The predicted UL144 product homology with tumor necrosis factor receptor family members, along with the unexpectedly high level of sequence variability of the UL144 ORF, suggests that the predicted product may play a role in HCMV infectivity and subsequent host disease.
Induction of Transformation and p53-Dependent Apoptosis by Adenovirus Type 5 E4orf6/7 cDNA
Authors: Yamano Shigeru , Tokino Takashi , Yasuda Motoaki , Kaneuchi Masanori , Takahashi Minoru , Niitsu Yoshiro , Fujinaga Kei , Yamashita Toshiharu
Published: December 2019

Adenovirus (Ad) E4orf6/7, one of the early gene products of human Ads, forms a stable complex with the cellular transcription factor E2F to activate transcription from the Ad E2 promoter. E2F cDNAs have growth-promoting and apoptosis-inducing activities when overexpressed in cells. We cloned Ad5 E4orf6/7 cDNA in both simian virus 40- and human cytomegalovirus-based expression vectors to examine its transforming and apoptotic activities. The cloned E4orf6/7 collaborated with a retinoblastoma protein (RB)-nonbinding and therefore E2F-nonreleasing mutant of Ad5 E1A (
dl
922/947) to morphologically transform primary rat cells, suggesting that E2F is an important cellular protein functioning downstream of E1A for transformation. In a G418 colony formation assay, E4orf6/7 was shown to suppress growth of untransformed rat cells. Moreover, a recombinant Ad expressing Ad5 E4orf6/7 induced apoptosis in rat cells when coinfected with wild-type p53-expressing Ad. Mutational analysis of E4orf6/7 revealed that both of the domains required for growth inhibition and transformation by E4orf6/7 lay in the C-terminal region, which is essential for transactivation from the upstream sequence of an E2a promoter containing E2F-binding sites. However, the smallest mutant of E4orf6/7, encoding the C-terminal 59 amino acids, failed to complement the RB-nonbinding
dl
922/947 mutant despite showing growth inhibition and E2F transactivation activities. Thus, it is suggested that a subregion of E4orf6/7 which is required for growth inhibition and transformation in collaboration with
dl
922/947 overlaps the transactivation domain of E4orf6/7.
Mutational Analysis of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase
Authors: Lai Vicky C. H. , Kao C. Cheng , Ferrari Eric , Park Justin , Uss Annette S. , Wright-Minogue Jacquelyn , Hong Zhi , Lau Johnson Y. N.
Published: December 2019

Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in
Escherichia coli
failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BΔCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (ΔCT179 and ΔCT218) that lacked RdRp activities.
Physical and Functional Interaction between the Y-Box Binding Protein YB-1 and Human Polyomavirus JC Virus Large T Antigen
Authors: Safak Mahmut , Gallia Gary L. , Ansari Sameer A. , Khalili Kamel
Published: December 2019

Y-box binding protein YB-1 is a member of a family of DNA and RNA binding proteins which have been shown to affect gene expression at both the transcriptional and translational levels. We have previously shown that YB-1 modulates transcription from the promoters of the ubiquitous human polyomavirus JC virus (JCV). Here we investigate the physical and functional interplay between YB-1 and the viral regulatory protein large T antigen (T-antigen), using JCV as a model system. Results of mobility band shift assays demonstrated that the efficiency of binding of YB-1 to a 23-bp single-stranded viral target sequence was significantly increased when T-antigen was included in the binding reaction mixture. Affinity chromatography and coimmunoprecipitation assays demonstrated that YB-1 and T-antigen physically interact with each other. Additionally, results of transcription studies demonstrated that these two proteins interact functionally on the JCV early and late gene promoters. Whereas ectopic expression of YB-1 and T-antigen results in synergistic transactivation of the viral late promoter, YB-1 alleviates T-antigen-mediated transcriptional suppression of the viral early promoter activity. Furthermore, we have localized, through the use of a series of deletion mutants, the sequences of these proteins which are important for their interaction. The T-antigen-interacting region of YB-1 is located in the cold shock domain of YB-1 and its immediate flanking sequences, and the YB-1-interacting domain of T-antigen maps to the carboxy-terminal half of T-antigen. Results of transient transfection assays with various YB-1 mutants and T-antigen expression constructs confirm the specificity of the functional interaction between YB-1 and T-antigen. Taken together, these data demonstrate that the cellular factor YB-1 and the viral regulatory protein T-antigen interact both physically and functionally and that this interaction modulates transcription from the JCV promoters.
Mutations Abrogating the RNase Activity in Glycoprotein Ernsof the Pestivirus Classical Swine Fever Virus Lead to Virus Attenuation
Authors: Meyers Gregor , Saalmüller Armin , Büttner Mathias
Published: December 2019

Classical swine fever (CSF) is a severe hemorrhagic disease of swine caused by the pestivirus CSF virus (CSFV). Amino acid exchanges or deletions introduced by site-directed mutagenesis into the putative active site of the RNase residing in the glycoprotein Ernsof CSFV abolished the enzymatic activity of this protein, as demonstrated with an RNase test suitable for detection of the enzymatic activity in crude cell extracts. Incorporation of the altered sequences into an infectious CSFV clone resulted in recovery of viable viruses upon RNA transfection, except for a variant displaying a deletion of the histidine codon at position 297 of the long open reading frame. These RNase-negative virus mutants displayed growth characteristics in tissue culture that were undistinguishable from wild-type virus and were stable for at least seven passages. In contrast to animals inoculated with an RNase-positive control virus, infection of piglets with an RNase-negative mutant containing a deletion of the histidine codon 346 of the open reading frame did not lead to CSF. Neither fever nor extended viremia could be detected. Animals infected with this mutant did not show decrease of peripheral B cells, a characteristic feature of CSF in swine. Animal experiments with four other mutants with either exchanges of codons 297 or 346 or double exchanges of both codons 297 and 346 showed that all these RNase-negative mutants were attenuated. All viruses with mutations affecting codon 346 were completely apathogenic, whereas those containing only changes of codon 297 consistently induced clinical symptoms for several days, followed by sudden recovery. Analyses of reisolated viruses gave no indication for the presence of revertants in the infected animals.
Dendritic Cells Transduced with an Adenovirus Vector Encoding Epstein-Barr Virus Latent Membrane Protein 2B: a New Modality for Vaccination
Authors: Ranieri E. , Herr W. , Gambotto A. , Olson W. , Rowe D. , Robbins P. D. , Kierstead L. Salvucci , Watkins S. C. , Gesualdo L. , Storkus W. J.
Published: December 2019

Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignancies, particularly in immunocompromised hosts. As a strategy for stimulating immunity against EBV for the treatment of EBV-associated tumors, we have genetically engineered dendritic cells (DC) to express EBV antigens, such as latent membrane protein 2B (LMP2B), using recombinant adenovirus vectors. CD8+T lymphocytes from HLA-A2.1+, EBV-seropositive healthy donors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or AdLMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC were positive for EGFP expression as assessed by fluorescence-activated cell sorting analysis, indicating efficient gene transfer. AdLMP2-transduced DC were used to stimulate CD8+T cells. Responder CD8+T cells were tested for gamma interferon (IFN-γ) release by enzyme-linked spot (ELISPOT) assay and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-337 and LMP2 426-434) were very low as assessed by IFN-γ spot formation (T-cell frequency, <0.003%). IFN-γ ELISPOT assays performed at day 14 showed a significant (2-log) increase of the day 0 frequency of T cells reactive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P< 0.001). Moreover, specific cytolytic activity was observed against the autologous EBV B-lymphoblastoid cell lines after 21 days of stimulation of T-cell responders with AdLMP2-transduced DC (P< 0.01). In summary, autologous mature DC genetically modified with an adenovirus encoding EBV antigens stimulate the generation of EBV-specific CD8+effector T cells in vitro, supporting the potential application of EBV-based adenovirus vector vaccination for the immunotherapy of the EBV-associated malignancies.
Single-Step Conversion of Cells to Retrovirus Vector Producers with Herpes Simplex Virus–Epstein-Barr Virus Hybrid Amplicons
Authors: Sena-Esteves Miguel , Saeki Yoshinaga , Camp Sara M. , Chiocca E. Antonio , Breakefield Xandra O.
Published: December 2019

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV–Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes
gag-pol
and
env
(GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding
lacZ
were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10
6
and 10
7
transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.
Effect of Cytoplasmic Tail Truncations on the Activity of the M
2
Ion Channel of Influenza A Virus
Authors: Tobler Kurt , Kelly Marie L. , Pinto Lawrence H. , Lamb Robert A.
Published: December 2019

The M
2
protein of influenza A virus forms a proton channel that is required for viral replication. The M
2
ion channel is a homotetramer and has a 24-residue N-terminal extracellular domain, a 19-residue transmembrane domain, and a 54-residue cytoplasmic tail. We show here that the N-terminal methionine residue is cleaved from the mature protein. Translational stop codons were introduced into the M
2
cDNA at residues 46, 52, 62, 72, 77, 82, 87, and 92. The deletion mutants were designated trunc
x
, according to the amino acid position that was changed to a stop codon. We studied the role of the cytoplasmic tail by measuring the ion channel activity (the current sensitive to the M
2
-specific inhibitor amantadine) of the cytoplasmic tail truncation mutants expressed in oocytes of
Xenopus laevis
. When their conductance was measured over time, mutants trunc72, trunc77, and trunc92 behaved comparably to wild-type M
2
protein (a decrease of only 4% over 30 min). In contrast, conductance decreased by 28% for trunc82, 27% for trunc62, and 81% for trunc52 channels. Complete closure of the channel could be observed in some cells for trunc62 and trunc52 within 30 min. These data suggest that a role of the cytoplasmic tail region of the M
2
ion channel is to stabilize the pore against premature closure while the ectodomain is exposed to low pH.
Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
Authors: Yang Wengang , Summers Jesse
Published: December 2019

DNA of the avian hepadnavirus, duck hepatitis B virus, was found to be integrated at low abundance into the cellular DNA extracted from the livers of infected ducklings. The frequency of integration was estimated to be at least one viral genome per 10
3
to 10
4
cells by 6 days postinfection. The structures of virus-cell junctions determined by sequencing were compared with those of virus-virus junctions formed by nonhomologous recombination between the ends of linear viral DNA forms. This comparison allowed us to conclude that linear viral DNA was the preferential form used as an integration substrate. Potential factors promoting viral DNA integration during chronic infection are discussed.
Interaction of Hepatitis C Virus Core Protein with Viral Sense RNA and Suppression of Its Translation
Authors: Shimoike Takashi , Mimori Shigetaka , Tani Hideki , Matsuura Yoshiharu , Miyamura Tatsuo
Published: December 2019

To clarify the binding properties of hepatitis C virus (HCV) core protein and its viral RNA for the encapsidation, morphogenesis, and replication of HCV, the specific interaction of HCV core protein with its genomic RNA synthesized in vitro was examined in an in vivo system. The positive-sense RNA from the 5′ end to nucleotide (nt) 2327, which covers the 5′ untranslated region (5′UTR) and a part of the coding region of HCV structural proteins, interacted with HCV core protein, while no interaction was observed in the same region of negative-sense RNA and in other regions of viral and antiviral sense RNAs. The internal ribosome entry site (IRES) exists around the 5′UTR of HCV; therefore, the interaction of the core protein with this region of HCV RNA suggests that there is some effect on its cap-independent translation. Cells expressing HCV core protein were transfected with reporter RNAs consisting of nt 1 to 709 of HCV RNA (the 5′UTR of HCV and about two-thirds of the core protein coding regions) followed by a firefly luciferase gene (HCV07Luc RNA). The translation of HCV07Luc RNA was suppressed in cells expressing the core protein, whereas no significant suppression was observed in the case of a reporter RNA possessing the IRES of encephalomyocarditis virus followed by a firefly luciferase. This suppression by the core protein occurred in a dose-dependent manner. The expression of the E1 envelope protein of HCV or β-galactosidase did not suppress the translation of both HCV and EMCV reporter RNAs. We then examined the regions that are important for suppression of translation by the core protein and found that the region from nt 1 to 344 was enough to exert this suppression. These results suggest that the HCV core protein interacts with viral genomic RNA at a specific region to form nucleocapsids and regulates the expression of HCV by interacting with the 5′UTR.
The Major Neutralizing Antigenic Site on Herpes Simplex Virus Glycoprotein D Overlaps a Receptor-Binding Domain
Authors: Whitbeck J. Charles , Muggeridge Martin I. , Rux Ann H. , Hou Wangfang , Krummenacher Claude , Lou Huan , van Geelen Albert , Eisenberg Roselyn J. , Cohen Gary H.
Published: December 2019

Herpes simplex virus (HSV) entry is dependent on the interaction of virion glycoprotein D (gD) with one of several cellular receptors. We previously showed that gD binds specifically to two structurally dissimilar receptors, HveA and HveC. We have continued our studies by using (i) a panel of baculovirus-produced gD molecules with various C-terminal truncations and (ii) a series of gD mutants with nonoverlapping 3-amino-acid deletions between residues 222 and 254. Binding of the potent neutralizing monoclonal antibody (MAb) DL11 (group Ib) was unaffected in forms of gD containing residues 1 to 250 but was greatly diminished in molecules truncated at residue 240 or 234. Both receptor binding and blocking of HSV infection were also affected by these C-terminal truncations. gD-1(234t) bound weakly to both HveA and HveC as determined by enzyme-linked immunosorbent assay (ELISA) and failed to block infection. Interestingly, gD-1(240t) bound well to both receptors but blocked infection poorly, indicating that receptor binding as measured by ELISA is not the only gD function required for blocking. Optical biosensor studies showed that while gD-1(240t) bound HveC with an affinity similar to that of gD-1(306t), the rates of complex formation and dissociation were significantly faster than for gD-1(306t). Complementation analysis showed that any 3-amino-acid deletion between residues 222 and 251 of gD resulted in a nonfunctional protein. Among this set of proteins, three had lost DL11 reactivity (those with deletions between residues 222 and 230). One of these proteins (deletion 222–224) was expressed as a soluble form in the baculovirus system. This protein did not react with DL11, bound to both HveA and HveC poorly as shown by ELISA, and failed to block HSV infection. Since this protein was bound by several other MAbs that recognize discontinuous epitopes, we conclude that residues 222 to 224 are critical for gD function. We propose that the potent virus-neutralizing activity of DL11 (and other group Ib MAbs) likely reflects an overlap between its epitope and a receptor-binding domain of gD.
Activation of the PAK-Related Kinase by Human Immunodeficiency Virus Type 1 Nef in Primary Human Peripheral Blood Lymphocytes and Macrophages Leads to Phosphorylation of a PIX-p95 Complex
Authors: Brown Amanda , Wang Xia , Sawai Earl , Cheng-Mayer Cecilia
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication in both primary T lymphocytes and monocyte-derived macrophages. This enhancement phenotype has been linked to the ability of Nef to modulate the activity of cellular kinases. We find that despite the reported high-affinity interaction between Nef and the Src kinase Hck in vitro, a Nef-Hck interaction in the context of HIV-1-infected primary macrophages is not detectable. However, Nef binding and activation of the PAK-related kinase and phosphorylation of its substrate could be readily detected in both infected primary T lymphocytes and macrophages. Furthermore, we show that this substrate is a complex composed of the recently characterized PAK interacting partner PIX (PAK-interacting guanine nucleotide exchange factor) and its tightly associated p95 protein. PAK and PIX-p95 appear to be differentially activated and phosphorylated depending on the intracellular environment in whichnefis expressed. These results identify the PIX-p95 complex as a novel effector of Nef in primary cells and suggest that the regulation of the PAK signaling pathway may differ in T cells and macrophages.
The Residues between the Two Transformation Effector Sites of Epstein-Barr Virus Latent Membrane Protein 1 Are Not Critical for B-Lymphocyte Growth Transformation
Authors: Izumi Kenneth M. , McFarland Ellen Cahir , Riley Elisabeth A. , Rizzo Danielle , Chen Yuzhi , Kieff Elliott
Published: December 2019

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-κB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.
Transcriptional Up-Regulation of the Cyclin D2 Gene and Acquisition of New Cyclin-Dependent Kinase Partners in Human T-Cell Leukemia Virus Type 1-Infected Cells
Authors: Santiago Francisco , Clark Elizabeth , Chong Siewyen , Molina Carlos , Mozafari Fariba , Mahieux Renaud , Fujii Masahiro , Azimi Nazli , Kashanchi Fatah
Published: December 2019

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax1is a 40-kDa phosphoprotein, predominantly localized in the nucleus of the host cell, which functions to transactivate both viral and cellular promoters. It seems likely that HTLV-1, through expression of the viral regulatory protein Tax1, provides some initial alteration in cell metabolism predisposing the development of ATL. Here, we demonstrate that HTLV-1 infection in T-cell lines and patient samples causes overexpression of an early G1cyclin, cyclin D2. The transcriptional up-regulation of the cyclin D2 gene is due to activation of Tax on the cyclin D2 gene. More important, we find that overexpression of cyclin D2 is accompanied by acquisition of new partners such as cyclin-dependent kinase 2 (cdk2), cdk4, and cdk6 in infected cells. This is in contrast to uninfected T cells, where cyclin D2 associates only with cdk6. Functional effects of these cyclin-cdk complexes in infected cells are shown by hyperphosphorylation of Rb and histone H1, indicators of active progression into S phase as well as changes in cellular chromatin and transcription machinery. These studies link HTLV-1 infection with changes of cellular cyclin gene expression, hence providing clues to development of T-cell leukemia.
Hyperphosphorylation of the Hepatitis C Virus NS5A Protein Requires an Active NS3 Protease, NS4A, NS4B, and NS5A Encoded on the Same Polyprotein
Authors: Neddermann Petra , Clementi Angelica , De Francesco Raffaele
Published: December 2019

The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in
cis
). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in
trans
. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.
Characterization of a Novel Simian Immunodeficiency Virus (SIV) from L’Hoest Monkeys (
Cercopithecus l’hoesti
): Implications for the Origins of SIVmnd and Other Primate Lentiviruses
Authors: Hirsch Vanessa M. , Campbell Barbara J. , Bailes Elizabeth , Goeken Robert , Brown Charles , Elkins William R. , Axthelm Michael , Murphey-Corb Michael , Sharp Paul M.
Published: December 2019

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) appear to have originated by cross-species transmission of simian immunodeficiency virus (SIV) from asymptomatically infected African primates. Few of the SIVs characterized to date efficiently infect human primary lymphocytes. Interesting, two of the three identified to infect such cultures (SIVsm and SIVcpz) have appeared in human populations as genetically related HIVs. In the present study, we characterized a novel SIV isolate from an East African monkey of the
Cercopithecus
genus, the l’hoest monkey (
C. l’hoesti
), which we designated SIVlhoest. This SIV isolate efficiently infected both human and macaque lymphocytes and resulted in a persistent infection of macaques, characterized by high primary virus load and a progressive decline in circulating CD4 lymphocytes, consistent with progression to AIDS. Phylogenetic analyses showed that SIVlhoest is genetically distinct from other previously characterized primate lentiviruses but clusters in the same major lineage as SIV from mandrills (SIVmnd), a West African primate species. Given the geographic distance between the ranges of l’hoest monkeys and mandrills, this may indicate that SIVmnd arose through cross-species transmission from close relatives of l’hoest monkeys that are sympatric with mandrills. These observations lend support to the hypothesis that the primate lentiviruses originated and coevolved within monkeys of the
Cercopithecus
genus. Regarded in this light, lentivirus infections of primates not belonging to the
Cercopithecus
genus may have resulted from cross-species transmission in the not-too-distant past.
Two-Helper RNA System for Production of Recombinant Semliki Forest Virus Particles
Authors: Smerdou C. , Liljeström P.
Published: December 2019

Alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. However, present strategies for production of such particles have biosafety limitations due to the generation, by RNA recombination, of replication-proficient viruses (RPVs). Here we describe a new packaging system for Semliki Forest virus (SFV) based on a the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 polyprotein are expressed from two independent RNA molecules. The capsid gene contains a translational enhancer and therefore that sequence was also engineered in front of the spike sequence p62-6K-E1. A sequence coding for the foot-and-mouth disease virus 2A autoprotease was inserted in frame between the capsid translational enhancer and the spike genes. This allows production of the spike proteins at high levels with cotranslational removal of the enhancer sequence and normal biosynthesis of the spike complex. The autoprotease activity of the capsid protein was abolished by mutation, further increasing the biosafety of the system. Cotransfection of cells with both helper RNAs and an SFV vector replicon carrying the LacZ gene led to production of recombinant particles with titers of up to 8 × 10
8
particles per 10
6
cells. Extensive analysis failed to demonstrate the presence of any RPVs, emphasizing the high biosafety of the system based on two-helper RNAs.
Genetic Instability of Live, Attenuated Human Immunodeficiency Virus Type 1 Vaccine Strains
Authors: Berkhout Ben , Verhoef Koen , van Wamel Jeroen L. B. , Back Nicole K. T.
Published: December 2019

Live, attenuated viruses have been the most successful vaccines in monkey models of human immunodeficiency virus type 1 (HIV-1) infection. However, there are several safety concerns about using such an anti-HIV vaccine in humans, including reversion of the vaccine strain to virulence and recombination with endogenous retroviral sequences to produce new infectious and potentially pathogenic viruses. Because testing in humans would inevitably carry a substantial risk, we set out to test the genetic stability of multiply deleted HIV constructs in perpetuated tissue culture infections. The Δ3 candidate vaccine strain of HIV-1 contains deletions in the viral long terminal repeat (LTR) promoter and the
vpr
and
nef
genes. This virus replicates with delayed kinetics, but a profound enhancement of virus replication was observed after approximately 2 months of culturing. Analysis of the revertant viral genome indicated that the three introduced deletions were maintained but a 39-nucleotide sequence was inserted in the LTR promoter region. This insert was formed by duplication of the region encoding three binding sites for the Sp1 transcription factor. The duplicated Sp1 region was demonstrated to increase the LTR promoter activity, and a concomitant increase in the virus replication rate was measured. In fact, duplication of the Sp1 sites increased the fitness of the Δ3 virus (Vpr/Nef/U3) to levels higher than that of the singly deleted ΔVpr virus. These results indicate that deleted HIV-1 vaccine strains can evolve into fast-replicating variants by multiplication of remaining sequence motifs, and their safety is therefore not guaranteed. This insight may guide future efforts to develop more stable anti-HIV vaccines.
Molecular Characterization and Placental Expression of HERV-W, a New Human Endogenous Retrovirus Family
Authors: Blond Jean-Luc , Besème Frédéric , Duret Laurent , Bouton Olivier , Bedin Frédéric , Perron Hervé , Mandrand Bernard , Mallet François
Published: December 2019

The multiple sclerosis-associated retrovirus (MSRV) isolated from plasma of MS patients was found to be phylogenetically and experimentally related to human endogenous retroviruses (HERVs). To characterize the MSRV-related HERV family and to test the hypothesis of a replication-competent HERV, we have investigated the expression of MSRV-related sequences in healthy tissues. The expression of MSRV-related transcripts restricted to the placenta led to the isolation of overlapping cDNA clones from a cDNA library. These cDNAs spanned a 7.6-kb region containing
gag
,
pol
, and
env
genes; RU5 and U3R flanking sequences; a polypurine tract; and a primer binding site (PBS). As this PBS showed similarity to avian retrovirus PBSs used by tRNA
Trp
, this new HERV family was named HERV-W. Several genomic elements were identified, one of them containing a complete HERV-W unit, spanning all cDNA clones. Elements of this multicopy family were not replication competent, as
gag
and
pol
open reading frames (ORFs) were interrupted by frameshifts and stop codons. A complete ORF putatively coding for an envelope protein was found both on the HERV-W DNA prototype and within an RU5-
env
-U3R polyadenylated cDNA clone. Placental expression of 8-, 3.1-, and 1.3-kb transcripts was observed, and a putative splicing strategy was described. The apparently tissue-restricted HERV-W long terminal repeat expression is discussed with respect to physiological and pathological contexts.
The Humoral Immune Response to Human T-Cell Lymphotropic Virus Type 1 Envelope Glycoprotein gp46 Is Directed Primarily against Conformational Epitopes
Authors: Hadlock Kenneth G. , Rowe Judy , Foung Steven K. H.
Published: December 2019

Individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1) develop a robust immune response to the surface envelope glycoprotein gp46 that is partially protective. The relative contribution of antibodies to conformation-dependent epitopes, including those mediating virus neutralization as part of the humoral immune response, is not well defined. We assess in this report the relationship between defined linear and conformational epitopes and the antibodies elicited to these domains. First, five monoclonal antibodies to linear epitopes within gp46 were evaluated for their ability to abrogate binding of three human monoclonal antibodies that inhibit HTLV-1-mediated syncytia formation and recognize conformational epitopes. Binding of antibodies to conformational epitopes was unaffected by antibodies to linear epitopes throughout the carboxy-terminal half and central domain of HTLV-1 gp46. Second, an enzyme-linked immunoadsorbent assay was developed and used to measure serum antibodies to native and denatured gp46 from HTLV-1-infected individuals. In sera from infected individuals, reactivity to denatured gp46 had an average of 15% of the reactivity observed to native gp46. Third, serum antibodies from 24 of 25 of HTLV-1-infected individuals inhibited binding of a neutralizing human monoclonal antibody, PRH-7A, to a conformational epitope on gp46 that is common to HTLV-1 and -2. Thus, antibodies to conformational epitopes comprise the majority of the immune response to HTLV-1 gp46, and the epitopes recognized by these antibodies do not appear to involve sequences in previously described immunodominant linear epitopes.
Limited Protection from a Pathogenic Chimeric Simian-Human Immunodeficiency Virus Challenge following Immunization with Attenuated Simian Immunodeficiency Virus
Authors: Lewis Mark G. , Yalley-Ogunro Jake , Greenhouse Jack J. , Brennan Terry P. , Jiang Jennifer Bo , VanCott Thomas C. , Lu Yichen , Eddy Gerald A. , Birx Deborah L.
Published: December 2019

Two live attenuated single-deletion mutant simian immunodeficiency virus (SIV) constructs, SIV239Δnefand SIVPBj6.6Δnef, were tested for their abilities to stimulate protective immunity in macaques. During the immunization period the animals were examined for specific immune responses and virus growth. Each construct generated high levels of specific immunity in all of the immunized animals. The SIV239Δnefconstruct was found to grow to high levels in all immunized animals, with some animals remaining positive for virus isolation and plasma RNA throughout the immunization period. The SIVPBj6.6Δnefwas effectively controlled by all of the immunized animals, with virus mostly isolated only during the first few months following immunization and plasma RNA never detected. Following an extended period of immunization of over 80 weeks, the animals were challenged with a pathogenic simian-human immunodeficiency virus (SHIV) isolate, SIV89.6PD, by intravenous injection. All of the SIV239Δnef-immunized animals became infected with the SHIV isolate; two of five animals eventually controlled the challenge and three of five animals, which failed to check the immunizing virus, progressed to disease state before the unvaccinated controls. One of five animals immunized with SIVPBj6.6Δneftotally resisted infection by the challenge virus, while three others limited its growth and the remaining animal became persistently infected and eventually died of a pulmonary thrombus. These data indicate that vaccination with attenuated SIV can protect macaques from disease and in some cases from infection by a divergent SHIV. However, if animals are unable to control the immunizing virus, potential damage that can accelerate the disease course of a pathogenic challenge virus may occur.
The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions
Authors: Inabe Kazunori , Nishizawa Masako , Tajima Shigeru , Ikuta Kazuyoshi , Aida Yoko
Published: December 2019

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)
2
motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.
Coxsackievirus and Adenovirus Receptor Amino-Terminal Immunoglobulin V-Related Domain Binds Adenovirus Type 2 and Fiber Knob from Adenovirus Type 12
Authors: Freimuth Paul , Springer Karen , Berard Chris , Hainfeld Jim , Bewley Maria , Flanagan John
Published: December 2019

The extracellular region of the coxsackievirus and adenovirus receptor (CAR) is predicted to consist of two immunoglobulin (Ig)-related structural domains. We expressed the isolated CAR amino-terminal domain (D1) and a CAR fragment containing both extracellular Ig domains (D1/D2) in
Escherichia coli
. Both D1 and D1/D2 formed complexes in vitro with the recombinant knob domain of adenovirus type 12 (Ad12) fiber, and D1 inhibited adenovirus type 2 (Ad2) infection of HeLa cells. These results indicate that the adenovirus-binding activity of CAR is localized in the amino-terminal IgV-related domain and confirm our earlier observation that Ad2 and Ad12 bind to the same cellular receptor. Preliminary crystallization studies suggest that complexes of Ad12 knob bound to D1 will be suitable for structure determination.