bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cellular Transcription Factors Enhance Herpes Simplex Virus Type 1 oriS-Dependent DNA Replication
Authors: Nguyen-Huynh Anh Tuan , Schaffer Priscilla A. 
Published: December 2019

ABSTRACT
The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains three binding sites for the viral origin binding protein (OBP) flanked by transcriptional regulatory elements of the immediate-early genes encoding ICP4 and ICP22/47. To assess the role of flanking sequences in oriS function, plasmids containing oriS and either wild-type or mutant flanking sequences were tested in transient DNA replication assays. Although the ICP4 and ICP22/47 regulatory regions were shown to enhance oriS function, most individual elements in these regions, including the VP16-responsive TAATGARAT elements, were found to be dispensable for oriS function. In contrast, two oriS core-adjacent regulatory (Oscar) elements, OscarL and OscarR, at the base of the oriS palindrome were shown to enhance oriS function significantly and additively. Specifically, mutational disruption of either element reduced oriS-dependent DNA replication by 60 to 70%, and disruption of both elements reduced replication by 90%. The properties of protein-DNA complexes formed in gel mobility shift assays using uninfected and HSV-1-infected Vero cell nuclear extracts demonstrated that both OscarL and OscarR are binding sites for cellular proteins. Whereas OscarR does not correspond to the consensus binding site of any known transcription factor, OscarL contains a consensus binding site for the transcription factor Sp1. Gel mobility shift and supershift experiments using antibodies directed against members of the Sp1 family of transcription factors demonstrated the presence of Sp1 and Sp3, but not Sp2 or Sp4, in the protein-DNA complexes formed at OscarL. The abilities of OscarL and OscarR to bind their respective cellular proteins correlated directly with the efficiency of oriS-dependent DNA replication. Cooperative interactions between the Oscar-binding factors and proteins binding to adjacent OBP binding sites were not observed. Notably, Oscar element mutations that impaired oriS-dependent DNA replication had no detectable effect on either basal or induced levels of transcription from the ICP4 and ICP22/47 promoters, as determined by RNase protection assays. The Oscar elements thus appear to provide binding sites for cellular proteins that facilitate oriS-dependent DNA replication but have no effect on transcription of oriS-flanking genes.
Substitution ofrasfor the Herpesvirus Saimiri STP Oncogene in Lymphocyte Transformation
Authors: Guo Jie , Williams Kenneth , Duboise S. Monroe , Alexander Louis , Veazey Ronald , Jung Jae U. 
Published: December 2019

ABSTRACTSTP-C488 (STP of herpesvirus saimiri [HVS] group C strain 488 [C488]) is the only virus-encoded protein found to associate with cellularrasand activaterassignal transduction pathways. To investigate an important role forrassignal transduction in STP-dependent growth transformation, we constructed recombinant strains of HVS C488 in which the STP-C488 oncogene was replaced with cellular normal ras (c-ras) or viral oncogenic ras (v-ras). Recombinant HVSΔSTP/v-rasimmortalized primary common marmoset T lymphocytes to interleukin-2-independent growth as efficiently as wild-type HVS C488 (wt HVS), while recombinant HVSΔSTP/c-rasdid so with low efficiency. Whereas wt HVS immortalized CD4−CD8+single-positive T lymphocytes, HVSΔSTP/c-ras- and HVSΔSTP/v-ras-immortalized cells were principally CD4+CD8+double-positive T lymphocytes. In addition, HVSΔSTP/v-ras-immortalized T cells showed a high level ofrasexpression and exhibited an adherent macrophage-like morphology. These phenotypes were likely caused by the drastic activation of AP-1 transcriptional factor activity. Finally, HVSΔSTP/v-rasand HVSΔSTP/c-raseach induced lymphoma in one of two common marmosets, although onset of disease was more rapid with the v-rasvirus. These results demonstrate thatrascan substitute for the STP oncogene of HVS C488 to allow immortalized growth of primary lymphoid cells and that an activated form ofrasdoes so more efficiently than the normal cellular form ofras.
The Herpes Simplex Virus Type 1 U
L
17 Gene Encodes Virion Tegument Proteins That Are Required for Cleavage and Packaging of Viral DNA
Authors: Salmon Brandy , Cunningham Charles , Davison Andrew J. , Harris Wendy J. , Baines Joel D. 
Published: December 2019

ABSTRACT

Previous studies have suggested that the U
L
17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a
lacZ
expression cassette in place of 1,490 bp of the 2,109-bp U
L
17 open reading frame [HSV-1(ΔU
L
17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5′ end of the U
L
17 open reading frame [HSV-1(U
L
17-stop)] were plaque purified on engineered cell lines containing the U
L
17 gene. A virus derived from HSV-1(U
L
17-stop) but containing a restored U
L
17 gene was also constructed and was designated HSV-1(U
L
17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(ΔU
L
17) nor HSV-1(U
L
17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(ΔU
L
17) or HSV-1(U
L
17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(ΔU
L
17) or HSV-1(U
L
17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(ΔU
L
17) compared to wild-type virus show no detectable differences. These data indicate that the U
L
17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U
L
17 gene product, an anti-U
L
17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent
M
r
77,000 and weakly with a protein of apparent
M
r
72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U
L
17 protein. We therefore conclude that the U
L
17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.
Effect of Cleavage Mutants on Syncytium Formation Directed by the Wild-Type Fusion Protein of Newcastle Disease Virus
Authors: Li Zengji , Sergel Theresa , Razvi Enal , Morrison Trudy 
Published: December 2019

ABSTRACT
The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.
Hepatitis C Virus Structural Proteins Assemble into Viruslike Particles in Insect Cells
Authors: Baumert Thomas F. , Ito Susumu , Wong David T. , Liang T. Jake 
Published: December 2019

ABSTRACT
Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.
A Cellular Protein Binds Vaccinia Virus Late Promoters and Activates Transcription In Vitro
Authors: Zhu Min , Moore Trisha , Broyles Steven S. 
Published: December 2019

ABSTRACT
Available evidence indicates that the transcription of the late class of vaccinia virus genes requires the participation of several virus-encoded proteins in addition to the viral RNA polymerase. In this report we describe the identification of a protein present in extracts of uninfected HeLa cells that binds avidly to viral late promoter DNA. The protein bound specifically to several different vaccinia virus late promoters but not an early nor an intermediate promoter. DNase I footprinting localized the protein’s binding site to nucleotides surrounding the transcriptional start site of the I1L promoter. Optimal promoter binding required sequences in the highly conserved TAAAT motif at the transcriptional start site as well as sequences immediately upstream; however, one variation on the motif’s sequence did not affect promoter binding by the protein. Partially purified late promoter binding protein (LPBP) was capable of stimulating the transcription activity of extracts depleted of LPBP on a late promoter-driven template, establishing LPBP as a transcription activator in vitro. These results suggest that a cellular protein is responsible for targeting vaccinia virus late promoters for initiation of transcription.
Mutational Scan of the Human Immunodeficiency Virus Type 2 Integrase Protein
Authors: van den Ent Fusinita M. I. , Vos Arnold , Plasterk Ronald H. A. 
Published: December 2019

ABSTRACT
Retroviral integrase (IN) cleaves linear viral DNA specifically near the ends of the DNA (cleavage reaction) and subsequently couples the processed ends to phosphates in the target DNA (integration reaction). In vitro, IN catalyzes the disintegration reaction, which is the reverse of the integration reaction. Ideally, we would like to test the role of each amino acid in the IN protein. We mutagenized human immunodeficiency virus type 2 IN in a random way using PCR mutagenesis and generated a set of mutants in which 35% of all residues were substituted. Mutant proteins were tested for in vitro activity, e.g., site-specific cleavage of viral DNA, integration, and disintegration. Changes in 61 of the 90 proteins investigated showed no phenotypic effect. Substitutions that changed the choice of nucleophile in the cleavage reaction were found. These clustered around the active-site residues Asp-116 and Glu-152. We also found alterations of amino acids that affected cleavage and integration differentially. In addition, we analyzed the disintegration activity of the proteins and found substitutions of amino acids close to the dimer interface that enhanced intermolecular disintegration activity, whereas other catalytic activities were present at wild-type levels. This study shows the feasibility of investigating the role of virtually any amino acid in a protein the size of IN.
Predominance of HLA-Restricted Cytotoxic T-Lymphocyte Responses to Serotype-Cross-Reactive Epitopes on Nonstructural Proteins following Natural Secondary Dengue Virus Infection
Authors: Mathew Anuja , Kurane Ichiro , Green Sharone , Stephens Henry A. F. , Vaughn David W. , Kalayanarooj Siripen , Suntayakorn Saroj , Chandanayingyong Dasnayanee , Ennis Francis A. , Rothman Alan L. 
Published: December 2019

ABSTRACTWe examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4+and CD8+CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8+NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8+NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4+CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8+CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.
Role of Matrix in an Early Postentry Step in the Human Immunodeficiency Virus Type 1 Life Cycle
Authors: Kiernan Rosemary E. , Ono Akira , Englund George , Freed Eric O. 
Published: December 2019

ABSTRACT
The matrix protein of human immunodeficiency virus type 1 (HIV-1) has been reported to play a crucial role in the targeting of the Gag polyprotein precursor to the plasma membrane and in the incorporation of viral envelope glycoproteins into budding virions. In this report, we present evidence that mutation of a highly conserved Leu at matrix amino acid 20 blocks or markedly delays virus replication in a range of cell types, including T-cell lines, primary human peripheral blood mononuclear cells, and monocyte-derived macrophages. These mutations do not impair virus assembly and release, RNA encapsidation, or envelope glycoprotein incorporation into virions but rather cause significant defects in an early step in the virus life cycle, as measured by single-cycle infectivity assays and the analysis of viral DNA synthesis early postinfection. This infectivity defect is independent of the type of envelope glycoprotein carried on mutant virions; similar results are obtained in pseudotyping experiments using wild-type or truncated HIV-1 envelope glycoproteins, the amphotropic murine leukemia virus envelope, or the vesicular stomatitis G protein. Intriguingly, matrix residue 20 mutations also increase the apparent binding of Gag to membrane, accelerate the kinetics of Gag processing, and induce defects in endogenous reverse transcriptase activity without affecting virion density or morphology. These results help elucidate the function of matrix in HIV-1 replication.
Recombinant Vaccine-Induced Protection against the Highly Pathogenic Simian Immunodeficiency Virus SIVmac251: Dependence on Route of Challenge Exposure
Authors: Benson J. , Chougnet C. , Robert-Guroff M. , Montefiori D. , Markham P. , Shearer G. , Gallo R. C. , Cranage M. , Paoletti E. , Limbach K. , Venzon D. , Tartaglia J. , Franchini G. 
Published: December 2019

ABSTRACTVaccine protection from infection and/or disease induced by highly pathogenic simian immunodeficiency virus (SIV) strain SIVmac251in the rhesus macaque model is a challenging task. Thus far, the only approach that has been reported to protect a fraction of macaques from infection following intravenous challenge with SIVmac251was the use of a live attenuated SIV vaccine. In the present study, thegag,pol, andenvgenes of SIVK6Wwere expressed in the NYVAC vector, a genetically engineered derivative of the vaccinia virus Copenhagen strain that displays a highly attenuated phenotype in humans. In addition, the genes for the α and β chains of interleukin-12 (IL-12), as well as the IL-2 gene, were expressed in separate NYVAC vectors and inoculated intramuscularly, in conjunction with or separate from the NYVAC-SIV vaccine, in 40 macaques. The overall cytotoxic T-lymphocyte (CTL) response was greater, at the expense of proliferative and humoral responses, in animals immunized with NYVAC-SIV and NYVAC–IL-12 than in animals immunized with the NYVAC-SIV vaccine alone. At the end of the immunization regimen, half of the animals were challenged with SIVmac251by the intravenous route and the other half were exposed to SIVmac251intrarectally. Significantly, five of the eleven vaccinees exposed mucosally to SIVmac251showed a transient peak of viremia 1 week after viral challenge and subsequently appeared to clear viral infection. In contrast, all 12 animals inoculated intravenously became infected, but 5 to 6 months after viral challenge, 4 animals were able to control viral expression and appeared to progress to disease more slowly than control animals. Protection did not appear to be associated with any of the measured immunological parameters. Further modulation of immune responses by coadministration of NYVAC-cytokine recombinants did not appear to influence the outcome of viral challenge. The fact that the NYVAC-SIV recombinant vaccine appears to be effective per se in the animal model that best mirrors human AIDS supports the idea that the development of a highly attenuated poxvirus-based vaccine candidate can be a valuable approach to significantly decrease the spread of human immunodeficiency virus (HIV) infection by the mucosal route.
Adenovirus Type 12-Induced Fragility of the Human
RNU2
Locus Requires p53 Function
Authors: Li Zengji , Yu Adong , Weiner Alan M. 
Published: December 2019

ABSTRACT

Adenovirus type 12 (Ad12) infection of human cells induces four chromosomal fragile sites corresponding to the U1 small nuclear RNA (snRNA) genes (the
RNU1
locus), the U2 snRNA genes (
RNU2
), the U1 snRNA pseudogenes (
PSU1
), and the 5S rRNA genes (
RN5S
). Ad12-induced fragility of the
RNU2
locus requires U2 snRNA transcriptional regulatory elements and viral early functions but not viral replication or integration, or chromosomal sequences flanking the
RNU2
locus. We now show that Ad12 cannot induce the
RNU1
,
RNU2
, or
PSU1
fragile sites in Saos-2 cells lacking the p53 and retinoblastoma (Rb) proteins but that viral induction of fragility is rescued in these cells when the expression of wild-type p53 or selected hot-spot mutants (i.e., V143A, R175H, R248W, and R273H) is restored by transient expression or stable retroviral transduction. We also observed weak constitutive fragility of the
RNU1
and
RNU2
loci in cells belonging to xeroderma pigmentosum complementation groups B and D (XPB and XPD) which are partially defective in the ERCC2 (XPD) and ERCC3 (XPB) helicase activities shared between the repairosome and the RNA polymerase II basal transcription factor TFIIH. We propose a model for Ad12-induced chromosome fragility in which interaction of p53 with the Ad12 E1B 55-kDa transforming protein (and possibly E4orf6) induces a p53 gain of function which ultimately perturbs the RNA polymerase II basal transcription apparatus. The p53 gain of function could interfere with chromatin condensation either by blocking mitotic shutdown of U1 and U2 snRNA transcription or by phenocopying global or local DNA damage. Specific fragilization of the
RNU1
,
RNU2
, and
PSU1
loci could reflect the unusually high local concentration of strong transcription units or the specialized nature of the U1 and U2 snRNA transcription apparatus.
Transduction of Dendritic Cells by DNA Viral Vectors Directs the Immune Response to Transgene Products in Muscle Fibers
Authors: Jooss Karin , Yang Yiping , Fisher Krishna J. , Wilson James M. 
Published: December 2019

ABSTRACTImmune responses to vector-corrected cells have limited the application of gene therapy for treatment of chronic disorders such as inherited deficiency states. We have found that recombinant adeno-associated virus (AAV) efficiently transduces muscle fibers in vivo without activation of cellular and humoral immunity to neoantigenic transgene products such as β-galactosidase, which differs from the experience with recombinant adenovirus, where vibrant T-cell responses to the transgene product destroy the targeted muscle fibers. T cells activated following intramuscular administration of adenovirus expressinglacZ(AdlacZ) can destroy AAVlacZ-transduced muscle fibers, indicating a prior state of immunologic nonresponsiveness in the context of AAV gene therapy. Adoptive transfer of dendritic cells infected with AdlacZleads to immune mediated elimination of AAVlacZ-transduced muscle fibers. AAVlacZ-transduced antigen-presenting cells fail to demonstrate β-galactosidase activity and are unable to elicit transgene immunity in adoptive transfer experiments. These studies indicate that vector-mediated transduction of dendritic cells is necessary for cellular immune responses to muscle gene therapy, a step which AAV avoids, providing a useful biological niche for its use in gene therapy.