bluuebunny/crossref_metadata_2025 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

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

STP-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

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

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

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

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

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

We 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

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

Vaccine 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

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

Immune 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.
Effectiveness of Postinoculation (
R
)-9-(2-Phosphonylmethoxypropyl)Adenine Treatment for Prevention of Persistent Simian Immunodeficiency Virus SIV
mne
Infection Depends Critically on Timing of Initiation and Duration of Treatment
Authors: Tsai Che-Chung , Emau Peter , Follis Kathryn E. , Beck Thomas W. , Benveniste Raoul E. , Bischofberger Norbert , Lifson Jeffrey D. , Morton William R.
Published: December 2019

(
R
)-9-(2-Phosphonylmethoxypropyl)adenine (PMPA), an acyclic nucleoside phosphonate analog, is one of a new class of potent antiretroviral agents. Previously, we showed that PMPA treatment for 28 days prevented establishment of persistent simian immunodeficiency virus (SIV) infection in macaques even when therapy was initiated 24 h after intravenous virus inoculation. In the present study, we tested regimens involving different intervals between intravenous inoculation with SIV and initiation of PMPA treatment, as well as different durations of treatment, for the ability to prevent establishment of persistent infection. Twenty-four cynomolgus macaques (
Macaca fascicularis
) were studied for 46 weeks after inoculation with SIV. All mock-treated control macaques showed evidence of productive infection within 2 weeks postinoculation (p.i.). All macaques that were treated with PMPA for 28 days beginning 24 h p.i. showed no evidence of viral replication following discontinuation of PMPA treatment. However, extending the time to initiation of treatment from 24 to 48 or 72 h p.i. or decreasing the duration of treatment reduced effectiveness in preventing establishment of persistent infection. Only half of the macaques treated for 10 days, and none of those treated for 3 days, were completely protected when treatment was initiated at 24 h. Despite the reduced efficacy of delayed and shortened treatment, all PMPA-treated macaques that were not protected showed delays in the onset of cell-associated and plasma viremia and antibody responses compared with mock controls. These results clearly show that both the time between virus exposure and initiation of PMPA treatment as well as the duration of treatment are crucial factors for prevention of acute SIV infection in the macaque model.
A Chimeric Ty3/Moloney Murine Leukemia Virus Integrase Protein Is Active In Vivo
Authors: Dildine Sandra L. , Respess James , Jolly Doug , Sandmeyer Suzanne B.
Published: December 2019

This report describes the results of experiments to determine whether chimeras between a retrovirus and portions of Ty3 are active in vivo. A chimera between Ty3 and a Neo
r
-marked Moloney murine leukemia virus (M-MuLV) was constructed. The C-terminal domain of M-MuLV integrase (IN) was replaced with the C-terminal domain of Ty3 IN. The chimeric retroviruses were expressed from an amphotrophic envelope packaging cell line. The virus generated was used to infect the human fibrosarcoma cell line HT1080, and cells in which integration had occurred were selected by G418 resistance. Three independently integrated viruses were rescued. In each case, the C-terminal Ty3 IN sequences were maintained and short direct repeats of the genomic DNA flanked the integration site. Sequence analysis of the genomic DNA flanking the insertion did not identify a tRNA gene; therefore, these integration events did not have Ty3 position specificity. This study showed that IN sequences from the yeast retrovirus-like element Ty3 can substitute for M-MuLV IN sequences in the C-terminal domain and contribute to IN function in vivo. It is also one of the first in vivo demonstrations of activity of a retrovirus encoding an integrase chimera. Studies of chimeras between IN species with distinctive integration patterns should complement previous work by expanding our understanding of the roles of nonconserved domains.
Deletion Analysis of a Defective Interfering Semliki Forest Virus RNA Genome Defines a Region in the nsP2 Sequence That Is Required for Efficient Packaging of the Genome into Virus Particles
Authors: White Christine L. , Thomson Michael , Dimmock Nigel J.
Published: December 2019

The 1,244-nucleotide genome of Semliki Forest virus (SFV) defective interfering (DI) RNA 19 (DI-19) is coterminal with the infectious genome and contains two major deletions. One deletion removes the end of the nsP1 gene and the beginning of the nsP2 gene, and the other removes the end of the nsP2 gene, the nsP3 and nsP4 genes, and all of the structural protein genes (M. Thomson and N. J. Dimmock, Virology 199:354–365, 1994). Like all DI SFV RNAs, DI-19 contains three regions that are conserved. Region a comprises the 5′ terminus continuous with part of the nsP1 gene, region b comprises a central part of the nsP2 gene, and region c comprises the 3′ terminus and the associated untranslated region. A deletion analysis of the 265-nucleotide b region (nucleotides 679 to 943, inclusive) was undertaken to determine its role in genome replication and packaging into DI virus particles. Deleted plasmids were constructed and transcribed, and the resulting DI RNAs were transfected into SFV-infected BHK cells. Putative progeny DI virus particles that had been released into the tissue culture fluid were then serially passaged in new monolayers together with added high-multiplicity SFV, and cells and tissue culture fluids were tested for the presence of DI RNA by reverse transcription-PCR. DI RNA that had all of the b region deleted was replicated well in BHK-21 cells, as shown by the presence of large amounts of negative-sense DI RNA and an increase in the amount of positive-sense RNA in the cytoplasm, but was packaged very inefficiently, as indicated by very low amounts of DI RNA in the tissue culture fluid. The genome of a deletion mutant that retained the 3′ 224 nucleotides of region b was packaged successfully, but one that retained only the 5′ 41 nucleotides was not detected in the tissue culture fluid. These and other data suggest that nucleotides 720 to 777 of region b are of particular importance in the packaging process. This finding agrees with data obtained with Ross River virus and contrasts with the well-studied Sindbis alphavirus major packaging signal that is located within the nsP1 gene.
African Origin of Human T-Lymphotropic Virus Type 2 (HTLV-2) Supported by a Potential New HTLV-2d Subtype in Congolese Bambuti Efe Pygmies
Authors: Vandamme Anne-Mieke , Salemi Marco , Van Brussel Marianne , Liu Hsin-Fu , Van Laethem Kristel , Van Ranst Marc , Michels Ludovic , Desmyter Jan , Goubau Patrick
Published: December 2019

We identified a potential new subtype within human T-cell lymphotropic virus type 2 (HTLV-2), HTLV-2d, present in members of an isolated Efe Bambuti Pygmy tribe. Two of 23 Efe Pygmies were HTLV-2 seropositive, with HTLV-2 Western blot and enzyme-linked immunosorbent assay reactivities. From one of them the entire genome of the HTLV-2 strain Efe2 could be amplified and sequenced. In all gene regions analyzed, this strain was the most divergent HTLV-2 strain, differing by 2.4% (
tax/rex
) to 10.7% (long terminal repeat) from both subtypes HTLV-2a and HTLV-2b, yet major functional elements are conserved. The similarity between the HTLV-2 Efe2 Gag and Env proteins and the corresponding HTLV-2a and -2b proteins is consistent with the observed serological reactivity. In the proximal pX region, one of the two alternative splice acceptor sites is abolished in HTLV-2 Efe2. Another interesting feature of this potential new subtype is that it has a Tax protein of 344 amino acids (aa), which is intermediate in length between the HTLV-2a Tax protein (331 aa) and the HTLV-2b and -2c Tax proteins (356 aa) and similar to the simian T-cell lymphotropic virus type 2 (STLV-2) PP1664 Tax protein. Together these two findings suggest a different phenotype for the HTLV-2 Efe2 strain. Phylogenetic analyses confirmed that the Pygmy Efe2 strain potentially belonged to a new and quite divergent subtype, HTLV-2d. When the STLV-2 bonobo viruses PP1664 and PanP were used as an outgroup, it was clear that the Pygmy HTLV-2 Efe2 strain had the longest independent evolution and that HTLV-2 evolution is consistent with an African origin.
Borna Disease Virus-Induced Neurological Disorder in Mice: Infection of Neonates Results in Immunopathology
Authors: Hallensleben Wiebke , Schwemmle Martin , Hausmann Jürgen , Stitz Lothar , Volk Benedikt , Pagenstecher Axel , Staeheli Peter
Published: December 2019

Borna disease virus (BDV) is a neurotropic nonsegmented negative-stranded RNA virus that persistently infects warm-blooded animals. In horses and other natural animal hosts, infections with BDV cause meningoencephalitis and behavioral disturbances. Experimental infection of adult mice takes a nonsymptomatic course, an observation previously believed to indicate that this animal species is not suitable for pathogenesis studies. We now demonstrate that BDV frequently induces severe neurological disease in infected newborn mice. Signs of neurological disease were first observed 4 to 6 weeks after intracerebral infection. They included a characteristic nonphysiological position of the hind limbs at an early stage of the disease and paraparesis at a later stage. Histological examination revealed large numbers of perivascular and meningeal inflammatory cells in brains of diseased mice and, unexpectedly, no increase in immunoreactivity to glial fibrillar acidic protein. The incidence and severity of BDV-induced disease varied dramatically among mouse strains. While only 13% of the infected C57BL/6 mice showed disease symptoms, which were mostly transient, more than 80% of the infected MRL mice developed severe neurological disorder. In spite of these differences in susceptibility to disease, BDV replicated to comparable levels in the brains of mice of the various strains used. Intracerebral infections of newborn β2-microglobulin-deficient C57BL/6 and MRL mice, which both lack CD8+T cells, did not result in meningoencephalitis or neurological disease, indicating that the BDV-induced neurological disorder in mice is a cytotoxic T-cell-mediated immunopathological process. With this new animal model it should now be possible to characterize the disease-inducing immune response to BDV in more detail.
Role of the N-Terminal Zinc Finger of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein in Virus Structure and Replication
Authors: Tanchou Valérie , Decimo Didier , Péchoux Christine , Lener Daniela , Rogemond Véronique , Berthoux Lionel , Ottmann Michèle , Darlix Jean-Luc
Published: December 2019

Nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 is found covering the genomic RNA in the interior of the viral particle. It is a highly basic protein with two zinc fingers of the form CX2CX4HX4C which exhibit strong affinity for a zinc cation. To study the structure-function relationship of the N-terminal zinc finger of NCp7, this domain was either deleted or changed to CX2CX4
C
X4C. We examined virus formation and structure as well as proviral DNA synthesis. Our data show that these two NC mutations result in the formation of particles with an abnormal core morphology and impair the end of proviral DNA synthesis, leading to noninfectious viruses.
Identification of a Putative Receptor for Porcine Reproductive and Respiratory Syndrome Virus on Porcine Alveolar Macrophages
Authors: Duan Xiaobo , Nauwynck Hans J. , Favoreel Herman W. , Pensaert Maurice B.
Published: December 2019

To identify the receptor which may determine the macrophage tropism of porcine reproductive and respiratory syndrome virus (PRRSV), monoclonal antibodies (MAbs) against porcine alveolar macrophages (PAM) were produced. Two MAbs (41D3 and 41D5) which completely blocked PRRSV infection of PAM were further characterized. It was found that they reduce the attachment of PRRSV to PAM and immunoprecipitate a 210-kDa membrane protein from PAM. This protein was detected on the cell membranes of PAM but not of PRRSV-nonpermissive cells. A colocalization was found between the reactive sites of MAb 41D3 and PRRSV on PAM membranes. All PRRSV-infected cells in tissues of experimentally infected pigs reacted with MAb 41D3. Taken together, all these data suggest that the identified 210-kDa membrane protein is a putative receptor for PRRSV on porcine macrophages.
Molecular Evidence for Nosocomial Transmission of Human Immunodeficiency Virus from a Surgeon to One of His Patients
Authors: Blanchard Alain , Ferris Stéphane , Chamaret Sophie , Guétard Denise , Montagnier Luc
Published: December 2019

We have investigated the molecular evidence in favor of the transmission of human immunodeficiency virus (HIV) from an HIV-infected surgeon to one of his patients. After PCR amplification, the
env
and
gag
sequences from the viral genome were cloned and sequenced. Phylogenetic analysis revealed that the viral sequences derived from the surgeon and his patient are closely related, which strongly suggests that nosocomial transmission occurred. In addition, these viral sequences belong to group M of HIV type 1 but are divergent from the reference sequences of the known subtypes.
Role of Envelope Protein gE Endocytosis in the Pseudorabies Virus Life Cycle
Authors: Tirabassi R. S. , Enquist L. W.
Published: December 2019

Several groups have reported that certain herpesvirus envelope proteins do not remain on the surface of cells that express them but rather are internalized by endocytosis in a recycling process. The biological function of membrane protein endocytosis in the virus life cycle remains a matter of speculation and debate. In this report, we demonstrate that some, but not all, membrane proteins encoded by the alphaherpesvirus pseudorabies virus (PRV) are internalized after reaching the plasma membrane. Glycoproteins gE and gB are internalized from the plasma membrane of cells, while gI and gC are not internalized efficiently. We show for gE that the cytoplasmic domain of the protein is required for endocytosis. While the gI protein is incapable of endocytosis on its own, it can be internalized when complexed with gE. We demonstrate that endocytosis of the gE-gI complex and gB occurs early after infection of tissue culture cells but that this process stops completely after 6 h of infection, a time that correlates with significant shutoff of host protein synthesis. We also show that gE protein internalized at 4 h postinfection is not present in virions formed at a later time. We discuss the differences in PRV gE and gI endocytosis compared to that of the varicella-zoster virus homologs and the possible roles of glycoprotein endocytosis in the virus life cycle.
Ligand Induction of Retinoic Acid Receptors Alters an Acute Infection by Murine Cytomegalovirus
Authors: Angulo Ana , Chandraratna Roshantha A. S. , LeBlanc James F. , Ghazal Peter
Published: December 2019

Here we report that administration of retinoids can alter the outcome of an acute murine cytomegalovirus (MCMV) infection. We show that a crucial viral control element, the major immediate-early enhancer, can be activated by retinoic acid (RA) via multiple RA-responsive elements (DR2) that bind retinoid X receptor-retinoic acid receptor (RAR) heterodimers with apparent dissociation constants ranging from 15 to 33 nM. Viral growth is dramatically increased upon RA treatment of infected tissue culture cells. Using synthetic retinoid receptor-specific agonists and antagonists, we provide evidence that RAR activation in cells is required for mediating the response of MCMV to RA. Oral administration of RA to infected immunocompetent mice selectively exacerbates an infection by MCMV, while cotreatment with an RAR antagonist protects against the adverse effects of RA on MCMV infection. In conclusion, these chemical genetic experiments provide evidence that an RAR-mediated pathway can modulate in vitro and in vivo infections by MCMV.
Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity
Authors: Mendez Ernesto , Ruggli Nicolas , Collett Marc S. , Rice Charles M.
Published: December 2019

Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10
5
PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.
Cell-Specific Expression of RANTES, MCP-1, and MIP-1α by Lower Airway Epithelial Cells and Eosinophils Infected with Respiratory Syncytial Virus
Authors: Olszewska-Pazdrak Barbara , Casola Antonella , Saito Tadahito , Alam Rafeul , Crowe Sheila E. , Mei Fang , Ogra Pearay L. , Garofalo Roberto P.
Published: December 2019

Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infancy, a syndrome characterized by wheezing, respiratory distress, and the pathologic findings of peribronchial mononuclear cell infiltration and release of inflammatory mediators by basophil and eosinophil leukocytes. Composition and activation of this cellular response are thought to rely on the discrete target cell selectivity of C-C chemokines. We demonstrate that infection in vitro of human epithelial cells of the lower respiratory tract by RSV induced dose- and time-dependent increases in mRNA and protein secretion for RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α). Production of MCP-1 and MIP-1α was selectively localized only in epithelial cells of the small airways and lung. Exposure of epithelial cells to gamma interferon (IFN-γ), in combination with RSV infection, induced a significant increase in RANTES production that was synergistic with respect to that obtained by RSV infection or IFN-γ treatment alone. Epithelial cell-derived chemokines exhibited a strong chemotactic activity for normal human blood eosinophils. Furthermore, eosinophils were susceptible to RSV and released RANTES and MIP-1α as a result of infection. Therefore, the inflammatory process in RSV-induced bronchiolitis appears to be triggered by the infection of epithelial cells and further amplified via mechanisms driven by IFN-γ and by the secretion of eosinophil chemokines.
Activation of Papillomavirus Late Gene Transcription and Genome Amplification upon Differentiation in Semisolid Medium Is Coincident with Expression of Involucrin and Transglutaminase but Not Keratin-10
Authors: Ruesch Margaret N. , Stubenrauch Frank , Laimins Laimonis A.
Published: December 2019

The life cycle of the papillomaviruses is closely linked to host cell differentiation, as demonstrated by the fact that amplification of viral DNA and transcription of late genes occur only in the suprabasal cells of a differentiated epithelium. Previous studies examining the pathogenesis of papillomavirus infections have relied on the use of organotypic raft cultures or lesions from patients to examine these differentiation-dependent viral activities. In this study, we used a simple system for epithelial differentiation to study human papillomavirus (HPV) late functions. We demonstrate that the suspension of HPV-infected keratinocytes in semisolid medium containing 1.6% methylcellulose for 24 h was sufficient for the activation of the late promoter, transcription of late genes, and amplification of viral DNA. These activities were shown to be linked to and coincide with cellular differentiation. Expression of the late protein E1
∧
E4 and amplification of viral DNA were detected in the identical set of cells after suspension in methylcellulose. This technique was also used to analyze the differentiation properties of the cells which expressed the late protein E1
∧
E4. While induction of the spinous layer markers involucrin and transglutaminase was compatible with late promoter induction, expression of the differentiation-specific keratin-10 was shown not to be required for HPV late functions. Interestingly, while the majority of normal human keratinocytes induced filaggrin expression by 24 h, this marker of the granular layer was induced in a smaller subset of HPV type 31 (HPV-31)-positive cells at this time point. The HPV-31-positive cells which expressed filaggrin did not induce the late protein E1
∧
E4. Use of the methylcellulose system to induce epithelial differentiation coupled with the ability to perform a genetic analysis of HPV functions by using transfection of cloned viral DNA will facilitate the study of the regulation of the papillomavirus life cycle.
Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus–Adeno-Associated Virus Vector
Authors: Palombo Fabio , Monciotti Andrea , Recchia Alessandra , Cortese Riccardo , Ciliberto Gennaro , La Monica Nicola
Published: December 2019

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the β-galactosidase (β-Gal) reporter gene and the hygromycin resistance (Hyg
r
) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV
rep
gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hyg
r
–β-Gal sequence was obtained with insect cells only when the
rep
gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the
rep
gene results in a 10- to 50-fold increase in the number of Hyg
r
stable cell clones. Additionally,
rep
expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.
Template-Independent Repair of the 3′ End of Cucumber Mosaic Virus Satellite RNA Controlled by RNAs 1 and 2 of Helper Virus
Authors: Burgyán József , García-Arenal Fernando
Published: December 2019

RNA viruses which do not have a poly(A) tail or a tRNA-like structure for the protection of their vulnerable 3′ termini may have developed a different strategy to maintain their genome integrity. We provide evidence that deletions of up to 7 nucleotides from the 3′ terminus of cucumber mosaic cucumovirus (CMV) satellite RNA (satRNA) were repaired in planta in the presence of the helper virus (HV) CMV. Sequence comparison of 3′-end-repaired satRNA progenies, and of satRNA and HV RNA, suggested that the repair was not dependent on a viral template. The 3′ end of CMV satRNA lacking the last three cytosines was not repaired in planta in the presence of tomato aspermy cucumovirus (TAV), although TAV is an efficient helper for the replication of CMV satRNA. With use of pseudorecombinants constructed by the interchange of RNAs 1 and 2 of TAV and CMV, evidence was provided that the 3′-end repair was controlled by RNAs 1 and 2 of CMV, which encode subunits of the viral RNA replicase. These results, and the observation of short repeated sequences close to the 3′ terminus of repaired molecules, suggest that the HV replicase maintains the integrity of the satRNA genome, playing a role analogous to that of cellular telomerases.