bluuebunny/crossref_metadata_2025_split huggingface dataset:
R Region Sequences in the Long Terminal Repeat of a Murine Retrovirus Specifically Increase Expression of Unspliced RNAs
Authors: Trubetskoy Alla M. , Okenquist Sharon A. , Lenz Jack 
Published: December 2019

ABSTRACT

A stem-loop structure at the 5′ end of the R region of the long terminal repeat (LTR) of the murine leukemia virus SL3 and other type C mammalian retroviruses is important for maximum levels of expression of a reporter gene under the control of the viral LTR. This element, termed the R region stem-loop (RSL), has a small effect on transcriptional initiation and no effect on RNA polymerase processivity. Its major effect is on posttranscriptional processing of LTR-driven transcripts. Here we tested whether the RSL affected the production of RNAs from a full-length SL3 genome. Mutation of the RSL in the 5′ LTR of SL3 reduced the cytoplasmic levels of full-length viral transcripts but not those of spliced,
env
mRNA transcripts. Thus, the RSL specifically affected the cytoplasmic levels of the unspliced viral RNA. To test further whether the effect was specific for unspliced transcripts, a system was devised in which the expression of a reporter gene under the control of the viral LTR was tested in the presence or absence of an intron. Mutation of the RSL resulted in only about a twofold decline in the level of reporter gene expression when the transcripts contained an intron. However, when the intron was removed, mutation of the RSL reduced expression of the reporter gene about 10- to 60-fold in various cell lines. The secondary structure of the RSL was essential for its activity on the intronless transcript. Thus, the RSL appears to be important for the cytoplasmic accumulation of unspliced viral RNA and unspliced RNA from chimeric transcription units under the control of the viral LTR.
Two Novel Promoters in the Upstream Regulatory Region of Human Papillomavirus Type 31b Are Negatively Regulated by Epithelial Differentiation
Authors: Ozbun Michelle A. , Meyers Craig 
Published: December 2019

ABSTRACT
Organotypic cultures support the stratification and differentiation of keratinocytes and the human papillomavirus (HPV) life cycle. We report transcription from four novel promoters in the HPV31b upstream regulatory region during the viral life cycle in organotypic cultures. Promoter initiation was not differentiation dependent; two promoters were down-regulated upon epithelial differentiation.
Regulation of Vaccinia Virus Morphogenesis: Phosphorylation of the A14L and A17L Membrane Proteins and C-Terminal Truncation of the A17L Protein Are Dependent on the F10L Kinase
Authors: Betakova Tatiana , Wolffe Elizabeth J. , Moss Bernard 
Published: December 2019

ABSTRACTThis study focused on three vaccinia virus-encoded proteins that participate in early steps of virion morphogenesis: the A17L and A14L membrane proteins and the F10L protein kinase. We found that (i) the A17L protein was cleaved at or near an AGX consensus motif at amino acid 185, thereby removing its acidic C terminus; (ii) the nontruncated form was associated with immature virions, but only the C-terminal truncated protein was present in mature virions; (iii) the nontruncated form of the A17L protein was phosphorylated on serine, threonine, and tyrosine residues, whereas the truncated form was unphosphorylated; (iv) nontruncated and truncated forms of the A17L protein existed in a complex with the A14L membrane protein; (v) C-terminal cleavage of the A17L protein and phosphorylation of the A17L and A14L proteins failed to occur in cells infected with a F10L kinase mutant at the nonpermissive temperature; and (vi) the F10L kinase was the only viral late protein that was necessary for phosphorylation of the A17L protein, whereas additional proteins were needed for C-terminal cleavage. We suggest that phosphorylation of the A17L and A14L proteins is mediated by the F10L kinase and is required to form the membranes associated with immature virions. Removal of phosphates and the A17L acidic C-terminal peptide occur during the transition to mature virions.
Enhanced Measles Virus cDNA Rescue and Gene Expression after Heat Shock
Authors: Parks Christopher L. , Lerch Robert A. , Walpita Pramila , Sidhu Mohinderjit S. , Udem Stephen A. 
Published: December 2019

ABSTRACT
Rescue of negative-stranded RNA viruses from full-length genomic cDNA clones is an essential technology for genetic analysis of this class of viruses. Using this technology in our studies of measles virus (MV), we found that the efficiency of the measles virus rescue procedure (F. Radecke et al., EMBO J. 14:5773–5784, 1995) could be improved by modifying the procedure in two ways. First, we found that coculture of transfected 293-3-46 cells with a monolayer of Vero cells increased the number of virus-producing cultures about 20-fold. Second, we determined that heat shock treatment increased the average number of transfected cultures that produced virus another two- to threefold. In addition, heat shock increased the number of plaques produced by positive cultures. The effect of heat shock on rescue led us to test the effect on transient expression from an MV minireplicon. Heat shock increased the level of reporter gene expression when either minireplicon DNA or RNA was used regardless of whether complementation was provided by cotransfection with expression plasmids or infection with MV helper virus. In addition, we found that MV minireplicon gene expression could be stimulated by cotransfection with an Hsp72 expression plasmid, indicating that hsp72 likely plays a role in the effect of heat shock.
Human Follicular Dendritic Cells Remain Uninfected and Capture Human Immunodeficiency Virus Type 1 through CD54-CD11a Interaction
Authors: Fujiwara Masatoshi , Tsunoda Rikiya , Shigeta Shiro , Yokota Tomoyuki , Baba Masanori 
Published: December 2019

ABSTRACTIt has been reported that human immunodeficiency virus type 1 (HIV-1) bound to follicular dendritic cells (FDCs) remains highly infectious to CD4+T cells even when it forms immune complexes with neutralizing antibody (HIV-1/IC). To elucidate the role of FDCs in HIV-1 transmission to CD4+T cells in lymph nodes, we have isolated and purified FDCs from human tonsils and examined whether the HIV-1/IC trapped on their surface is infectious to CD4+T cells. To our surprise, not the HIV-1/IC but the antibody-free HIV-1 on FDCs could be transmitted to CD4+T cells. Furthermore, in contrast to previous studies showing that FDCs are productively infected with HIV-1, the present study clearly demonstrated that FDCs were not the target cells for HIV-1 infection. FDCs could capture the viral particles on their surface; however, the binding of HIV-1 to FDCs was strongly inhibited by the presence of anti-CD54 (ICAM-1) monoclonal antibody (MAb) and anti-CD11a (LFA-1) MAb, suggesting that the adhesion molecules play an important role in the interaction between HIV-1 and FDCs.
Mutational Analysis of Heptad Repeats in the Membrane-Proximal Region of Newcastle Disease Virus HN Protein
Authors: Stone-Hulslander Judith , Morrison Trudy G. 
Published: December 2019

ABSTRACT
For most paramyxoviruses, syncytium formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (X. Hu et al., J. Virol. 66:1528–1534, 1992). The stalk region of the Newcastle disease virus (NDV) HN protein has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (R. Buckland et al., J. Gen. Virol. 73:1703–1707, 1992; T. Sergel-Germano et al., J. Virol. 68:7654–7658, 1994; J. Reitter et al., J. Virol. 69:5995–6004, 1995). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic a-position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein activity and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.
Transduction of Human Progenitor Hematopoietic Stem Cells by Human Immunodeficiency Virus Type 1-Based Vectors Is Cell Cycle Dependent
Authors: Sutton Richard E. , Reitsma Michael J. , Uchida Nobuko , Brown Patrick O. 
Published: December 2019

ABSTRACTHuman immunodeficiency virus (HIV) type 1 vectors are highly efficient in their ability to transduce human progenitor hematopoietic stem cells (PHSC). Although mitosis was not required for transduction of these cells, transduction rates were much greater once cells had been cultured in the presence of cytokines. Transduction rates, however, rarely exceeded 70%. We demonstrate here that there is a distinct subpopulation that is more easily transduced by HIV vectors. These cells were distinguished by a disproportionate population in the S/G2/M phases of the cell cycle. By sorting them prior to transduction, we found that those cells in either the G1or S/G2/M fraction were more readily transduced than G0cells. Maintaining the cells in G0by omitting cytokines from the medium reduced transduction rates by up to 10-fold. Addition of cytokines to the medium immediately after transduction did not improve the transduction efficiency as measured by expression of the transgene. Analysis of replication intermediates indicated that the block to transduction of G0cells operated near the time of initiation of reverse transcription. These results suggest that although lentivirus vectors can transduce nondividing PHSC, transduction efficiency is severalfold greater once the cells exit G0and enter G1. Further characterization of these more transducible cells and identification of the cellular factors responsible may enhance transduction while maintaining the pluripotentiality of the PHSC.
Genetic Divergence with Emergence of Novel Phenotypic Variants of Equine Arteritis Virus during Persistent Infection of Stallions
Authors: Hedges Jodi F. , Balasuriya Udeni B. R. , Timoney Peter J. , McCollum William H. , MacLachlan N. James 
Published: December 2019

ABSTRACTThe persistently infected carrier stallion is the critical natural reservoir of equine arteritis virus (EAV), as venereal infection of mares frequently occurs after breeding to such stallions. Two Thoroughbred stallions that were infected during the 1984 outbreak of equine viral arteritis in central Kentucky subsequently became long-term EAV carriers. EAV genomes amplified from the semen of these two stallions were compared by sequence analysis of the six 3′ open reading frames (ORFs 2 through 7), which encode the four known structural proteins and two uncharacterized glycoproteins. The major variants of the EAV population that sequentially arose within the reproductive tract of each carrier stallion varied by approximately 1% per year, and the heterogeneity of the viral quasispecies increased during the course of long-term persistent infection. The various ORFs of the dominant EAV variants evolved independently, and there was apparently strong selective pressure on the uncharacterized GP3 protein during persistent infection. Amino acid changes also occurred in the V1 variable region of the GLprotein. This region has been previously identified as a crucial neutralization domain, and selective pressures exerted on the V1 region during persistent EAV infection led to the emergence of virus variants with distinct neutralization properties. Thus, evolution of the EAV quasispecies that occurs during persistent infection of the stallion clearly can influence viral phenotypic properties such as neutralization and perhaps virulence.
High Major Histocompatibility Complex-Unrestricted Lysis of Simian Immunodeficiency Virus Envelope-Expressing Cells Predisposes Macaques to Rapid AIDS Progression
Authors: Yin Cheng , Wu Mary S. , Pauza C. David , Salvato Maria S. 
Published: December 2019

ABSTRACTBefore the development of virus-specific immune responses, peripheral blood mononuclear cells (PBMC) from uninfected rhesus monkeys and human beings have the capacity to lyse target cells expressing simian immunodeficiency virus (SIV) or human immunodeficiency virus-1 (HIV) envelope (gp130 and gp120) antigens. Lysis by naive effector cells does not require major histocompatibility complex (MHC)-restricted antigen presentation, is equally effective for allogeneic and xenogeneic targets, and is designated MHC-unrestricted (UR) lysis. UR lysis is not sensitive to EGTA and does not require de novo RNA or protein synthesis. Several kinds of envelope-expressing targets, including cells that poorly express MHC class I antigens, can be lysed. CD4+effectors are responsible for most of the lytic activity. High lysis is correlated with high expression of HIV or SIV envelope, specifically, the central one-third of the gp130 molecule, and lysis is completely inhibited by a monoclonal antibody against envelope. Our work extends observations of human lymphocytes expressing HIV gp120 to the SIV/rhesus monkey model for AIDS. Additionally, we address the relevance of UR lysis in vivo. A survey of PBMC from 56 uninfected rhesus monkeys indicates that 59% of the individuals had peak UR lytic activity above 15% specific lysis. Eleven of these monkeys were subsequently infected with SIV. Animals with UR lytic activity above 15% specific lysis were predisposed to more rapid disease progression than animals with low UR lytic activity, suggesting a strong correlation between this form of innate immunity and disease progression to AIDS.
Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus
Authors: Klausegger Alfred , Strobl Birgit , Regl Gerhard , Kaser Alexandra , Luytjes Willem , Vlasak Reinhard 
Published: December 2019

ABSTRACT

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates
p
-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-
O
-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse α
1
macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.
Antiretroviral Cytolytic T-Lymphocyte Nonresponsiveness: FasL/Fas-Mediated Inhibition of CD4+and CD8+Antiviral T Cells by Viral Antigen-Positive Veto Cells
Authors: Rich Robert F. , Green William R. 
Published: December 2019

ABSTRACTC57BL/6 (H-2b) mice generate type-specific cytolytic T-lymphocyte (CTL) responses to an immunodominant Kb-restricted epitope, KSPWFTTL located in the membrane-spanning domain of p15TM of AKR/Gross murine leukemia viruses (MuLV). AKR.H-2bcongenic mice, although carrying the responderH-2bmajor histocompatibility complex (MHC) haplotype, are low responders or nonresponders for AKR/Gross MuLV-specific CTL, apparently due to the presence of inhibitory AKR.H-2bcells. Despite their expression of viral antigens and Kb, untreated viable AKR.H-2bspleen cells cause dramatic inhibition of the C57BL/6 (B6) antiviral CTL response to in vitro stimulation with AKR/Gross MuLV-induced tumor cells. This inhibition is specific (AKR.H-2bmodulator spleen cells do not inhibit allogeneic MHC or minor histocompatibility antigen-specific CTL production), dependent on direct contact of AKR.H-2bcells in a dose-dependent manner with the responder cell population, and not due to soluble factors. Here, the mechanism of inhibition of the antiviral CTL response is shown to depend on Fas/Fas-ligand interactions, implying an apoptotic effect on B6 responder cells. Although B6.gld (FasL−) responders were as sensitive to inhibition by AKR.H-2bmodulator cells as were B6 responders, B6.lpr (Fas−) responders were largely insensitive to inhibition, indicating that the responder cells needed to express Fas. A Fas-Ig fusion protein, when added to the in vitro CTL stimulation cultures, relieved the inhibition caused by the AKR.H-2bcells if the primed responders were from either B6 or B6.gld mice, indicating that the inhibitory AKR.H-2bcells express FasL. Because of the antigen specificity of the inhibition, these results collectively implicate a FasL/Fas interaction mechanism: viral antigen-positive AKR.H-2bcells expressing FasL inhibit antiviral T cells (“veto” them) when the AKR.H-2bcells are recognized. Consistent with this model, inhibition by AKR.H-2bmodulator cells was MHC restricted, and resulted in approximately a 10- to 70-fold decrease in the in vitro expansion of pCTL/CTL. Both CD8+CTL and CD4+Th responder cells were susceptible to inhibition by FasL+AKR.H-2binhibitory cells as the basis for inhibition. The CTL response in the presence of inhibitory cells could be restored by several cytokines or agents that have been shown by others to interfere with activation-induced cell death (e.g., interleukin-2 [IL-2], IL-15, transforming growth factor β, lipopolysaccharide, 9-cis-retinoic acid) but not others (e.g., tumor necrosis factor alpha). These results raise the possibility that this type of inhibitory mechanism is generalized as a common strategy for retrovirus infected cells to evade immune T-cell recognition.
Selection of RNA Replicons Capable of Persistent Noncytopathic Replication in Mammalian Cells
Authors: Frolov Ilya , Agapov Eugene , Hoffman Thomas A. , Prágai Béla M. , Lippa Mara , Schlesinger Sondra , Rice Charles M. 
Published: December 2019

ABSTRACT
The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989–12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments.
Intracellular Formation and Processing of the Heterotrimeric gH-gL-gO (gCIII) Glycoprotein Envelope Complex of Human Cytomegalovirus
Authors: Huber Mary T. , Compton Teresa 
Published: December 2019

ABSTRACT

The human cytomegalovirus (HCMV) gCIII complex contains glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and glycoprotein O (gO; gpUL74). To examine how gH, gL, and gO interact within HCMV-infected cells to assemble the tripartite complex, pulse-chase experiments were performed. These analyses demonstrated that gH and gL associate by the end of the pulse period to form a disulfide dependent gH-gL complex. Subsequently, the gH-gL complex interacts with a 100-kDa precursor form of gO to form a 220-kDa precursor of the mature gH-gL-gO complex that contains a 125-kDa form of gO. The 220-kDa precursor complex (pgCIII) was sensitive to treatment with endoglycosidase H (endo H), while the mature gCIII complex was essentially resistant to digestion with this enzyme, suggesting that formation of pgCIII complex occurs in the endoplasmic reticulum (ER) and is processed to mature gH-gL-gO (gCIII) in a post-ER compartment. While the N-linked glycans on the 100-kDa form of gO were modified to endo H-resistant states as the 125-kDa gO formed, additional posttranslational modifications were detected on gO. These processing alterations were non-N-linked oligosaccharide modifications that could not be accounted for by phosphorylation or by O-glycosylation of the type sensitive to
O
-glycanase. Of gH, gL, gO, and the various complexes that they form, only the mature form of the complex was detectable at the infected cell membrane, as judged by surface biotinylation studies.
Iκκ Mediates NF-κB Activation in Human Immunodeficiency Virus-Infected Cells
Authors: Asin Susana , Taylor Julie A. , Trushin Sergey , Bren Gary , Paya Carlos V. 
Published: December 2019

ABSTRACTHuman monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-κB in the nucleus secondary to increased IκBα, IκBβ, and IκBɛ degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IκB, we have sought to identify the regulatory domains of IκBα targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IκBα, we determined that persistent HIV infection of these cells targets the NH2but not the COOH terminus of IκBα. Further analysis demonstrated that phosphorylation at S32and S36is necessary for HIV-dependent IκBα degradation and NF-κB activation. Of the putative N-terminal IκBα kinases, we demonstrated that the Iκκ complex, but not p90rsk, is activated by HIV infection and mediates HIV-dependent NF-κB activation. Analysis of viral replication in cells that constitutively express IκBα negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-κB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.