bluuebunny/crossref_metadata_2025 huggingface dataset:
Progression to the G1b Phase of the Cell Cycle Is Required for Completion of Human Immunodeficiency Virus Type 1 Reverse Transcription in T Cells
Authors: Korin Yael D. , Zack Jerome A.
Published: December 2019

Successful infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile, reverse transcripts. In the present study, we isolated highly purified quiescent T cells and utilized the CD3/CD28 activation pathways as well as cell cycle inhibitors to further define the role of costimulation and cell cycle progression in HIV-1 reverse transcription. Activation with αCD3 alone resulted in cell cycle progression into only G1a and incomplete HIV-1 reverse transcription. Costimulation through the CD28 receptor and transition into G1b was required to efficiently complete the reverse transcription process. These findings have relevance to immune activation in vivo, since lymphocytes rendered anergic by a single activation signal would be nonpermissive for productive infection with HIV-1. Importantly, these data also suggest that HIV vector-based genetic transduction strategies might be successful only in target cells that transition into the G1b phase of the cell cycle.
Loss of Viral Fitness Associated with Multiple Gag and Gag-Pol Processing Defects in Human Immunodeficiency Virus Type 1 Variants Selected for Resistance to Protease Inhibitors In Vivo
Authors: Zennou Véronique , Mammano Fabrizio , Paulous Sylvie , Mathez Dominique , Clavel François
Published: December 2019

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.
Efficient Expression and Rapid Purification of Human T-Cell Leukemia Virus Type 1 Protease
Authors: Ding Y. Shirley , Owen Sherry M. , Lal Renu B. , Ikeda Richard A.
Published: December 2019

Human T-cell leukemia virus type 1 (HTLV-1) is an oncovirus that is clinically associated with adult T-cell leukemia. We report here the construction of a pET19-based expression clone containing HTLV-1 protease fused to a decahistidine-containing leader peptide. The recombinant protein is efficiently expressed in
Escherichia coli
, and the fusion protein can be easily purified by affinity chromatography. Active mature protease in yields in excess of 3 mg/liter of culture can then be obtained by a novel two-step refolding and autoprocessing procedure. The purified enzyme exhibited

K
m

and
K
cat
values of 0.3 mM and 0.143 sec
−1
at pH 5.3 and was inhibited by pepstatin A.
cis
-Acting Sequences Required for Simian Foamy Virus Type 1 Vectors
Authors: Wu Min , Chari Soumya , Yanchis Tina , Mergia Ayalew
Published: December 2019

We have constructed a series of vectors based on simian foamy virus type 1 (SFV-1) to define the minimum
cis
-acting elements required for gene transfer. To characterize these vectors, we inserted the coding sequence of the bacterial
lacZ
gene linked to the cytomegalovirus immediate-early gene promoter. Introduction of a deletion mutation in the leader region between the 5′ long terminal repeat and the start of the
gag
gene at position 1659 to 1694 completely abrogated gene transfer by the SFV-1 vector. Deletion of 39 nucleotides from position 1692 to 1731 in the leader region resulted in a significant reduction in the transducing-particle titer. Furthermore, we have identified a second
cis
-acting element located at the 3′ end of the
pol
gene between position 6486 and 6975 to be critical for SFV-1 vector transduction. These results identify the two important
cis
-acting elements required for SFV-1 vector construction, and the finding of a
cis
-acting element in the
pol
gene is unique among retroviruses.
Coding Sequences Enhance Internal Initiation of Translation by Hepatitis A Virus RNA In Vitro
Authors: Graff Judith , Ehrenfeld Ellie
Published: December 2019

Hepatitis A virus (HAV), unlike other picornaviruses, has a slow-growth phenotype in permissive cell lines and in general does not induce host cell cytopathology. Although there are no published reports of productive infection of HeLa cells by HAV, HAV RNA appears to be readily translated in HeLa cells when transcribed by T7 RNA polymerase provided by a recombinant vaccinia virus. The 5′ noncoding region of HAV was fused to poliovirus (PV) coding sequences to determine the effect on translation efficiency in HeLa cell extracts in vitro. Conditions were optimized for utilization of the HAV internal ribosome entry segment (IRES). Transcripts from chimeric constructs fused precisely at the initiation codon were translated very poorly. However, chimeric RNAs which included 114 or more nucleotides from the HAV capsid coding sequences downstream of the initiation codon were translated much more efficiently than those lacking these sequences, making HAV-directed translation efficiency similar to that directed by the PV IRES. Sixty-six nucleotides were insufficient to confer increased translation efficiency. The most 5′-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an inhibitory effect on translation efficiency. Constructs lacking these terminal sequences, or those in which the PV 5′-terminal sequences replaced those from HAV, translated three- to fourfold better than those with the intact HAV 5′-terminal end.
Structured-Tree Topology and Adaptive Evolution of the Simian Immunodeficiency Virus SIVsm Envelope during Serial Passage in Rhesus Macaques According to Likelihood Mapping and Quartet Puzzling
Authors: Spencer Valli P. J. , Goudsmit Jaap
Published: December 2019

Species-specific strains of simian immunodeficiency virus (SIV) are nonpathogenic in African primates. The SIV strain most closely related to human immunodeficiency virus type 2 (HIV-2) is SIVsm, the strain specific to the sooty mangabey (
Cercocebus atys
). Infection of Asian primates with SIV causes AIDS and allows the study of the adaptive evolution of a lentivirus to replicate efficiently in a new host, providing a useful animal model of HIV infection and AIDS in humans. Serial passage of SIVsm from sooty mangabeys in rhesus macaques drastically shortened the time of disease progression from 1.5 years to 1 month as the retrovirus adapted to these Asian hosts. In the present study we analyzed the quasispecies nature of the SIVsm envelope gene (
env
) during serial population passage in rhesus macaques. We asked ourselves if phylogenetic evidence could be provided for the structured topology of the SIVsm
env
tree and subsequently for the adaptive evolution of SIVsm
env
. Likelihood mapping showed that phylogenetic reconstruction of the passage was possible because a high percentage of the sequence data had a “tree-like” form. Subsequently, quartet puzzling was used and produced a phylogeny with a structure parallel to the known infection history. The adaptation of SIVsm to Asian rhesus macaques appears to be an ordered process in which the
env
evolves in a tree-like manner, particularly in its constant regions.
Absence of Macrophage Inflammatory Protein-1α Prevents the Development of Blinding Herpes Stromal Keratitis
Authors: Tumpey Terrence M. , Cheng Hao , Cook Donald N. , Smithies Oliver , Oakes John E. , Lausch Robert N.
Published: December 2019

Prior studies in our laboratory have suggested that the CC chemokine macrophage inflammatory protein-1α (MIP-1α) may be an important mediator in the blinding ocular inflammation which develops following herpes simplex virus type 1 (HSV-1) infection of the murine cornea. To directly test this hypothesis, MIP-1α-deficient (−/−) mice and their wild-type (+/+) counterparts were infected topically on the scarified cornea with 2.5 × 105PFU of HSV-1 strain RE and subsequently graded for corneal opacity. Four weeks postinfection (p.i.), the mean corneal opacity score of −/− mice was 1.1 ± 0.3 while that of the +/+ mice was 3.7 ± 0.5. No detectable infiltrating CD4+T cells were seen histologically at 14 or 21 days p.i. in −/− animals, whereas the mean CD4+T-cell count per field (36 fields counted) in +/+ hosts was 26 ± 2 (P< 0.001). In addition, neutrophil counts in the −/− mouse corneas were reduced by >80% in comparison to the wild-type controls. At 2 weeks p.i., no interleukin-2 or gamma interferon could be detected in six of seven −/− mice, whereas both T-cell cytokines were readily demonstrable in +/+ mouse corneas. Also, MIP-2 and monocyte chemoattractant protein-1 protein levels were significantly lower in MIP-1α −/− mouse corneas than in +/+ host corneas, suggesting that MIP-1α directly, or more likely indirectly, influences the expression of other chemokines. Interestingly, despite the paucity of infiltrating cells, HSV-1 clearance from the eyes of −/− mice was not significantly different from that observed in +/+ hosts. We conclude that MIP-1α is not needed to control virus growth in the cornea but is essential for the development of severe stromal keratitis.
Antiviral Activity of the Proteasome on Incoming Human Immunodeficiency Virus Type 1
Authors: Schwartz Olivier , Maréchal Valérie , Friguet Bertrand , Arenzana-Seisdedos Fernando , Heard Jean-Michel
Published: December 2019

Following cell surface receptor binding and membrane fusion, human immunodeficiency virus (HIV) virion cores are released in the cytoplasm. Incoming viral proteins represent potential targets for cytosolic proteases. We show that treatment of target cells with the proteasome inhibitors MG132 and lactacystin increased the efficiency of HIV infection. Proteasome inhibitors were active at the early steps of the viral cycle. Incoming p24
Gag
proteins accumulated in the cytosol, and larger amounts of proviral DNA were synthesized. In vitro, purified 20S proteasome degraded HIV virion components. Thus, degradation of incoming viral proteins by the proteasome represents an early intracellular defense against infection.
Retrotransposition of Nonviral RNAs in an Avian Packaging Cell Line
Authors: Lum Richard , Linial Maxine L.
Published: December 2019

Retroviruses produced from the quail packaging cell line SE21Q1b predominantly contain cellular RNAs instead of viral RNAs. These RNAs can be reverse transcribed and integrated into the genomes of newly infected cells and are thereafter referred to as newly formed retrogenes. We investigated whether retrogene formation can occur within SE21Q1b cells themselves and whether this occurs intracellularly or via extracellular reinfection. By using packaging cell line mutants derived from the SE21Q1b provirus and selectable reporter constructs, we found that the process requires envelope glycoproteins and a retroviral packaging signal. Our results suggest that extracellular reinfection is the primary route of retrotransposition of nonviral RNAs.
Insertion of a Sequence Encoding Light Chain 3 of Microtubule-Associated Proteins 1A and 1B in a Pestivirus Genome: Connection with Virus Cytopathogenicity and Induction of Lethal Disease in Cattle
Authors: Meyers Gregor , Stoll Dieter , Gunn Michael
Published: December 2019

Pestiviruses represent the first RNA viruses for which recombination with cellular protein-coding sequences has been reported. As a result of such recombinations cytopathogenic (cp) pestiviruses can develop from noncytopathogenic (noncp) viruses. In the case of bovine viral diarrhea virus (BVDV), the generation of cp mutants is linked to the induction of the lethal syndrome mucosal disease (MD) in cattle. The cp BVDV JaCP was isolated from an animal which had come down with MD. The genome of JaCP contains a novel kind of cellular insertion (LC3*) which is flanked by duplicated pestivirus sequences. Neither insertion nor duplication is present in the genome of the accompanying noncp virus JaNCP. As part of the viral polyprotein, the insertion in the JaCP genome is translated into a polypeptide almost identical to a fragment of light chain 3, a subunit of the microtubule-associated proteins 1A and 1B from the rat. Transient-expression studies revealed that the LC3* sequence is able to induce an additional cleavage of the viral polyprotein. The respective cleavage occurs directly downstream of the LC3*-encoded sequence and is not dependent on the NS3 serine protease. Insertion of LC3* into an infectious noncp pestivirus cDNA clone without duplicated viral sequences resulted in recovery of a defective cp virus able to replicate only in the presence of a noncp helper virus. In contrast, introduction of both insertion and duplication led to an autonomously replicating cp virus.
Epidemiology of Infection with Epstein-Barr Virus Types 1 and 2: Lessons from the Study of a T-Cell-Immunocompromised Hemophilic Cohort
Authors: Yao Q. Y. , Croom-Carter D. S. G. , Tierney R. J. , Habeshaw G. , Wilde J. T. , Hill F. G. H. , Conlon C. , Rickinson A. B.
Published: December 2019

In apparent contrast to earlier work on Epstein-Barr virus (EBV) carriage in the general Caucasian population, in vitro virus isolations from human immunodeficiency virus (HIV)-positive male homosexual cohorts have shown frequent examples of multiple EBV infection and an overall prevalence of type 2 EBV strains exceeding 30%. Here we ask to what extent these findings might hold true in another T-cell-immunocompromised cohort, HIV-positive hemophilic patients. Resident EBV strains were rescued within lymphoblastoid cell lines derived from the blood and throat washings of 39 such individuals, using the same in vitro protocols of virus isolation as for the homosexual cohort. A mean of 19 independent cell lines was made per patient, and in each case the resident virus was characterized by PCR-based viral genomic analysis and by immunoblotting to reveal the viral “EBNAprint.” By these criteria a significant proportion (14 of 39) of the hemophilic cohort carried more than one EBV strain, suggesting that T-cell impairment does indeed sensitize virus carriers to reinfection with new strains of exogenously transmitted virus. However, the overall incidence of type 2 EBV infection was 10%, which is close to that observed in the earlier work with healthy carriers and substantially lower than that seen in HIV-positive homosexuals. We infer that type 2 EBV is relatively rare in the general Caucasian population but has become endemic in the homosexual community.
Structure of a Neutralizing Antibody Bound Monovalently to Human Rhinovirus 2
Authors: Hewat Elizabeth A. , Marlovits Thomas C. , Blaas Dieter
Published: December 2019

The structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-Å resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45° to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2–Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.
Bovine Leukemia Virus-Induced Lymphocytosis and Increased Cell Survival Mainly Involve the CD11b+B-Lymphocyte Subset in Sheep
Authors: Chevallier Nathalie , Berthelemy Madeleine , Le Rhun Danielle , Lainé Véronique , Levy Daniel , Schwartz-Cornil Isabelle
Published: December 2019

In this study, we show that bovine leukemia virus (BLV)-induced persistent lymphocytosis (PL) results from the in vivo expansion of the CD11b+B-lymphocyte population. This subset shares phenotypic characteristics with murine and human B-1 cells. BLV interactions with the sheep B-1-like subset were explored. We found that B-1- and B-2-like cells are initially infected to similar extents. However, in long-term-infected sheep, the viral load is higher in B-1-like cells and only B-1- and not B-2-like cells show increased ex vivo survival compared to that in uninfected sheep. Ex vivo viral expression was found in both B-1- and B-2-like cells, indicating that both cell types support viral replication. Finally, cycloheximide and a protein kinase C inhibitor (H7) that blocks the ex vivo activation of viral expression did not affect the increased survival in B-1-like cells, suggesting that resistance to apoptosis is acquired in vivo. Collectively, these results indicate a peculiar susceptibility of sheep B-1-like cells to BLV transforming effects and further support the involvement of increased survival in BLV pathogenesis.
Biological Characterization of Rev Variation in Equine Infectious Anemia Virus
Authors: Belshan Michael , Harris Matthew E. , Shoemaker Anne E. , Hope Thomas J. , Carpenter Susan
Published: December 2019

Sequence analysis identified significant variation in the second exon of equine infectious anemia virus (EIAV)
rev
. Functional analysis indicated that limited amino acid variation in Rev significantly altered the export activity of the protein but did not affect Rev-dependent alternative splicing. EIAV Rev can mediate export through two independent
cis
-acting Rev-responsive elements (RREs), and differences among Rev variants were more pronounced when both RREs were present. Variation in Rev may be an important mechanism for regulation of virus replication in vivo and may contribute to changes in clinical disease.
Functions of Conserved Motifs in the RNA-Dependent RNA Polymerase of a Yeast Double-Stranded RNA Virus
Authors: Routhier Eric , Bruenn Jeremy A.
Published: December 2019

At least eight conserved motifs are visible in the totivirus RNA-dependent RNA polymerase (RDRP). We have systematically altered each of these in the
Saccharomyces cerevisiae
double-stranded RNA virus ScVL1 by substituting the conserved motifs from a giardiavirus. The results help define the conserved regions of the RDRP involved in polymerase function and those essential for other reasons.
Different Patterns of Neuronal Infection after Intracerebral Injection of Two Strains of Pseudorabies Virus
Authors: Card J. Patrick , Levitt Pat , Enquist Lynn W.
Published: December 2019

Pseudorabies virus (PRV), a swine neurotropic alphaherpesvirus, is known to invade the central nervous system (CNS) of a variety of animal species through peripherally projecting axons, replicate in the parent neurons, and then pass transsynaptically to infect other neurons of a circuit. Studies of the human pathogen herpes simplex virus type 1 have reported differences in the direction of transport of two strains of this virus after direct injection into the primate motor cortex. In the present study we examined the direction of transport of virulent and attenuated strains of PRV, utilizing injections into the rat prefrontal cortex to evaluate specific movement of virus through CNS circuitry. The data demonstrate strain-dependent patterns of infection consistent with bidirectional (anterograde and retrograde) transport of virulent virus and unidirectional (retrograde) transport of attenuated PRV from the site of injection. The distribution of infected neurons and the extent of transsynaptic passage also suggest that a release defect in the attenuated strain reduces the apparent rate of viral transport through neuronal circuitry. Finally, injection of different concentrations of virus influenced the onset of replication within a neural circuit. Taken together, these data suggest that viral envelope glycoproteins and virus concentration at the site of injection are important determinants of the rate and direction of viral transport through a multisynaptic circuit in the CNS.
Feline Coronavirus Type II Strains 79-1683 and 79-1146 Originate from a Double Recombination between Feline Coronavirus Type I and Canine Coronavirus
Authors: Herrewegh Arnold A. P. M. , Smeenk Ingrid , Horzinek Marian C. , Rottier Peter J. M. , de Groot Raoul J.
Published: December 2019

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to recombinant viruses which encode a CCV-like S protein and the M, N, 7a, and 7b proteins of FCoV type I (K. Motowaka, T. Hoh- datsu, H. Hashimoto, and H. Koyama, Microbiol. Immunol. 40:425–433, 1996; H. Vennema, A. Poland, K. Floyd Hawkins, and N. C. Pedersen, Feline Pract. 23:40–44, 1995). In the present study, we have looked for additional FCoV-CCV recombination sites. Four regions in thepolgene were selected for comparative sequence analysis of the type II FCoV strains 79-1683 and 79-1146, the type I FCoV strains TN406 and UCD1, the CCV strain K378, and the TGEV strain Purdue. Our data show that the type II FCoVs have arisen from double recombination events: additional crossover sites were mapped in the ORF1ab frameshifting region of strain 79-1683 and in the 5′ half of ORF1b of strain 79-1146.
Antibody-Mediated Neutralization of Human Rhinovirus 14 Explored by Means of Cryoelectron Microscopy and X-Ray Crystallography of Virus-Fab Complexes
Authors: Che Zhiwei , Olson Norman H. , Leippe Donna , Lee Wai-ming , Mosser Anne G. , Rueckert Roland R. , Baker Timothy S. , Smith Thomas J.
Published: December 2019

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the β-B–β-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.
Infection of Glial Cells by the Human Polyomavirus JC Is Mediated by an N-Linked Glycoprotein Containing Terminal α(2-6)-Linked Sialic Acids
Authors: Liu Christine K. , Wei Grant , Atwood Walter J.
Published: December 2019

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49–58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both α(2-3)- and α(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the α(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the α(2-3) or the α(2-6) linkage revealed that JCV preferentially interacts with α(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl
N
-acetyl-α-
d
-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.
Vpr Stimulates Viral Expression and Induces Cell Killing in Human Immunodeficiency Virus Type 1-Infected Dividing Jurkat T Cells
Authors: Yao Xiao-Jian , Mouland Andrew J. , Subbramanian Ramu A. , Forget Janique , Rougeau Nicole , Bergeron Dominique , Cohen Eric A.
Published: December 2019

In this study we investigated the effects of Vpr during human immunodeficiency virus (HIV) infection of proliferating Jurkat T cells by using a vesicular stomatitis virus envelope G glycoprotein pseudotyped HIV superinfection system. We observe that the expression of Vpr results in a severe reduction in the life span of HIV type 1 (HIV-1)-infected dividing T cells in culture. In agreement with a recent report (S. A. Stewart, B. Poon, J. B. M. Jowett, and I. S. Chen, J. Virol. 71:5579–5592, 1997), we show that events characteristic of apoptotic cell death are involved in the Vpr-mediated cytopathic effects. Our results also show that infection with viruses expressing the wild-typevprgene results in an increase in viral gene expression and production. Interestingly, the effects of Vpr on cell viability and on viral gene expression both correlate with the ability of the protein to induce a cell cycle arrest in the G2/M phase. Mutagenesis analyses show that the C terminus of Vpr is essential for these biological activities. Although the role of Vpr is currently associated with the infection of nondividing cells, our results suggest that Vpr can also directly increase viral replication in vivo in infected dividing T cells. Furthermore, these in vitro observations suggest that Vpr-mediated cytotoxic effects could contribute to the CD4+depletion associated with AIDS progression.
CD4-Induced Conformational Changes in the Human Immunodeficiency Virus Type 1 gp120 Glycoprotein: Consequences for Virus Entry and Neutralization
Authors: Sullivan Nancy , Sun Ying , Sattentau Quentin , Thali Markus , Wu Dona , Denisova Galina , Gershoni Jonathan , Robinson James , Moore John , Sodroski Joseph
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.
Specific Interaction of Eukaryotic Translation Initiation Factor 3 with the 5′ Nontranslated Regions of Hepatitis C Virus and Classical Swine Fever Virus RNAs
Authors: Sizova Daria V. , Kolupaeva Victoria G. , Pestova Tatyana V. , Shatsky Ivan N. , Hellen Christopher U. T.
Published: December 2019

Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap-independent attachment (internal entry) of ribosomes to the ∼350-nucleotide internal ribosomal entry segment (IRES) at the 5′ end of both RNAs. Eukaryotic initiation factor 3 (eIF3) binds specifically to HCV and CSFV IRESs and plays an essential role in the initiation process on them. Here we report the results of chemical and enzymatic footprinting analyses of binary eIF3-IRES complexes, which have been used to identify the eIF3 binding sites on HCV and CSFV IRESs. eIF3 protected an internal bulge in the apical stem IIIb of domain III of the CSFV IRES from chemical modification and protected bonds in and adjacent to this bulge from cleavage by RNases ONE and V
1
. eIF3 protected an analagous region in domain III of the HCV IRES from cleavage by these enzymes. These results are consistent with the results of primer extension analyses and were supported by observations that deletion of stem-loop IIIb or of the adjacent hairpin IIIc from the HCV IRES abrogated the binding of eIF3 to this RNA. This is the first report that eIF3 is able to bind a eukaryotic mRNA in a sequence- or structure-specific manner. UV cross-linking of eIF3 to [
32
P]UTP-labelled HCV and CSFV IRES elements resulted in strong labelling of 4 (p170, p116, p66, and p47) of the 10 subunits of eIF3, 1 or more of which are likely to be determinants of these interactions. In the cytoplasm, eIF3 is stoichiometrically associated with free 40S ribosomal subunits. The results presented here are consistent with a model in which binding of these two translation components to separate, specific sites on both HCV and CSFV IRESs enhances the efficiency and accuracy of binding of these RNAs to 40S subunits in an orientation that promotes entry of the initiation codon into the ribosomal P site.
An Evolutionarily Conserved Splice Generates a Secreted Env-Bet Fusion Protein during Human Foamy Virus Infection
Authors: Giron Marie-Louise , de Thé Hugues , Saïb Ali
Published: December 2019

Foamy viruses (spumaretroviruses) represent a retroviral genus which exhibits unusual features relating it to pararetroviruses. Previously, we reported the existence of a protein species harboring Env, Bel, and Bet epitopes in human foamy virus (HFV)-infected cells (M. L. Giron, F. Rozain, M. C. Debons-Guillemin, M. Canivet, J. Périès, and R. Emanoil-Ravier, J. Virol. 67:3596–3600, 1993). Here, we identify this protein as a 160-kDa Env-Bet fusion glycoprotein (gp160) translated from an mRNA species harboring a highly conserved splice site which deletes the membrane anchor domain of Env and fuses the
env
open reading frame with that of
bel1/bet
. While gp160 and Bet proteins were both secreted into the supernatant, only Bet was taken up by recipient cells. Since Bet plays a key role in the switch from lytic to chronic infection, secretion of Bet and gp160, together with cellular uptake of Bet, could be highly relevant for both immune response and development of HFV infection in vivo.
Transcription Factor YY1 Represses Cell-Free Replication from Human Papillomavirus Origins
Authors: Lee Kyung-Yeol , Broker Thomas R. , Chow Louise T.
Published: December 2019

We have established cell-free replication for the human papillomavirus type 18 (HPV-18) origin of replication (
ori
)-containing DNA by using purified HPV-18 E1 and E2 gene products expressed as fusion proteins in
Escherichia coli
. The transcription factor YY1 has been shown to regulate RNA transcription by binding to a sequence overlapping the putative E1 protein binding site in the HPV-18
ori
. We show that exogenously added YY1 fusion protein inhibited HPV-18
ori
replication. Cotransfection of YY1 expression vectors also inhibited transient replication in 293 cells. However, inhibition did not appear to be mediated by binding to its cognate site in the
ori
as YY1 also inhibited the replication of the HPV-11
ori
, which does not have a known or suspected YY1 binding site. Moreover, inhibition was not alleviated by the inclusion of YY1 binding oligonucleotides in the replication reaction mixtures. Rather, we demonstrated a direct interaction between purified fusion E2 protein and fusion YY1 protein by the pull-down assay and a partial restoration of replication activity by an elevated E2 protein concentration. These results suggest that YY1 can inhibit HPV
ori
replication by interfering with E2 protein functions.
Transmissible Gastroenteritis Coronavirus Induces Programmed Cell Death in Infected Cells through a Caspase-Dependent Pathway
Authors: Eleouet Jean-François , Chilmonczyk Stefan , Besnardeau Lydia , Laude Hubert
Published: December 2019

In this report, we show that apoptosis (or programmed cell death) is induced in different cell lines infected with a coronavirus, the porcine transmissible gastroenteritis virus (TGEV). Kinetic analysis of internucleosomal DNA cleavage by agarose gel electrophoresis and flow cytometry or cytometric monitoring of the mitochondrial transmembrane potential showed that, for ST cells infected with TGEV, the first overt signs of apoptosis appeared from 10 to 12 h postinfection on. They preceded morphological changes characteristic of cells undergoing apoptosis, as observed by light and electron microscopy. The tripeptide pan-ICE (caspase) inhibitor
N
-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone blocked TGEV-induced apoptosis with no effect on virus production. The thiol agent pyrrolidine dithiocarbamate inhibited apoptosis, suggesting that TGEV infection may lead to apoptosis via cellular oxidative stress. The effect of TGEV infection on activation of NF-κB, a transcription factor known to be activated by oxidative stress, was examined. NF-κB DNA binding was shown to be strongly and quickly induced by TGEV infection. However, transcription factor decoy experiments showed that NF-κB activation is not critical for TGEV-induced apoptosis.
Identification of Kaposin (Open Reading Frame K12) as a Human Herpesvirus 8 (Kaposi’s Sarcoma-Associated Herpesvirus) Transforming Gene
Authors: Muralidhar Sumitra , Pumfery Anne M. , Hassani Morad , Sadaie M. Reza , Azumi Norio , Kishishita Masamichi , Brady John N. , Doniger Jay , Medveczky Peter , Rosenthal Leonard J.
Published: December 2019

The recently identified human herpesvirus 8 (HHV-8, or Kaposi’s sarcoma-associated herpesvirus) has been implicated in the etiology of both Kaposi’s sarcoma (KS) and primary effusion (body cavity-based) lymphoma (PEL) (Y. Chang et al., Science 266:1865–1869, 1994; P. S. Moore et al., J. Virol. 70:549–558, 1996). An important feature of the association of HHV-8 with these malignancies is the expression of an abundant, latency-associated 0.7-kb transcript, T0.7 (W. Zhong et al., Proc. Natl. Acad. Sci. USA 93:6641–6646, 1996). T0.7 is found in all stages in nearly all KS tumors of different epidemiologic origin, including AIDS-associated, African endemic, and classical KS (K. A. Staskus et al., J. Virol. 71:715–719, 1997), as well as in a body cavity-based lymphoma-derived cell line, BCBL-1, that is latently infected with HHV-8 (R. Renne et al., Nat. Med. 2:342–346, 1996). T0.7 encodes a unique HHV-8 open reading frame, K12, also known as kaposin. In this study, we report that the kaposin gene induced tumorigenic transformation. Constructs with kaposin expressed either from its endogenous promoter or from a heterologous promoter induced focal transformation upon transfection into Rat-3 cells. All transformed Rat-3 cell lines containing kaposin sequences produced high-grade, highly vascular, undifferentiated sarcomas upon subcutaneous injection of athymic
nu/nu
mice. Tumor-derived cell lines expressed kaposin mRNA, suggesting a role in the maintenance of the transformed phenotype. Furthermore, kaposin protein was detected in transformed and tumor-derived cells by immunofluorescence and localized to the cytoplasm. More importantly, expression of kaposin protein was also detected in the PEL cell lines BCBL-1 and KS-1. These findings demonstrate the oncogenic potential of kaposin and suggest its possible role in the development of KS and other HHV-8-associated malignancies.
Role of Rta in the Translation of Bicistronic BZLF1 of Epstein-Barr Virus
Authors: Chang Pey-Jium , Chang Yu-Sun , Liu Shih-Tung
Published: December 2019

The BZLF1 gene of Epstein-Barr virus (EBV), which encodes a transcription factor, Zta, is transcribed into monocistronic and bicistronic mRNAs from two different promoters during the immediate-early stage of the EBV lytic cycle. It is generally accepted that the Zta protein translated from the monocistronic mRNA profoundly influences the activation of the EBV lytic cycle. In this study, we constructed a plasmid, pCMV-RZLUC, which can transcribe a bicistronic mRNA consisting of BRLF1 and a BZLF1-
luc
fusion gene under latent conditions. P3HR1 cells transfected with this plasmid produce a luciferase activity which is approximately 17-fold higher than the activity exhibited by pRZLUC, a plasmid incapable of transcribing the bicistronic mRNA. Genetic analyses indicated that mutations in BRLF1 not only can decrease the translation of the fusion gene from the bicistronic mRNA but can also be complemented by a functional BRLF1 gene in
cis
. This observation implies that the product of BRLF1, Rta, is involved in the translation of the downstream gene. Results presented herein also demonstrate that these mutations cannot be complemented in
trans
with a plasmid overexpressing Rta, suggesting that the amount of Rta in the vicinity of the intercistronic region may be crucial for the translation. Furthermore, our results correspond to those of previous investigations indicating that the Zta protein can be translated from the bicistronic mRNA and that, similar to the translation of bicistronic ZLUC, mutations in BRLF1 also hinder the translation of Zta from the BRLF1-BZLF1 bicistronic mRNA. Translation of Zta from the bicistronic mRNA may play an essential role in the activation of the EBV lytic cycle.
Complete Genomic Sequence of Border Disease Virus, a Pestivirus from Sheep
Authors: Becher Paul , Orlich Michaela , Thiel Heinz-Jürgen
Published: December 2019

The genus
Pestivirus
of the family
Flaviviridae
comprises three established species, namely, bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus from sheep (BDV). In this study, we report the first complete nucleotide sequence of BDV, that of strain X818. The genome is 12,333 nucleotides long and contains one long open reading frame encoding 3,895 amino acids. The 5′ noncoding region (NCR) of BDV X818 consists of 372 nucleotides and is thus similar in length to the 5′ NCR reported for other pestiviruses. The 3′ NCR of X818 is 273 nucleotides long and thereby at least 32 nucleotides longer than the 3′ NCR of pestiviruses analyzed thus far. Within the 3′ NCR of BDV X818, the sequence motif TATTTATTTA was identified at four locations. The same repeat was found at two or three locations within the 3′ NCR of different CSFV isolates but was absent in the 3′ NCR of BVDV. Analysis of five additional BDV strains showed that the 3′ NCR sequences are highly conserved within this species. Comparison of the deduced amino acid sequence of X818 with the ones of other pestiviruses allowed the prediction of polyprotein cleavage sites which were conserved with regard to the structural proteins. It has been reported for two BVDV strains that cleavage at the nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B is mediated by the NS3 serine protease and for each site a conserved leucine was found at the P1 position followed by either serine or alanine at P1′ (N. Tautz, K. Elbers, D. Stoll, G. Meyers, and H.-J. Thiel, J. Virol. 71:5415–5422, 1997; J. Xu, E. Mendez, P. R. Caron, C. Lin, M. A. Murcko, M. S. Collett, and C. M. Rice, J. Virol. 71:5312–5322). Interestingly, P1′ of the predicted NS5A/5B cleavage site of BDV is represented by an asparagine residue. Transient expression studies demonstrated that this unusual NS5A/5B processing site is efficiently cleaved by the NS3 serine protease of BDV.
Functional Interaction of Human Immunodeficiency Virus Type 1 Vpu and Gag with a Novel Member of the Tetratricopeptide Repeat Protein Family
Authors: Callahan Michael A. , Handley Mark A. , Lee Yung-Hui , Talbot Katrin J. , Harper J. Wade , Panganiban Antonito T.
Published: December 2019

Viral protein U (Vpu) is a protein encoded by human immunodeficiency virus type 1 (HIV-1) that promotes the degradation of the virus receptor, CD4, and enhances the release of virus particles from cells. We isolated a cDNA that encodes a novel cellular protein that interacts with Vpu in vitro, in vivo, and in yeast cells. This Vpu-binding protein (UBP) has a molecular mass of 41 kDa and is expressed ubiquitously in human tissues at the RNA level. UBP is a novel member of the tetratricopeptide repeat (TPR) protein family containing four copies of the 34-amino-acid TPR motif. Other proteins that contain TPR motifs include members of the immunophilin superfamily, organelle-targeting proteins, and a protein phosphatase. UBP also interacts directly with HIV-1 Gag protein, the principal structural component of the viral capsid. However, when Vpu and Gag are coexpressed, stable interaction between UBP and Gag is diminished. Furthermore, overexpression of UBP in virus-producing cells resulted in a significant reduction in HIV-1 virion release. Taken together, these data indicate that UBP plays a role in Vpu-mediated enhancement of particle release.
Measles Virus Infection and Replication in Undifferentiated and Differentiated Human Neuronal Cells in Culture
Authors: McQuaid S. , Campbell S. , Wallace I. J. C. , Kirk J. , Cosby S. L.
Published: December 2019

Measles virus (MV) infection of the human central nervous system (CNS) typically involves widespread infection of neurons. However, little is known about how they become infected, how defective virus arises and accumulates, or how virus spreads among the cells of the CNS. In vitro studies of viral interactions with human neuronal cells may contribute to the resolution of such issues. In mixed cultures containing differentiated human neuronal (hNT2) cells and neuroepithelial cells, immunofluorescence studies show that the neurons, unlike both their NT2 progenitors and the neuroepithelial cells, are not initially susceptible to MV infection. This is possibly due to their lack of expression of CD46, a known cell surface receptor for MV. Later in the course of infection, however, both MV proteins and genomic RNA become detectable in their processes, where they contact infected, fully permissive neuroepithelial cells. Such a mechanism of virus transfer may be involved in the initiation and spread of persistent MV infection in diseases such as subacute sclerosing panencephalitis. Furthermore, mutated defective virus may readily accumulate and spread without the need, at any stage, for viral maturation and budding.
Actin-Dependent Receptor Colocalization Required for Human Immunodeficiency Virus Entry into Host Cells
Authors: Iyengar Sujatha , Hildreth James E. K. , Schwartz David H.
Published: December 2019

Human immunodeficiency virus (HIV) envelope binds CD4 and a chemokine receptor in sequence, releasing hydrophobic viral gp41 residues into the target membrane. HIV entry required actin-dependent concentration of coreceptors, which could be disrupted by cytochalasin D (CytoD) without an effect on cell viability or mitosis. Pretreatment of peripheral blood mononuclear cells, but not virus, inhibited entry and infection. Immunofluorescent confocal microscopy of activated cells revealed CD4 and CXCR4 in nonoverlapping patterns. Addition of gp120 caused polarized cocapping of both molecules with subsequent pseudopod formation, while CytoD pretreatment blocked these membrane changes completely.
The N-Terminal Extension of the Influenza B Virus Nucleoprotein Is Not Required for Nuclear Accumulation or the Expression and Replication of a Model RNA
Authors: Stevens Mark P. , Barclay Wendy S.
Published: December 2019

The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues. We have deleted the N-terminal 51 and 69 residues of the influenza B/Ann Arbor/1/66 virus NP and show that nuclear accumulation of the protein is unaffected. This indicates that the nuclear localization signal is not located at the extreme N terminus, as in influenza A virus NP. To determine if the N-terminal mutants could support the expression and replication of a model influenza B virus RNA, the genes encoding the subunits of the viral RNA-dependent RNA polymerase (PA, PB1, and PB2) were cloned. Coexpression of NP and the P proteins in 293 cells was found to permit the expression and replication of a transfected model RNA based on segment 4 of B/Maryland/59, in which the hemagglutinin-coding region was replaced by a chloramphenicol acetyltransferase gene. The expression and replication of the synthetic RNA were not affected by the replacement of NP with NP mutants lacking the N-terminal 51 or 69 residues, indicating that the N-terminal extension is not required for transcription or replication of the viral RNA. In addition, we report that the influenza B virus NP cannot be functionally replaced by type A virus NP in this system.