bluuebunny/crossref_metadata_2025_split huggingface dataset:
B Cells Regulate Murine Gammaherpesvirus 68 Latency
Authors: Weck Karen E. , Kim Susanne S. , Virgin Herbert W. , Speck Samuel H. 
Published: December 2019

ABSTRACT
The dynamics of the establishment of, and reactivation from, gammaherpesviruses latency has not been quantitatively analyzed in the natural host. Gammaherpesvirus 68 (γHV68) is a murine gammaherpesvirus genetically related to primate gammaherpesviruses that establishes a latent infection in infected mice. We used limiting dilution reactivation (frequency of cells reactivating γHV68 in vitro) and limiting dilution PCR (frequency of cells carrying γHV68 genome) assays to compare γHV68 latency in normal (C57BL/6) and B-cell-deficient (MuMT) mice. After intraperitoneal (i.p.) inoculation, latent γHV68 was detected in the spleen, bone marrow, and peritoneal cells. Both B-cell-deficient and C57BL/6 mice established latent infection in peritoneal cells after either i.p. or intranasal (i.n.) inoculation. In contrast, establishment of splenic latency was less efficient in B-cell-deficient than in C57BL/6 mice after i.n. inoculation. Analysis of reactivation efficiency (reactivation frequency compared to frequency of cells carrying γHV68 genome) revealed that (i) regardless of route or mouse strain, splenic cells reactivated γHV68 less efficiently than peritoneal cells, (ii) the frequency of cells carrying γHV68 genome was generally comparable over the course of infection between C57BL/6 and B-cell-deficient mice, (iii) between 28 and 250 days after infection, cells from B-cell-deficient mice reactivated γHV68 10- to 100-fold more efficiently than cells from C57BL/6 mice, (iv) at 7 weeks postinfection, B-cell-deficient mice had more genome-positive peritoneal cells than C57BL/6 mice, and (v) mixing cells (ratio of 3 to 1) that reactivated inefficiently with cells that reactivated efficiently did not significantly decrease reactivation efficiency. Consistent with a failure to normally regulate chronic γHV68 infection, the majority of infected B-cell-deficient mice died between 100 and 200 days postinfection. We conclude that (i) B cells are not required for establishment of γHV68 latency, (ii) there are organ-specific differences in the efficiency of γHV68 reactivation, (iii) B cells play a crucial role in regulating reactivation of γHV68 from latency, and (iv) B cells are important for controlling chronic γHV68 infection.
Hormonal Regulation of CD4+T-Cell Responses in Coxsackievirus B3-Induced Myocarditis in Mice
Authors: Huber S. A. , Kupperman J. , Newell M. K. 
Published: December 2019

ABSTRACTCoxsackievirus B3 infection causes significant cardiac inflammation in male, but not female, B1.Tg.Eα mice. This gender difference in disease susceptibility correlates with selective induction of CD4+Th1 (gamma interferon-positive) cell responses in animals with testosterone, whereas estradiol promotes preferential CD4+Th2 (interleukin-4 positive [IL-4+]) cell responses. Differences in immune deviation of CD4+T cells cannot be explained by variation in B7-1 or B7-2 expression. Infection significantly upregulated both molecules, but no differences were detected between estradiol- and testosterone-treated groups. Significantly increased numbers of activated (CD69+) T cells expressing the γδ T-cell receptor were found in male and testosterone-treated male and female mice. In vivo depletion of γδ+cells by using monoclonal antibodies inhibited myocarditis and resulted in a shift from a Th1 to Th2 response phenotype. Taken together, our results indicate that testosterone promotes a CD4+Th1 cell response and myocarditis by promoting increased γδ+cell activation.
Reversion of a Human Immunodeficiency Virus Type 1 Matrix Mutation Affecting Gag Membrane Binding, Endogenous Reverse Transcriptase Activity, and Virus Infectivity
Authors: Kiernan Rosemary E. , Ono Akira , Freed Eric O. 
Published: December 2019

ABSTRACT
We previously characterized mutations in the human immunodeficiency virus type 1 matrix (MA) protein that displayed reduced infectivity in single-round assays, defects in the stable synthesis of viral DNA in infected cells, and impaired endogenous reverse transcriptase activity. The mutants, which contained substitutions in a highly conserved Leu at MA amino acid 20, also increased binding of Gag to membrane. To elucidate further the role of MA in the virus replication cycle, we have characterized a viral revertant of an amino acid 20 mutant (20LK). The revertant virus, which replicates with essentially wild-type kinetics in H9 cells, contains second-site compensatory changes at MA amino acids 73 (E→K) and 82 (A→T), while retaining the original 20LK mutation. Single-cycle infectivity assays, performed with luciferase-expressing viruses, show that the 20LK/73EK/82AT triple mutant displays markedly improved infectivity relative to the original 20LK mutant. The stable synthesis of viral DNA in infected cells is also significantly increased compared with that of 20LK DNA. Furthermore, activity of revertant virions in endogenous reverse transcriptase assays is restored to near-wild-type-levels. Interestingly, although 20LK/73EK/82AT reverses the defects in replication kinetics, postentry events, and endogenous reverse transcriptase activity induced by the 20LK mutation, the reversion does not affect the 20LK-imposed increase in Gag membrane binding. Mutants containing single and double amino acid substitutions were constructed, and their growth kinetics were examined. Only virus containing all three changes (20LK/73EK/82AT) grew with significantly accelerated kinetics; 73EK, 73EK/82AT, and 20LK/82AT mutants displayed pronounced defects in virus particle production. Viral core-like complexes were isolated by sucrose density gradient centrifugation of detergent-treated virions. Intriguingly, the protein composition of wild-type and mutant detergent-resistant complexes differed markedly. In wild-type and 20LK complexes, MA was removed following detergent solubilization of the viral membrane. In contrast, in revertant preparations, the majority of MA cosedimented with the detergent-resistant complex. These results suggest that the 20LK/73EK/82AT mutations induced a significant alteration in MA-MA or MA-core interactions.
Susceptibility of Bovine Antigen-Presenting Cells to Infection by Bovine Herpesvirus 1 and In Vitro Presentation to T Cells: Two Independent Events
Authors: Renjifo Ximena , Letellier Carine , Keil Günther M. , Ismaili Jamila , Vanderplasschen Alain , Michel Patrick , Godfroid Jacques , Walravens Karl , Charlier Gérard , Pastoret Paul-Pierre , Urbain Jacques , Denis Martine , Moser Muriel , Kerkhofs Pierre 
Published: December 2019

ABSTRACTThe aim of the present study was to develop an in vitro system for presentation of bovine herpesvirus 1 (BHV-1) antigens to bovine T lymphocytes and to characterize the antigen-presenting cells (APC) which efficiently activate CD4+T cells. Two approaches were used to monitor the infection of APC by BHV-1 as follows: (i) detection of viral glycoproteins at the cell surface by immunofluorescence staining and (ii) detection of UL26 transcripts by reverse transcription-PCR. The monocytes were infected, while dendritic cells (DC) did not demonstrate any detectable viral expression. These data suggest that monocytes are one site of replication, while DC are not. The capacities of monocytes and DC to present BHV-1 viral antigens in vitro were compared. T lymphocytes (CD2+or CD4+) from BHV-1 immune cattle were stimulated in the presence of APC previously incubated with live or inactivated wild-type BHV-1. DC stimulated strong proliferation of Ag-specific T cells, while monocytes were poor stimulators of T-cell proliferation. When viral attachment to the surface of the APC was inhibited by virus pretreatment with soluble heparin, T-cell proliferation was dramatically decreased. Unexpectedly, incubation of DC and monocytes with the deletion mutant BHV-1 gD−/−, which displays impaired fusion capacity, resulted in strong activation of T lymphocytes by both APC types. Collectively, these results indicate that presentation of BHV-1 antigens to immune T cells is effective in the absence of productive infection and suggest that BHV-1 gD−/−mutant virus could be used to induce virus-specific immune responses in cattle.
Polarized Human Immunodeficiency Virus Budding in Lymphocytes Involves a Tyrosine-Based Signal and Favors Cell-to-Cell Viral Transmission
Authors: Deschambeault Julie , Lalonde Jean-Philippe , Cervantes-Acosta Guillermo , Lodge Robert , Cohen Éric A. , Lemay Guy 
Published: December 2019

ABSTRACTMaturation and release of human immunodeficiency virus type 1 (HIV-1) is targeted at the pseudopod of infected mononuclear cells. However, the intracellular mechanism or targeting signals leading to this polarized viral maturation are yet to be identified. We have recently demonstrated the presence of a functional YXXL motif for specific targeting of HIV-1 virions to the basolateral membrane surface in polarized epithelial Madin-Darby canine kidney cells (MDCK). Site-directed mutagenesis was used to demonstrate that the membrane-proximal tyrosine in the intracytoplasmic tail of the HIV-1 transmembrane glycoprotein (gp41) is an essential component of this signal. In the present study, immunolocalization of viral budding allowed us to establish that this tyrosine-based signal is involved in determining the exact site of viral release at the surface of infected mononuclear cells. Substitution of the critical tyrosine residue was also shown to increase the amount of envelope glycoprotein at the cell surface, supporting previous suggestions that the tyrosine-based motif can promote endocytosis. Although alteration of the dual polarization-endocytosis motif did not affect the infectivity of cell-free virus, it could play a key role in cell-to-cell viral transmission. Accordingly, chronically infected lymphocytes showed a reduced ability to transmit the mutant virus to a cocultivated cell line. Overall, our data indicate that the YXXL targeting motif of HIV is active in various cell types and could play an important role in viral propagation; this may constitute an alternative target for HIV therapeutics and vaccine development.
Potent Inhibition of Human Immunodeficiency Virus Type 1 (HIV-1) Gene Expression and Virus Production by an HIV-2 Tat Activation-Response RNA Decoy
Authors: Browning Catherine M. , Cagnon Laurence , Good Paul D. , Rossi John , Engelke David R. , Markovitz David M. 
Published: December 2019

ABSTRACT
Tat activation-response region (TAR) decoys have been developed for use in gene therapy for people infected with human immunodeficiency virus type 1 (HIV-1). When a TAR RNA decoy is overexpressed, it will bind Tat, thus leaving less of this crucial protein to bind to and activate the natural transcriptional promoter of HIV-1. Previous TAR decoy constructs have used HIV-1 TAR. However, recent epidemiological and biological data began to suggest that the TAR region from the human immunodeficiency virus type 2 (HIV-2) may suppress HIV-1 transcription and hence replication. We created a vector which overexpresses TAR-2 under the control of the human U6 small nuclear RNA gene promoter and here show that the U6–TAR-2 decoy construct potently inhibits both HIV-2 and HIV-1 gene expression. Further, this decoy construct is able to markedly suppress HIV-1 replication. Thus, we have directly proven that TAR-2 can suppress HIV-1 replication and suggest that the HIV-2 TAR decoy may prove useful for combating HIV-1 infection.
Selection for Neutralization Resistance of the Simian/Human Immunodeficiency Virus SHIVSF33AVariant In Vivo by Virtue of Sequence Changes in the Extracellular Envelope Glycoprotein That Modify N-Linked Glycosylation
Authors: Cheng-Mayer Cecilia , Brown Amanda , Harouse Janet , Luciw Paul A. , Mayer Allen J. 
Published: December 2019

ABSTRACTWe previously reported on the in vivo adaptation of an infectious molecular simian/human immunodeficiency virus (SHIV) clone, SHIVSF33, into a pathogenic biologic viral variant, designated SHIVSF33A. In the present study, we show that SHIVSF33Ais resistant to neutralization by human immunodeficiency virus (HIV) and SHIV antisera. Multiple amino acid substitutions accumulated over time throughout theenvgene of SHIVSF33A; some of them coincided with the acquisition of the neutralization resistance of the virus. Of interest are changes that resulted in the removal, repositioning, and addition of potential glycosylation sites within the V1, V2, and V3 regions of envelope gp120. To determine whether potential glycosylation changes within these principal neutralization domains of HIV type 1 formed the basis for the resistance to serum neutralization of SHIVSF33A, mutant viruses were generated on the backbone of parental SHIVSF33and tested for their neutralization sensitivity. The mutations generated did not alter the in vitro replication kinetics or cytopathicity of the mutant viruses in T-cell lines. However, the removal of a potential glycosylation site in the V1 domain or the creation of such a site in the V3 domain did allow the virus to escape serum neutralization antibodies that recognized parental SHIVSF33. The combination of the V1 and V3 mutations conferred an additive effect on neutralization resistance over that of the single mutations. Taken together, these data suggest that (i) SHIV variants with changes in the Env SU can be selected in vivo primarily by virtue of their ability to escape neutralizing antibody recognition and (ii) carbohydrates play an important role in conferring neutralization escape, possibly by altering the structure of envelope gp120 or by shielding principal neutralization sites.
Elimination of Duck Hepatitis B Virus RNA-Containing Capsids in Duck Interferon-Alpha-Treated Hepatocytes
Authors: Schultz Ursula , Summers Jesse , Staeheli Peter , Chisari Francis V. 
Published: December 2019

ABSTRACT
Evidence is presented that the previously cloned type I duck interferon (DuIFN) cDNA encodes a homologue of mammalian interferon-alpha (IFN-α). Recombinant DuIFN-α was used to study the inhibition of duck hepatitis B virus (DHBV) replication in primary hepatocytes in order to determine the IFN-sensitive steps of the virus replication cycle. IFN-treated cells accumulated two- to threefold-lower amounts of viral RNA transcripts early during infection, when IFN was added before virus. This reduction was not due to inhibition of virus entry since initial covalently closed circular DNA levels were not decreased in IFN-treated cells. Interestingly, the inhibitory effect of IFN on viral RNA levels was not observed in cells infected with a mutant DHBV that fails to synthesize core protein, suggesting that an uncharacterized core protein-mediated enhancing effect is blocked by IFN. When IFN was added at 4 days postinfection, encapsidated viral RNA pregenomes disappeared from infected cells within 3 days. This depletion was not simply due to conversion of pregenomes to DNA since depletion was not blocked by phosphonoformic acid, an inhibitor of the viral reverse transcriptase. The intracellular concentration of intact nucleocapsids was reduced, suggesting that in the presence of IFN pregenome-containing capsids were selectively depleted in hepatocytes. Thus, two steps in DHBV replication that involve the viral core protein were inhibited by DuIFN-α.
Identification of Replication Specificity Determinants in Two Strains of Tomato Leaf Curl Virus from New Delhi
Authors: Chatterji Anju , Padidam Malla , Beachy Roger N. , Fauquet Claude M. 
Published: December 2019

ABSTRACT

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of symptoms on tomato and tobacco. Replication assays in tobacco protoplasts and plants showed that a single amino acid change, Asn10 to Asp in the N terminus of Rep protein, determines specificity for replication of the two strains based upon its interaction with the origin of replication (
ori
) sequences. The change of Asp10 to Asn in Rep protein of the mild strain coupled with point mutations at the 3rd and 10th nucleotides of the 13-mer binding site altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in
ori
and Asn10 of Rep protein suggested that Asn10 recognizes the third base pair of the putative binding site sequence GGTGTCGGAGTC in the severe strain.
Role of Naturally Occurring Basic Amino Acid Substitutions in the Human Immunodeficiency Virus Type 1 Subtype E Envelope V3 Loop on Viral Coreceptor Usage and Cell Tropism
Authors: Kato Kayoko , Sato Hironori , Takebe Yutaka 
Published: December 2019

ABSTRACT

To assess the role of naturally occurring basic amino acid substitutions in the V3 loop of human immunodeficiency virus type 1 (HIV-1) subtype E on viral coreceptor usage and cell tropism, we have constructed a panel of chimeric viruses with mutant V3 loops of HIV-1 subtype E in the genetic background of HIV-1
LAI
. The arginine substitutions naturally occurring at positions 8, 11, and 18 of the V3 loop in an HIV-1 subtype E X4 strain were systematically introduced into that of an R5 strain to generate a series of V3 loop mutant chimera. These chimeric viruses were employed in virus infectivity assays using HOS-CD4 cells expressing either CCR5 or CXCR4, peripheral blood mononuclear cells, T-cell lines, or macrophages. The arginine substitution at position 11 of the V3 loop uniformly caused the loss of infectivity in HOS-CD4-CCR5 cells, indicating that position 11 is critical for utilization of CCR5. CXCR4 usage was conferred by a minimum of two arginine substitutions, regardless of combination, whereas arginine substitutions at position 8 and 11 were required for T-cell line tropism. Nonetheless, macrophage tropism was not conferred by the V3 loop of subtype E R5 strain per se. We found that the specific combinations of amino acid changes in HIV-1 subtype E env V3 loop are critical for determining viral coreceptor usage and cell tropism. However, the ability to infect HOS-CD4 cells through either CXCR4 or CCR5 is not necessarily correlated with T-cell or macrophage tropism, suggesting that cellular tropism is not dictated solely by viral coreceptor utilization.
Optimal Replication Activity of Vesicular Stomatitis Virus RNA Polymerase Requires Phosphorylation of a Residue(s) at Carboxy-Terminal Domain II of Its Accessory Subunit, Phosphoprotein P
Authors: Hwang Leroy N. , Englund Nathan , Das Tapas , Banerjee Amiya K. , Pattnaik Asit K. 
Published: December 2019

ABSTRACT

The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P
226/227
) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-type (wt) protein. Substitution of alanine for Ser-233 did not have any adverse effect on replication activity of the protein. In contrast, all the mutant proteins showed activities similar to that of the wt protein in transcription. These results indicate that phosphorylation of the carboxy-terminal domain II residues of P protein are required for optimal replication activity but not for transcription activity. Furthermore, substitution of glutamic acid residues for Ser-226 and Ser-227 resulted in a protein that was only 14% active in replication but almost fully active in transcription. Taken together, these results, along with our earlier studies, suggest that phosphorylation of residues at two different domains in the P protein regulates its activity in transcription and replication of the VSV genome.
Failure To Cleave Murine Leukemia Virus Envelope Protein Does Not Preclude Its Incorporation in Virions and Productive Virus-Receptor Interaction
Authors: Zavorotinskaya Tatiana , Albritton Lorraine M. 
Published: December 2019

ABSTRACT
It is thought that complete cleavage of retroviral envelope protein into mature surface protein (SU) and transmembrane protein (TM) is critical for its assembly into virions and the formation of infectious virus particles. Here we report the identification of highly infectious, cleavage-deficient envelope mutant proteins. Substitution of aspartate for lysine 104, arginines 124 and 126, or arginines 223 and 225 strongly suppressed cleavage of the envelope precursor and yet allowed efficient incorporation of precursor molecules as the predominant species in virions that were almost as infectious as the wild-type virus. These results indicate that cleavage of the envelope precursor into mature SU and TM is not necessary for assembly into virions. Moreover, they call into question how many mature envelope protein subunits are required to complete virus entry, suggesting that a very few molecules suffice. The failure of host cell proteases to cleave these mutant proteins, whose substitutions are distal to the actual site of cleavage, suggests that the envelope precursor is misfolded, sequestering the cleavage site. In agreement with this, all cleavage mutant proteins exhibited significant losses of receptor binding, suggesting that these residues play roles in proper envelope protein folding. We also identified a charged residue, arginine 102, whose substitution suppressed envelope cleavage and allowed precursor incorporation but resulted in virions that were virtually noninfectious and that exhibited the greatest reduction in receptor binding. Placement of these cleavage mutations into envelope proteins of targeted retroviral vectors for human gene therapy may prevent loss of the modified surface proteins from virions, improving their infectivity and storage hardiness.