bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intrinsic Human Immunodeficiency Virus Type 1 Resistance of Hematopoietic Stem Cells Despite Coreceptor Expression
Authors: Shen Hongmei , Cheng Tao , Preffer Frederick I. , Dombkowski David , Tomasson Michael H. , Golan David E. , Yang Otto , Hofmann Wolfgang , Sodroski Joseph G. , Luster Andrew D. , Scadden David T. 
Published: December 2019

ABSTRACTInteractions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1.
“Hidden” dUTPase Sequence in Human Immunodeficiency Virus Type 1 gp120
Authors: Abergel Chantal , Robertson David L. , Claverie Jean-Michel 
Published: December 2019

ABSTRACT

A coding region homologous to the sequence for essential eukaryotic enzyme dUTPase has been identified in different genomic regions of several viral lineages. Unlike the nonprimate lentiviruses (caprine arthritis- encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, and visna virus), where dUTPase is integrated into the
pol
coding region, this enzyme has never been demonstrated to be present in the primate lentivirus genomes (human immunodeficiency virus type 1 [HIV-1], HIV-2, or the related simian immunodeficiency virus). A novel approach allowed us to identify a weak but significant sequence similarity between HIV-1 gp120 and the human dUTPase. This finding was then extended to all of the primate lentivirus lineages. Together with the recently reported fragmentary structural similarity between the V3 loop region and the
Escherichia coli
dUTPase (P. D. Kwong, R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson, Nature 393:648–659, 1998), our results strongly suggest that an ancestral dUTPase gene has evolved into the present primate lentivirus CD4 and cytokine receptor interacting region of gp120.
New Mouse Model for Dengue Virus Vaccine Testing
Authors: Johnson Alison J. , Roehrig John T. 
Published: December 2019

ABSTRACTSeveral dengue (DEN) virus vaccines are in development; however, the lack of a reliable small animal model in which to test them is a major obstacle. Because evidence suggests that interferon (IFN) is involved in the human anti-DEN virus response, we tested mice deficient in their IFN functions as potential models. Intraperitoneally administered mouse-adapted DEN 2 virus was uniformly lethal in AG129 mice (which lack alpha/beta IFN and gamma IFN receptor genes), regardless of age. Immunized mice were protected from virus challenge, and survival times increased following passive transfer of anti-DEN polyclonal antibody. These results demonstrate that AG129 mice are a promising small animal model for DEN virus vaccine trials.
Decay Kinetics of Human Immunodeficiency Virus-Specific Effector Cytotoxic T Lymphocytes after Combination Antiretroviral Therapy
Authors: Ogg G. S. , Jin X. , Bonhoeffer S. , Moss P. , Nowak M. A. , Monard S. , Segal J. P. , Cao Y. , Rowland-Jones S. L. , Hurley A. , Markowitz M. , Ho D. D. , McMichael A. J. , Nixon D. F. 
Published: December 2019

ABSTRACTLittle is known of the changes in human immunodeficiency virus type 1 (HIV-1)-specific effector cytotoxic T lymphocytes (CTL) after potent antiretroviral therapy. Using HLA/peptide tetrameric complexes, we show that after starting treatment, there are early rapid fluctuations in the HIV-1-specific CTL response which last 1 to 2 weeks. These fluctuations are followed by an exponential decay (median half-life, 45 days) of HIV-1-specific CTL which continues while viremia remains undetectable. These data have implications for the immunological control of drug-resistant virus.
Molecular Characterization of a Bovine Enteric Calicivirus: Relationship to the Norwalk-Like Viruses
Authors: Liu B. L. , Lambden P. R. , Günther H. , Otto P. , Elschner M. , Clarke I. N. 
Published: December 2019

ABSTRACT

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5′ terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the
Caliciviridae
JV is most closely related to the group 1 NLVs.
The U
L
3 Protein of Herpes Simplex Virus 1 Is Translated Predominantly from the Second In-Frame Methionine Codon and Is Subject to at Least Two Posttranslational Modifications
Authors: Markovitz Nancy S. , Filatov Felix , Roizman Bernard 
Published: December 2019

ABSTRACT

The U
L
3 open reading frame (ORF) has the coding capacity of 235 codons. The proteins reacting with the anti-U
L
3 antibody form in denaturing polyacrylamide gel bands with apparent
M
r
s of 34,000, 35,000, 38,000, 40,000, 41,000, and 42,000 and designated 1 to 6, respectively. Studies on their origins revealed the following. (i) The U
L
3 proteins forming all six bands were present in lysates of cells infected with wild-type virus and treated with tunicamycin or monensin or in cells infected with the mutant lacking the gene encoding the U
S
3 protein kinase. (ii) The proteins contained in the slower-migrating bands were absent from cells infected with the mutant lacking the U
L
13 protein kinase. Bands 1 and 3, however were phosphorylated in cells infected with this mutant. (iii) Band 2 protein was absent from cells transfected with a plasmid carrying a substitution of the predicted first methionine codon of the U
L
3 ORF and superinfected with the U
L
3
−
mutant. (iv) Band 1 and 3 proteins were absent from lysates of cells transfected with a plasmid carrying a substitution of the second (M12) methionine codon of the U
L
3 ORF and superinfected with the U
L
3
−
mutant. (v) Cells superinfected with mutants lacking both methionine codons did not accumulate any of the proteins contained in the six bands. (vi) In vitro transcription-translation studies indicated that the translation of band 1 protein was initiated from the second (M12) methionine codon and that band 3 protein represented a U
L
13-independent, posttranslationally processed form of these proteins. The results indicate that the U
L
3 protein of herpes simplex virus 1 is translated predominantly from the second in-frame methionine codon and is subject to at least two posttranslational modifications.
Efficiency and Fidelity of Full-Site Integration Reactions Using Recombinant Simian Immunodeficiency Virus Integrase
Authors: Goodarzi Goodarz , Pursley Michael , Felock Peter , Witmer Marc , Hazuda Daria , Brackmann Karl , Grandgenett Duane 
Published: December 2019

ABSTRACTFull-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.
Amino Acid Substitutions within the Leucine Zipper Domain of the Murine Coronavirus Spike Protein Cause Defects in Oligomerization and the Ability To Induce Cell-to-Cell Fusion
Authors: Luo Zongli , Matthews Avery M. , Weiss Susan R. 
Published: December 2019

ABSTRACT
The murine coronavirus spike (S) protein contains a leucine zipper domain which is highly conserved among coronaviruses. To assess the role of this leucine zipper domain in S-induced cell-to-cell fusion, the six heptadic leucine and isoleucine residues were replaced with alanine by site-directed mutagenesis. The mutant S proteins were analyzed for cell-to-cell membrane fusion activity as well as for progress through the glycoprotein maturation process, including intracellular glycosylation, oligomerization, and cell surface expression. Single-alanine-substitution mutations had minimal, if any, effects on S-induced cell-to-cell fusion. Significant reduction in fusion activity was observed, however, when two of the four middle heptadic leucine or isoleucine residues were replaced with alanine. Double alanine substitutions that involved either of the two end heptadic leucine residues did not significantly affect fusion. All double-substitution mutant S proteins displayed levels of endoglycosidase H resistance and cell surface expression similar to those of the wild-type S. However, fusion-defective double-alanine-substitution mutants exhibited defects in S oligomerization. These results indicate that the leucine zipper domain plays a role in S-induced cell-to-cell fusion and that the ability of S to induce fusion may be dependent on the oligomeric structure of S.
Characterization of the Block in Replication of Nucleocapsid Protein Zinc Finger Mutants from Moloney Murine Leukemia Virus
Authors: Gorelick Robert J. , Fu William , Gagliardi Tracy D. , Bosche William J. , Rein Alan , Henderson Louis E. , Arthur Larry O. 
Published: December 2019

ABSTRACT

Mutagenesis studies have shown that retroviral nucleocapsid (NC) protein Zn
2+
fingers (-Cys-X
2
-Cys-X
4
-His-X
4
-Cys- [CCHC]) perform multiple functions in the virus life cycle. Moloney murine leukemia virus mutants His 34→Cys (CCCC) and Cys 39→His (CCHH) were able to package their genomes normally but were replication defective. Thermal dissociation experiments showed that the CCHH mutant was not defective in genomic RNA dimer structure. Primer tRNA placement on the viral genome and the ability of the tRNA to function in reverse transcription initiation in vitro also appear normal. Some “full-length” DNA copies of the viral genome were synthesized in mutant virus-infected cells. The CCCC and CCHH mutants produced these DNA copies at greatly reduced levels. Circle junction fragments, amplified from two-long-terminal-repeat viral DNA (vDNA) by PCR, were cloned and characterized. Remarkably, it was discovered that vDNA isolated from cells infected with mutant virions had a wide variety of abnormalities at the site at which the two ends of the linear precursor had been ligated to form the circle (i.e., the junction between the 5′ end of U3 and the 3′ end of U5). In some molecules, bases were missing from regions corresponding to the U3 and U5 linear vDNA termini; in others, the viral sequences extended either beyond the U5 sequences into the primer-binding site and 5′ leader or beyond the U3 sequences into the polypurine tract into the
env
coding region. Still other molecules contained nonviral sequences between the linear vDNA termini. Such defective genomes would certainly be unsuitable substrates for integration. Thus, strict conservation of the CCHC structure in NC is required for infection events prior to and possibly including integration.
Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in
Escherichia coli
: a New Approach for Construction of HCMV Mutants
Authors: Borst Eva-Maria , Hahn Gabriele , Koszinowski Ulrich H. , Messerle Martin 
Published: December 2019

ABSTRACT

We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in
Escherichia coli
(M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation in
E. coli
, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in
E. coli
. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.
Activation of cJUN N-Terminal Kinase by Herpes Simplex Virus Type 1 Enhances Viral Replication
Authors: McLean T. I. , Bachenheimer S. L. 
Published: December 2019

ABSTRACTSignal transduction pathways convey signals generated at the cell surface into the cell nucleus in order to initiate a program of gene expression that is characteristic for particular stimuli. Here we present evidence that infection by herpes simplex virus type 1 activated the two terminal kinases, cJUN N-terminal kinase (JNK) and p38, of stress-activated signal transduction kinase cascades. By using a solid-phase kinase assay, a phospho-specific antibody, and extracts prepared from a variety of infected cell types, we determined that activation of both kinases began 3 to 4 h postinfection (p.i.) and remained elevated out to 14 h p.i. Through the use of UV-irradiated or antibody-neutralized wild-type virus and the temperature-sensitive mutanttsB7, the high level of JNK activation was shown to be dependent on viral gene expression. Activation of JNK following infection by vi13, an ICP4 mutant virus that does not express early or late genes, suggested that only virus entry and immediate-early gene expression were necessary for JNK activation. The activation of JNK and p38 correlated with increased chloramphenicol acetyltransferase (CAT) activity in reporter assays dependent upon the activity of cJUN and ATF2trans-activation domains. Increased CAT activity dependent on TRE and CRE promoter sites was also observed in response to herpes simplex virus infection. The activities of ERK and ERK-dependent transcription factors were unchanged or depressed following infection, showing that activation of JNK and p38 was a specific event. Finally, the activation of JNK was important for the efficiency of viral replication. The yield of virus in NIH 3T3 cells stably expressing JIP-1, an inhibitor of JNK translocation to the nucleus, was reduced 70% compared to that of control cells, in single-step growth experiments.
Altering the Intracellular Environment Increases the Frequency of Tandem Repeat Deletion during Moloney Murine Leukemia Virus Reverse Transcription
Authors: Pfeiffer Julie K. , Topping Robert S. , Shin Nam-Hee , Telesnitsky Alice 
Published: December 2019

ABSTRACT

During retroviral DNA synthesis reverse transcriptase frequently performs nonrequired template switches that can lead to genetic rearrangements or recombination. It has been postulated that template switching occurs after pauses in the action of reverse transcriptase. Hence factors which affect pausing, such as polymerization rate, may affect the frequency of template switching. To address the hypothesis that increasing the time required to complete reverse transcription increases the frequency of template switching, we established conditions that lengthened the time required to complete a single round of intracellular Moloney murine leukemia virus reverse transcription approximately threefold. Under these conditions, which resulted from intracellular nucleotide pool imbalances generated with hydroxyurea, we examined template switching frequency using a
lacZ
-based tandem repeat deletion assay. We observed that the frequency of deletion during reverse transcription in hydroxyurea-treated cells was approximately threefold higher than that in untreated control cells. These findings suggest that rates of retroviral recombination may vary when the intracellular environment is altered.
Decreased Processivity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Containing Didanosine-Selected Mutation Leu74Val: a Comparative Analysis of RT Variants Leu74Val and Lamivudine-Selected Met184Val
Authors: Sharma Prem L. , Crumpacker Clyde S. 
Published: December 2019

ABSTRACTWe previously showed that a didanosine-selected mutation in pNL4-3 background conferred a replication disadvantage on human immunodeficiency virus type 1, resulting in a loss of replication fitness. This work has been extended by showing that a recombinant virus with the HXBc2 backbone and reverse transcriptase (RT) fragments from pNL4-3 containing the Leu74Val mutation produce decreasing amounts of p24 antigen over a 3-week period. The HXBc2 recombinant containing the wild-type RT from pNL4-3 replicated efficiently. When the virion-associated RT containing the Leu74Val mutation was used in an RT processivity assay with homopolymer RNA template-primer, poly(A), and oligo(dT), the RT with altered Leu74Val mutation was less processive, generating fewer cDNA products in comparison to wild-type pNL4-3 RT. The replication kinetics and RT processivity of the mutant with the Leu74Val mutation were compared to those of a lamivudine-selected mutant Met184Val. In replication kinetics assays, mutant Leu74Val replicated slower than the mutant Met184Val. In a processivity assay, the mutant RTs from both viruses show comparable decreases in processivity. These observations provide biochemical evidence of decreased processivity to support the decrease in replication fitness observed with the Leu74Val or Met184Val mutations.
Genetic Analysis of the Role of Herpes Simplex Virus Type 1 Glycoprotein K in Infectious Virus Production and Egress
Authors: Foster Timothy P. , Kousoulas Konstantin G. 
Published: December 2019

ABSTRACT
Herpes simplex virus type 1 (KOS)ΔgK is a mutant virus which lacks glycoprotein K (gK) and exhibits defects in virion egress (S. Jayachandra, A. Baghian, and K. G. Kousoulas, J. Virol. 69:5401–5413, 1997). To further understand the role of gK in virus egress, we constructed recombinant viruses, ΔgKhpd-1, -2, -3, and -4, that specified gK amino-terminal portions of 139, 239, 268, and 326 amino acids, respectively, corresponding to truncations immediately after each of the four putative membrane-spanning domains of gK. ΔgKhpd-1 and ΔgKhpd-2 viruses produced lower yields and smaller plaques than ΔgK. Numerous ΔgKhpd-1 capsids accumulated predominately within large double-membrane vesicles of which the inner membrane appeared to be derived from viral envelopes while the outer membrane appeared to originate from the outer nuclear membrane. The mutant virus ΔgKhpd-3 produced higher yields and larger plaques than the ΔgK virus. The mutant virus ΔgKhpd-4 produced yields and plaques similar to those of the wild-type virus strain KOS, indicating that deletion of the carboxy-terminal 12 amino acids did not adversely affect virus replication and egress. Comparisons of the gK primary sequences specified by alphaherpesviruses revealed the presence of a cysteine-rich motif (CXXCC), located within domain III in the lumen side of gK, and a tyrosine-based motif, YTKΦ (where Φ is any bulky hydrophobic amino acid), located between the second and third hydrophobic domains (domain II) in the cytoplasmic side of gK. The mutant virus gK/Y183S, which was constructed to specify gK with a single-amino-acid change (Y to S) within the YTKΦ motif, replicated less efficiently than the ΔgK virus. The mutant virus gK/C304S-C307S, which was constructed to specify two serine instead of cysteine residues within the cysteine-rich motif (CXXCC changed to SXXSC) of gK domain III, replicated more efficiently than the ΔgK virus. Our data suggests that gK contains domains in its amino-terminal portion that promote aberrant nucleocapsid envelopment and/or membrane fusion between different virion envelopes and contains domains within its domains II and III that function in virus replication and egress.