bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the Critical Domain in the V3 Loop of Human Immunodeficiency Virus Type 1 gp120 Involved in CCR5 Utilization
Authors: Hung Chia-Suei , Vander Heyden Nancy , Ratner Lee 
Published: December 2019

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) infection of CD4
+
lymphocytes and macrophages involves interaction of the surface subunit of the envelope protein (gp120) with coreceptors. Isolates have been found with specific tropism for macrophages and/or T-cell lines, through the utilization of chemokine receptor CCR5 (R5) or CXCR4 (X4). The third hypervariable loop (V3 loop) of gp120 is the major determinant of tropism. Using chimeric envelopes between HXB2 (X4) and ADA (R5), we found that the C-terminal half of the V3 loop was sufficient to confer on HXB2 the ability to infect CCR5-expressing cells. A sequence motif was identified at positions 289 to 292 allowing 30% of wild-type levels of infection, whereas full activity was achieved with the conversion of Lys to Glu at position 287 in addition to the above motif. Moreover, V3 loops from either SF2 (X4R5) or SF162 (R5) also allowed infection of CCR5-expressing cells, supporting the importance of V3 loops in influencing CCR5 utilization. The effects of amino acid changes at position 287 on the level of infection via CCR5 showed that negatively charged residues (Glu and Asp) were optimal for efficient interaction whereas only bulky hydrophobic residues drastically reduced infection. In addition, sequences at the N terminus of the V3 loop independently modulated the level of infection via CCR5. This study also examined the susceptibility of chimeric envelopes to neutralization by anticoreceptor antibodies and suggested the presence of differential interaction between the chimeric envelopes and CCR5. These findings highlight the critical residues in the V3 loop that mediate HIV-1 infection.
Perturbation of Cell Cycle Progression and Cellular Gene Expression as a Function of Herpes Simplex Virus ICP0
Authors: Hobbs William E. , DeLuca Neal A. 
Published: December 2019

ABSTRACT

Herpes simplex virus type 1 is capable of inhibiting host cell DNA synthesis following lytic infection. However, the mechanism and nature of potential effects on cell cycle progression have not been described. In this report, we characterize the dysregulation of the cell cycle following infection with the replication-incompetent virus
d
106, where immediate-early gene expression is restricted to infected-cell polypeptide 0 (ICP0) and the expression of all other viral genes is dramatically reduced or is not observed. Infection with
d
106 resulted in the accumulation of cells in both the G
1
/S and G
2
/M compartments, consistent with cell cycle arrest at both checkpoints. The isogenic variant
d
109, which does not express any viral proteins, failed to induce this phenotype, suggesting that the expression of ICP0 is crucial for cell cycle arrest. Analysis of global cellular gene expression patterns following infection with
d
106 and
d
109 revealed that a relatively small subset of cellular genes were induced as a consequence of ICP0 expression. A number of these genes induced in the presence of ICP0 are classically considered p53-responsive genes, including
p21
,
gadd45
, and
mdm-2
. However, infection with
d
106 of cells with both alleles of p53 deleted resulted in the same cell cycle arrest phenotype and similar cellular gene expression patterns, suggesting that the expression of ICP0 results in cell cycle arrest potentially via p53-dependent and p53-independent mechanisms. In addition, it was found that the effects of infection with
d
106 on viral and cellular gene expression were similar to the effects observed following treatment of cells with the histone deacetylase inhibitor trichostatin A.
Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia
Authors: Zheng Jialin , Ghorpade Anuja , Niemann Douglas , Cotter Robin L. , Thylin Michael R. , Epstein Leon , Swartz Jennifer M. , Shepard Robin B. , Liu Xiaojuan , Nukuna Adeline , Gendelman Howard E. 
Published: December 2019

ABSTRACTChemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis.
Stoichiometry of Monoclonal Antibody Neutralization of T-Cell Line-Adapted Human Immunodeficiency Virus Type 1
Authors: Schønning Kristian , Lund Ole , Søgaard Lund Ole , Stig Hansen John-Erik 
Published: December 2019

ABSTRACT

In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different
env
genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an
env
transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4
−
HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection.
Adeno-Associated Virus Type 2 Protein Interactions: Formation of Pre-Encapsidation Complexes
Authors: Dubielzig Ralf , King Jason A. , Weger Stefan , Kern Andrea , Kleinschmidt Jürgen A. 
Published: December 2019

ABSTRACT
The nonstructural adeno-associated virus type 2 Rep proteins are known to control viral replication and thus provide the single-stranded DNA genomes required for packaging into preformed capsids. In addition, complexes between Rep proteins and capsids have previously been observed in the course of productive infections. Such complexes have been interpreted as genome-linked Rep molecules associated with the capsid upon successful DNA encapsidation. Here we demonstrate via coimmunoprecipitation, cosedimentation, and yeast two-hybrid analyses that the Rep-VP association also occurs in the absence of packageable genomes, suggesting that such complexes could be involved in the preparation of empty capsids for subsequent encapsidation steps. The Rep domain responsible for the observed Rep-VP interactions is situated within amino acids 322 to 482. In the presence of all Rep proteins, Rep52 and, to a lesser extent, Rep78 are most abundantly recovered with capsids, whereas Rep68 and Rep40 vary in association depending on their expression levels. Rep78 and Rep52 are bound to capsids to roughly the same extent as the minor capsid protein VP2. Complexes of Rep78 and Rep52 with capsids differ in their respective detergent stabilities, indicating that they result from different types of interactions. Rep-VP interaction studies suggest that Rep proteins become stably associated with the capsid during the assembly process. Rep-capsid complexes can reach even higher complexity through additional Rep-Rep interactions, which are particularly detergent labile. Coimmunoprecipitation and yeast two-hybrid data demonstrate the interaction of Rep78 with Rep68, of Rep68 with Rep52, and weak interactions of Rep40 with Rep52 and Rep78. We propose that the large complexes arising from these interactions represent intermediates in the DNA packaging pathway.
SPI-1-Dependent Host Range of Rabbitpox Virus and Complex Formation with Cathepsin G Is Associated with Serpin Motifs
Authors: Moon Kristin B. , Turner Peter C. , Moyer Richard W. 
Published: December 2019

ABSTRACTSerpins are a superfamily of serine proteinase inhibitors which function to regulate a number of key biological processes including fibrinolysis, inflammation, and cell migration. Poxviruses are the only viruses known to encode functional serpins. While some poxvirus serpins regulate inflammation (myxoma virus SERP1 and cowpox virus [CPV] crmA/SPI-2) or apoptosis (myxoma virus SERP2 and CPV crmA/SPI-2), the function of other poxvirus serpins remains unknown. The rabbitpox virus (RPV) SPI-1 protein is 47% identical to crmA and shares all of the serpin structural motifs. However, no serpin-like activity has been demonstrated for SPI-1 to date. Earlier we showed that RPV with the SPI-1 gene deleted, unlike wild-type virus, fails to grow on A549 or PK15 cells (A. Ali, P. C. Turner, M. A. Brooks, and R. W. Moyer, Virology 202:306–314, 1994). Here we demonstrate that in the absence of a functional SPI-1 protein, infected nonpermissive cells which exhibit the morphological features of apoptosis fail to activate terminal caspases or cleave the death substrates PARP or lamin A. We show that SPI-1 forms a stable complex in vitro with cathepsin G, a member of the chymotrypsin family of serine proteinases, consistent with serpin activity. SPI-1 reactive-site loop (RSL) mutations of the critical P1 and P14 residues abolish this activity. Viruses containing the SPI-1 RSL P1 or P14 mutations also fail to grow on A549 or PK15 cells. These results suggest that the full virus host range depends on the serpin activity of SPI-1 and that in restrictive cells SPI-1 inhibits a proteinase with chymotrypsin-like activity and may function to inhibit a caspase-independent pathway of apoptosis.
Nonrandom Distribution of Hepatitis C Virus Quasispecies in Plasma and Peripheral Blood Mononuclear Cell Subsets
Authors: Afonso Anne Marie Roque , Jiang Jiaji , Penin François , Tareau Claire , Samuel Didier , Petit Marie-Anne , Bismuth Henri , Dussaix Elisabeth , Féray Cyrille 
Published: December 2019

ABSTRACT

The existence of an extrahepatic reservoir of hepatitis C virus (HCV) is suggested by differences in quasispecies composition between the liver, peripheral blood mononuclear cells, and serum. We studied HCV RNA compartmentalization in the plasma of nine patients, in CD19
+
, CD8
+
, and CD4
+
positively selected cells, and also in the negatively selected cell fraction (NF). HCV RNA was detected in all plasma samples, in seven of nine CD19
+
, three of eight CD8
+
, and one of nine CD4
+
cell samples, and in seven of eight NF cells. Cloning and sequencing of HVR1 in two patients showed a sequence grouping: quasispecies from a given compartment (all studied compartments for one patient and CD8
+
and NF for the other) were statistically more genetically like each other than like quasispecies from any other compartment. The characteristics of amino acid and nucleotide substitutions suggested the same structural constraints on HVR1, even in very divergent strains from the cellular compartments, and homogeneous selection pressure on the different compartments. These findings demonstrate the compartmental distribution of HCV quasispecies within peripheral blood cell subsets and have important implications for the study of extrahepatic HCV replication and interaction with the immune system.
Human Cytomegalovirus 86-Kilodalton IE2 Protein Blocks Cell Cycle Progression in G
1
Authors: Wiebusch Lüder , Hagemeier Christian 
Published: December 2019

ABSTRACT

The 86-kDa IE2 protein of human cytomegalovirus (HCMV) is an important regulator of viral and host cell gene expression. Still, besides its function as a transcription factor, little is known about the biological activities of IE2. Here, we show that IE2 can induce a G
1
arrest in several different cell lines, including HCMV-permissive U-373 cells. The known transcriptional activation domains of IE2 are dispensable for G
1
arrest, favoring a posttranscriptional mechanism mediating this cell cycle effect. We show that like human primary fibroblasts U-373 cells arrest in G
1
upon infection with HCMV. This G
1
arrest occurs within 24 h after infection and in proliferating cells depends on viral gene expression. Our data therefore suggest that IE2 is at least partially responsible for blocking the transition from G
1
to S phase, which is induced when cells are infected with HCMV.
Requirements for RNA Replication of a Poliovirus Replicon by Coxsackievirus B3 RNA Polymerase
Authors: Bell Yolanda C. , Semler Bert L. , Ehrenfeld Ellie 
Published: December 2019

ABSTRACT

A chimeric poliovirus type 1 (PV1) genome was constructed in which the 3D RNA polymerase (3D
pol
) coding sequences were replaced with those from coxsackievirus B3 (CVB3). No infectious virus was produced from HeLa cells transfected with the chimeric RNA. Processing of the PV1 capsid protein precursor was incomplete, presumably due to inefficient recognition of the P1 protein substrate by the chimeric 3CD proteinase containing CVB3 3D sequences. The ability of the chimeric RNA to replicate in the absence of capsid formation was measured after replacement of the P1 region with a luciferase reporter gene. No RNA synthesis was detected, despite efficient production of enzymatically active 3D
pol
from the 3D portion of the chimeric 3CD. The chimeric 3CD protein was unable to efficiently bind to the cloverleaf-like structure (CL) at the 5′ end of PV1 RNA, which has been demonstrated previously to be required for viral RNA synthesis. The CVB3 3CD protein bound the PV1 CL as well as PV1 3CD. An additional chimeric PV1 RNA that contained CVB3 3CD sequences also failed to produce virus after transfection. Since processing of PV1 capsid protein precursors by the CVB3 3CD was again incomplete, a luciferase-containing replicon was also analyzed for RNA replication. The 3CD chimera replicated at 33°C, but not at 37°C. Replacement of the PV1 5′-terminal CL with that of CVB3 did not rescue the temperature-sensitive phenotype. Thus, there is an essential interaction(s) between 3CD and other viral P2 or P3 protein products required for efficient RNA replication which is not fully achieved between proteins from the two different members of the same virus genus.
Repeated Delivery of Adeno-Associated Virus Vectors to the Rabbit Airway
Authors: Beck Suzanne E. , Jones Lori A. , Chesnut Kye , Walsh Scott M. , Reynolds Thomas C. , Carter Barrie J. , Askin Frederic B. , Flotte Terence R. , Guggino William B. 
Published: December 2019

ABSTRACTEfficient local expression from recombinant adeno-associated virus (rAAV)-cystic fibrosis (CF) transmembrane conductance regulator (CFTR) vectors has been observed in the airways of rabbits and monkeys for up to 6 months following a single bronchoscopic delivery. However, it is likely that repeated administrations of rAAV vectors will be necessary for sustained correction of the CF defect in the airways. The current study was designed to test the feasibility of repeated airway delivery of rAAV vectors in the rabbit lung. After two doses of rAAV-CFTR to the airways, rabbits generated high titers of serum anti-AAV neutralizing antibodies. Rabbits then received a third dose of a rAAV vector containing the green fluorescent protein (GFP) reporter gene packaged in either AAV serotype 2 (AAV2) or serotype 3 (AAV3) capsids. Each dose consisted of 1 ml containing 5 × 109DNase-resistant particles of rAAV vector, having no detectable replication-competent AAV or adenovirus. Three weeks later, GFP expression was observed in airway epithelial cells despite high anti-AAV neutralizing titers at the time of delivery. There was no significant difference in the efficiency of DNA transfer or expression between the rAAV3 and rAAV2 groups. No significant inflammatory responses to either repeated airway exposure to rAAV2-CFTR vectors or to GFP expression were observed. These experiments demonstrate that serum anti-AAV neutralizing antibody titers do not predict airway neutralizationin vivoand that repeated airway delivery rAAV allows for safe and effective gene transfer.
Construction and Preliminary Characterization of a Library of “Lethal” Preterminal Protein Mutant Adenoviruses
Authors: Schaack Jerome , Ho William Y. , Tolman Shawna , Ullyat Elizabeth , Guo Xiaoling , Frank Nina , Freimuth Paul I. , Roovers Dick J. , Sussenbach John S. 
Published: December 2019

ABSTRACT
Adenoviruses containing lethal in-frame insertion mutant alleles of the preterminal protein (pTP) gene were constructed with cell lines that express pTP. Thirty in-frame insertion mutant alleles, including 26 alleles previously characterized as lethal and 4 newly constructed mutant alleles, were introduced into the viral chromosome in place of the wild-type pTP gene. The viruses were tested for ability to form plaques at 37°C in HeLa-pTP cells and at 32°C and 39.5°C in HeLa cells. Two of the newly constructed viruses exhibited temperature sensitivity for plaque formation, one virus did not form plaques in the absence of complementation, seven additional mutants exhibited a greater than 10-fold reduction in plaque formation in the absence of complementation, and another eight mutants exhibited stronger phenotypes than did previously characterized in-frame insertion mutants in the plaque assay. These mutant viruses offer promise for analysis of pTP functions.
Molecular Characterization of a Porcine Enteric Calicivirus Genetically Related to Sapporo-Like Human Caliciviruses
Authors: Guo M. , Chang K. O. , Hardy M. E. , Zhang Q. , Parwani A. V. , Saif L. J. 
Published: December 2019

ABSTRACT

Porcine enteric calicivirus (PEC) is associated with diarrhea in pigs, and to date it is the only cultivable enteric calicivirus (tissue culture-adapted [TC] PEC/Cowden). Based on sequence analysis of cDNA clones and reverse transcription-PCR products, TC PEC/Cowden has an RNA genome of 7,320 bp, excluding its 3′ poly(A)
+
tail. The genome is organized in two open reading frames (ORFs), similar to the organizations of the human Sapporo-like viruses (SLVs) and the lagoviruses. ORF1 encodes the polyprotein that is fused to and contiguous with the capsid protein. ORF2 at the 3′ end encodes a small basic protein of 164 amino acids. Among caliciviruses, PEC has the highest amino acid sequence identities in the putative RNA polymerase (66%), 2C helicase (49.6%), 3C-like protease (43.7%), and capsid (39%) regions with the SLVs, indicating that PEC is genetically most closely related to the SLVs. The complete RNA genome of wild-type (WT) PEC/Cowden was also sequenced. Sequence comparisons revealed that the WT and TC PEC/Cowden have 100% nucleotide sequence identities in the 5′ terminus, 2C helicase, ORF2, and the 3′ nontranslated region. TC PEC/Cowden has one silent mutation in its protease, two amino acid changes and a silent mutation in its RNA polymerase, and five nucleotide substitutions in its capsid that result in one distant and three clustered amino acid changes and a silent mutation. These substitutions may be associated with adaptation of TC PEC/Cowden to cell culture. The cultivable PEC should be a useful model for studies of the pathogenesis, replication, and possible rescue of uncultivable human enteric caliciviruses.
rab5 GTPase Regulates Adenovirus Endocytosis
Authors: Rauma Tanja , Tuukkanen Juha , Bergelson Jeffrey M. , Denning Gerene , Hautala Timo 
Published: December 2019

ABSTRACT

Adenovirus interaction with αv integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca
2+
]
c
-mediated signaling or for tyrosine phosphorylation of pp125
FAK
, p130
CAS
, and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.
Rescue of Influenza A Virus from Recombinant DNA
Authors: Fodor Ervin , Devenish Louise , Engelhardt Othmar G. , Palese Peter , Brownlee George G. , García-Sastre Adolfo 
Published: December 2019

ABSTRACT
We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.