bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Immunodeficiency Virus Type 1 DNA Sequences Genetically Damaged by Hypermutation Are Often Abundant in Patient Peripheral Blood Mononuclear Cells and May Be Generated during Near-Simultaneous Infection and Activation of CD4
+
T Cells
Authors: Janini Mario , Rogers Melissa , Birx Deborah R. , McCutchan Francine E. 
Published: July 2002

ABSTRACT

G-to-A hypermutation has been sporadically observed in human immunodeficiency virus type 1 (HIV-1) proviral sequences from patient peripheral blood mononuclear cells (PBMC) and virus cultures but has not been systematically evaluated. PCR primers matched to normal and hypermutated sequences were used in conjunction with an agarose gel electrophoresis system incorporating an AT-binding dye to visualize, separate, clone, and sequence hypermutated and normal sequences in the 297-bp HIV-1 protease gene amplified from patient PBMC. Among 53 patients, including individuals infected with subtypes A through D and at different clinical stages, at least 43% of patients harbored abundant hypermutated, along with normal, protease genes. In 70 hypermutated sequences, saturation of G residues in the GA or GG dinucleotide context ranged from 20 to 94%. Levels of other mutants were not elevated, and G-to-A replacement was entirely restricted to GA or GG, and not GC or GT, dinucleotides. Sixty-nine of 70 hypermutated and 3 of 149 normal sequences had in-frame stop codons. To investigate the conditions under which hypermutation occurs in cell cultures, purified CD4
+
T cells from normal donors were infected with cloned NL4-3 virus stocks at various times before and after phytohemagglutinin (PHA) activation. Hypermutation was pronounced when HIV-1 infection occurred simultaneously with, or a few hours after, PHA activation, but after 12 h or more after PHA activation, most HIV-1 sequences were normal. Hypermutated sequences generated in culture corresponded exactly in all parameters to those obtained from patient PBMC. Near-simultaneous activation and infection of CD4
+
T cells may represent a window of susceptibility where the informational content of HIV-1 sequences is lost due to hypermutation.
Mutation of the Methylated tRNA3Lys Residue A58 Disrupts Reverse Transcription and Inhibits Replication of Human Immunodeficiency Virus Type 1
Authors: Renda Matthew J. , Rosenblatt Joseph D. , Klimatcheva Ekaterina , Demeter Lisa M. , Bambara Robert A. , Planelles Vicente 
Published: July 2002

ABSTRACT

Cellular tRNA

3

Lys

serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA

3

Lys

interacts directly with HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to initiate reverse transcription. Residue A58 of tRNA

3

Lys

, which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 1-methyladenosine 58 (M
1
A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA

3

Lys

residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA

3

Lys

derivative, tRNA

3

Lys

A58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M
1
A58 is necessary for the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M
1
A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M
1
A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1.
Role of CD8
+
Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
Authors: Lifson Jeffrey D. , Rossio Jeffrey L. , Piatak Michael , Parks Thomas , Li Li , Kiser Rebecca , Coalter Vicky , Fisher Brad , Flynn Bernard M. , Czajak Susan , Hirsch Vanessa M. , Reimann Keith A. , Schmitz Joern E. , Ghrayeb John , Bischofberger Norbert , Nowak Martin A. , Desrosiers Ronald C. , Wodarz Dominik 
Published: July 2002

ABSTRACT

Transient antiretroviral treatment with tenofovir, (
R
)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8
+
lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8
+
cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8
+
cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.
The UL6 Gene Product Forms the Portal for Entry of DNA into the Herpes Simplex Virus Capsid
Authors: Newcomb William W. , Juhas Rachel M. , Thomsen Darrell R. , Homa Fred L. , Burch April D. , Weller Sandra K. , Brown Jay C. 
Published: July 2002

ABSTRACTDuring replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 ± 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 ± 1.1 and 5.0 ± 0.7 nm, respectively, and a height of 19.5 ± 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as φ29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.
Critical Roles of Nuclear Receptor Response Elements in Replication of Hepatitis B Virus
Authors: Yu Xianming , Mertz Janet E. 
Published: July 2002

ABSTRACT

Functional analysis of the roles of the nuclear receptor response elements (NRREs) in the transcription and replication of hepatitis B virus (HBV) in the context of its whole genome has been hampered by the extensive overlapping of the NRREs with the regions encoding viral proteins. We introduced point mutations that inactivate the NRREs individually without altering the open reading frames of viral proteins. These mutations in the context of a plasmid containing 1.2 copies of the HBV genome were transiently transfected into the human hepatoma cell line Huh7. Inactivation of the NRRE in either the preC promoter (NRRE
preC
) or enhancer I (NRRE
enhI
) led to moderate reductions in synthesis of viral RNAs. Concurrent inactivation of both NRREs led to 7- to 8-fold reductions in synthesis of the preC, pregenomic, and preS RNAs and a 15-fold reduction in synthesis of the S RNA. The accumulation of viral DNA in the cytoplasmic nucleocapsids and virion particles in the culture medium was also reduced seven- to eightfold. These results suggest that these NRREs are critical for the efficient propagation of HBV in hepatocytes. In cotransfection experiments we also found that overexpression of PPARα-RXRα in the presence of their respective ligands led to a fourfold increase in pregenomic RNA synthesis and a four- to fivefold increase in viral DNA synthesis, while it had little or no effect on synthesis of the other viral RNAs. Similar effects were observed with overexpression of PPARγ-RXRα in the presence of their respective ligands. This activation was dependent on NRRE
preC
, because the increase in synthesis of viral RNA and DNA was not observed when this site was mutated. Likewise, no activation of synthesis of pregenomic RNA and viral DNA by PPARα-RXRα was observed in a naturally occurring NRRE
preC
−
mutant of HBV. Our results suggest that interactions between nuclear receptors and NRREs present in the HBV genome may play critical roles in regulating its transcription and replication during HBV infection of hepatocytes.
Rabies Virus Ocular Disease: T-Cell-Dependent Protection Is under the Control of Signaling by the p55 Tumor Necrosis Factor Alpha Receptor, p55TNFR
Authors: Camelo Serge , Castellanos Jaime , Lafage Mireille , Lafon Monique 
Published: July 2002

ABSTRACTFollowing brain infection, the Challenge Virus Standard strain of rabies virus infects the retina. Rabies virus ocular infection induces the infiltration of neutrophils and predominantly T cells into the eye. The role of tumor necrosis factor alpha (TNF-α)-lymphotoxin signaling in the control of rabies virus ocular infection and inflammatory cell infiltration was assessed using mice lacking the p55 TNF-α receptor (p55TNFR−/−mice). The incidence of ocular disease and the intensity of retinal infection were greater in p55TNFR−/−mice than in C57BL/6 mice: the aggravation correlated with less neutrophil and T-cell infiltration. This indicates that cellular infiltration is under the control of the p55 TNF-α receptor and suggests that inflammatory cells may protect the eye against rabies virus ocular infection. The role of T cells following rabies virus ocular disease was assessed by comparison of rabies virus infection in nude mice with their normal counterparts. Indeed, the incidence and severity of the rabies virus ocular disease were higher in athymic nude mice than in BALB/c mice, indicating that T lymphocytes are protective during rabies virus ocular infection. Moreover, few T cells and neutrophils underwent apoptosis in rabies virus-infected retina. Altogether, these data suggest that T lymphocytes and neutrophils are able to enter the eye, escape the immune privilege status, and limit rabies virus ocular disease. In conclusion, rabies virus-mediated eye disease provides a new model for studying mechanisms regulating immune privilege during viral infection.
Naturally Occurring Deletional Mutation in the C-Terminal Cytoplasmic Tail of CCR5 Affects Surface Trafficking of CCR5
Authors: Shioda Tatsuo , Nakayama Emi E. , Tanaka Yuetsu , Xin Xiaomi , Liu Huanliang , Kawana-Tachikawa Ai , Kato Atsushi , Sakai Yuko , Nagai Yoshiyuki , Iwamoto Aikichi 
Published: July 2002

ABSTRACTCCR5 is an essential coreceptor for the cellular entry of R5 strains of human immunodeficiency virus type 1 (HIV-1). CCR5-893(−) is a single-nucleotide deletion mutation which is observed exclusively in Asians (M. A. Ansari-Lari, et al., Nat. Genet. 16:221–222, 1997). This mutant gene produces a CCR5 which lacks the entire C-terminal cytoplasmic tail. To assess the effect of CCR5-893(−) on HIV-1 infection, we generated a recombinant Sendai virus expressing the mutant CCR5 and compared its HIV-1 coreceptor activity with that of wild-type CCR5. Although the mutant CCR5 has intact extracellular domains, its coreceptor activity was much less than that of wild-type CCR5. Flow cytometric analyses and confocal microscopic observation of cells expressing the mutant CCR5 revealed that surface CCR5 levels were greatly reduced in these cells, while cytoplasmic CCR5 levels of the mutant CCR5 were comparable to that of the wild type. Peripheral blood CD4+T cells obtained from individuals heterozygous for this allele expressed very low levels of CCR5. These data suggest that the CCR5-893(−) mutation affects intracellular transport of CCR5 and raise the possibility that this mutation also affects HIV-1 transmission and disease progression.
Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein
Authors: Shen Yuqiao , Kitzes Galila , Nye Julie A. , Fattaey Ali , Hermiston Terry 
Published: July 2002

ABSTRACT

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus
dl
309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.
The C-Terminal 88 Amino Acids of the Sendai Virus P Protein Have Multiple Functions Separable by Mutation
Authors: Tuckis Jeffery , Smallwood Sherin , Feller Joyce A. , Moyer Sue A. 
Published: July 2002

ABSTRACT

The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocapsid (NC) template through a P-NP interaction. To identify P amino acids responsible for binding we performed site-directed mutagenesis on the C-terminal 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein-protein interactions. All the mutants formed P oligomers and bound to L protein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others—JT4, JT6, and JT9—were completely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. NC binding was inhibited by combinations of the inactive mutations. The remaining P mutants were active in transcription but defective in various aspects of genome replication. Some P mutants were defective in NP
0
binding and abolished the reconstitution of replication from separate P-L and NP
0
-P complexes. In some of these cases the coexpression of the wt polymerase with the mutant NP
0
-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occurred in vivo, but not in vitro, suggesting that the intact cell is providing an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions besides NC binding that can be separated by mutation.
Regulation of Tumor Necrosis Factor Alpha Promoter by Human Parvovirus B19 NS1 through Activation of AP-1 and AP-2
Authors: Fu Yi , Ishii Keiko Kumura , Munakata Yasuhiko , Saitoh Takako , Kaku Mitsuo , Sasaki Takeshi 
Published: July 2002

ABSTRACTHuman parvovirus B19 frequently causes acute and chronic arthritis in adults. The molecular mechanism of B19 arthritis, however, remains poorly understood. We previously showed that the transmission of B19 from rheumatoid synoviocytes to monocytic cells is associated with enhanced secretion of tumor necrosis factor alpha (TNF-α), which triggers inflammation, and interleukin-6. To determine the role of B19 in the production of TNF-α, we focused on the function of its nonstructural protein, NS1, and established monocytic U937 lines transduced with the NS1 gene under the control of an inducible promoter. Production of TNF-α mRNA and protein was elevated in a manner associated with NS1 expression. Reporter assays revealed that AP-1 and AP-2 motifs on the TNF-α promoter were responsible for NS1-mediated up-regulation. Electrophoretic mobility shift assay showed specific binding of nuclear proteins from NS1 gene-transduced cells with the AP-1 or AP-2 probe. Antibodies against transcription factors AP-1 and AP-2 and anti-NS1 antibody inhibited the binding of nuclear proteins to the corresponding probes. These data indicate that NS1 up-regulates TNF-α transcription via activation of AP-1 and AP-2 in monocytic cells. The molecular mechanisms of NS1-mediated TNF-α expression would explain the pathogenesis of B19-associated inflammation.
Scrapie Infectivity in Hamster Blood Is Not Associated with Platelets
Authors: Holada Karel , Vostal Jaroslav G. , Theisen Patrick W. , MacAuley Claudia , Gregori Luisa , Rohwer Robert G. 
Published: July 2002

ABSTRACT
The infectivity of hamster scrapie strain 263K was measured in platelets isolated from blood pooled from six hamsters with clinical scrapie. The total number of infectious doses present in the blood pool was 220, out of which only 3.5 infectious doses were associated with platelets. A larger proportion of the total infectivity was recovered from the mononuclear leukocyte fraction. This result indicates that platelets are not the source of blood-borne infectivity in transmissible spongiform encephalopathy-infected hamsters.
Sport, age, and sex specific incidence of sports injuries in Western Australia
Authors: Stevenson Mark R , Hamer Peter , Finch Caroline F , Elliot Bruce , Kresnow Marcie-jo 
Published: July 2002

Objective—To describe the trends in recreational sports injury in Perth, Western Australia.Design—A prospective cohort study of sports injuries during the 1997 winter season (May to September).Setting—Sample of Australian football, field hockey, basketball, and netball players from the Perth metropolitan area, Western Australia.Methods—A cohort of sports participants was followed over the five month winter sports season. Before the season, participants completed a baseline questionnaire and during the season were interviewed every four weeks by telephone.Results—Overall, 92% of participants (n = 1391) who completed a baseline questionnaire completed at least one follow up telephone interview. About half (51%) of the cohort sustained one or more injuries during the winter season accounting for a total of 1034 injuries. Most injuries were of moderate (58%, n = 598) or minor (40%, n = 412) severity, with only 3% (n = 24) requiring emergency department treatment or a hospital stay. The injury incidence rate was greatest for football (20.3/1000 hours of participation), similar for field hockey and basketball (15.2/1000 hours and 15.1/1000 hours respectively), and lowest for netball (12.1/1000 hours). The incidence of injury was greatest in the first four weeks of the season, and participants aged between 26 and 30 years had about a 55% greater risk of injury than those aged less than 18 years.Conclusions—This is one of the first studies to show that recreational sports are safe. Although the likelihood of injury was greatest in the first month of the season, few injuries required admission to hospital or emergency department treatment. A greater emphasis on prevention in the early part of the season should help to reduce the elevated incidence of injury found at this time.