bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Recombinant Respiratory Syncytial Viruses with the Region Responsible for Type 2 T-Cell Responses and Pulmonary Eosinophilia Deleted from the Attachment (G) Protein
Authors: Elliott Matthew B. , Pryharski Karin S. , Yu Qingzhong , Boutilier L. A. , Campeol N. , Melville K. , Laughlin Todd S. , Gupta C. K. , Lerch Robert A. , Randolph Valerie B. , LaPierre Natisha A. , Dack Kristen M. Heers , Hancock Gerald E. 
Published: July 2004

ABSTRACT

It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cpΔG150-222 or rA2cpΔG177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37°C, but pinpoint morphology was observed at 39°C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log
10
-unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cpΔG150-222 or rA2cpΔG177-220. These findings are important for the development of attenuated RSV vaccines.
PKR-Dependent and -Independent Mechanisms Are Involved in Translational Shutoff during Sindbis Virus Infection
Authors: Gorchakov Rodion , Frolova Elena , Williams Bryan R. G. , Rice Charles M. , Frolov Ilya 
Published: July 2004

ABSTRACTThe replication of Sindbis virus (SIN) profoundly affects the metabolism of infected vertebrate cells. One of the main events during SIN infection is the strong inhibition of translation of cellular mRNAs. In this study, we used a combination of approaches, including the study of SIN replication in PKR−/−mouse embryo fibroblasts or in the presence of an excess of catalytically inactive PKR. We show that the PKR-dependent inhibition of translation is not the only and most likely not the major pathway mediating translational shutoff during SIN infection. The PKR-independent mechanism strongly affects the translation of cellular templates, whereas translation of SIN subgenomic RNA is resistant to inhibition, and this leads to a benefit for viral replication. Our findings suggest that both PKR-dependent and non-PKR-dependent mechanisms of SIN-induced translational shutoff can be manipulated by using SIN replicons expressing mutated SIN nsP2 or kinase-defective PKR. Specifically, we show that expression of heterologous genes from SIN-based and most likely other alphavirus-based replicons can be increased by downregulating both the PKR-dependent and PKR-independent translational shutoffs.
Development of a DNA Vaccine Targeting Human Papillomavirus Type 16 Oncoprotein E6
Authors: Peng Shiwen , Ji Hongxiu , Trimble Cornelia , He Liangmei , Tsai Ya-Chea , Yeatermeyer Jessica , Boyd David A. K. , Hung Chien-Fu , Wu T.-C. 
Published: July 2004

ABSTRACTHuman papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8+T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8+T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2Kb, is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8+T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.
Initial Cleavage of the Human Immunodeficiency Virus Type 1 GagPol Precursor by Its Activated Protease Occurs by an Intramolecular Mechanism
Authors: Pettit Steven C. , Everitt Lorraine E. , Choudhury Sumana , Dunn Ben M. , Kaplan Andrew H. 
Published: July 2004

ABSTRACT
Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.
Mutations in Multiple Domains Activate Paramyxovirus F Protein-Induced Fusion
Authors: Seth Shaguna , Goodman Andrew L. , Compans Richard W. 
Published: July 2004

ABSTRACT
SER virus, a paramyxovirus that is closely related to simian virus 5 (SV5), is unusual in that it fails to induce syncytium formation. The SER virus F protein has an unusually long cytoplasmic tail (CT), and it was previously observed that truncations or specific mutations of this domain result in enhanced syncytium formation. In addition to the long CT, the SER F protein has nine amino acid differences from the F protein of SV5. We previously observed only a partial suppression of fusion in a chimeric SV5 F protein with a CT derived from SER virus, indicating that these other amino acid differences between the SER and SV5 F proteins also play a role in regulating the fusion phenotype. To examine the effects of individual amino acid differences, we mutated the nine SER residues individually to the respective residues of the SV5 F protein. We found that most of the mutants were expressed well and were transported to the cell surface at levels comparable to that of the wild-type SER F protein. Many of the mutants showed enhanced lipid mixing, calcein transfer, and syncytium formation even in the presence of the long SER F protein CT. Some mutants, such as the I310 M, T438S, M489I, T516V, and N529K mutants, also showed fusion at lower temperatures of 32, 25, and 18°C. The residue Asn529 plays a critical role in the suppression of fusion activity, as the mutation of this residue to lysine caused a marked enhancement of fusion. The effect of the N529K mutation on the enhancement of fusion by a previously described mutant, L539,548A, as well as by chimeric SV5/SER F proteins was also dramatic. These results indicate that activation to a fusogenic conformation is dependent on the interplay of residues in the ectodomain, the transmembrane domain, and the CT domain of paramyxovirus F proteins.
Basal Core Promoter and Precore Mutations in the Hepatitis B Virus Genome Enhance Replication Efficacy of Lamivudine-Resistant Mutants
Authors: Tacke Frank , Gehrke Christina , Luedde Tom , Heim Albert , Manns Michael P. , Trautwein Christian 
Published: July 2004

ABSTRACT
During chronic hepatitis B virus (HBV) infection, mutations in the precore (PC) or basal core promoter (BCP) region affecting HBV e antigen (HBeAg) expression occur commonly and represent the predominant virus species in patients with HBeAg-negative chronic hepatitis B. The PC mutation (G1896A+C1858T) creates a translational stop codon resulting in absent HBeAg expression, whereas BCP mutations (A1762T/G1764A) reduce HBeAg expression by transcriptional mechanisms. Treatment of chronic HBV infection with lamivudine (LMV) often selects drug-resistant strains with single (rtM204I) or double (rtL180M+rtM204V) point mutations in the YMDD motif of HBV reverse transcriptase. We cloned replication-competent HBV vectors (genotype A, adw2) combining mutations in the core (wild type [wt], PC, and BCP) and polymerase gene (wt, rtM204I, and rtL180M/M204V) and analyzed virus replication and drug sensitivity in vitro. Resistance to LMV (rtM204I/rtL180M+rtM204V) was accompanied by a reduced replication efficacy as evidenced by reduced pregenomic RNA, encapsidated progeny DNA, polymerase activity, and virion release. PC mutations alone did not alter virus replication but restored replication efficacy of the LMV-resistant mutants without affecting drug resistance. BCP mutants had higher replication capacities than did the wt, also in combination with LMV resistance mutations. All nine HBV constructs showed similar sensitivities to adefovir. In conclusion, BCP-PC mutations directly impact the replication capacity of LMV-resistant mutants. PC mutations compensated for replication inefficiency of LMV-resistant mutants, whereas BCP mutations increased viral replication levels to above the wt baseline values, even in LMV-resistant mutants, without affecting drug sensitivity in vitro. Adefovir may be an effective treatment when combinations of core and polymerase mutations occur.
Identification of Protein Tyrosine Kinases Required for B-Cell- Receptor-Mediated Activation of an Epstein-Barr Virus Immediate-Early Gene Promoter
Authors: Lavens Sandra , Faust Emmanuel A. , Lu Fang , Jacob Michele , Leta Messele , Lieberman Paul M. , Puré Ellen 
Published: July 2004

ABSTRACTEpstein-Barr Virus (EBV) is a potentially oncogenic herpesvirus that infects >90% of the world's population. EBV exists predominantly as a latent infection in B lymphocytes, with periodic lytic-cycle reactivation essential for cellular and host transmission. Viral reactivation can be stimulated by ligand-induced activation of B-cell-receptor (BCR)-coupled signaling pathways. The critical first step in the transition from latency to the lytic cycle is the expression of the viral immediate-early gene BZLF1 through the transcription activation of its promoter, Zp. However, the BCR-coupled signal transduction cascade(s) leading to the induction of Zp and the expression of the BZLF1 gene product, Zta, is currently unclear. A major obstacle to delineating the relevant signal transduction events has been the lack of a model of EBV infection that is amenable to genetic manipulation. The use of the avian B-cell line DT40 has proven to be a powerful tool for delineating BCR-mediated signal transduction pathways that appear to be highly conserved between avian and mammalian systems. We demonstrate that the DT40 cell line is a robust and genetically tractable system for the study of BCR-mediated signaling pathways leading to transcriptional activation of BZLF1. Using this system, we demonstrate that activation of Zp requires the BCR-coupled protein tyrosine kinases Syk and Btk and that it is positively regulated by Lyn. Thus, the use of DT40 cells has allowed us to delineate the early signaling components required for BCR-dependent reactivation of latent EBV, and this system is likely to prove useful for further dissection of the downstream signaling cascades involved.
Dissecting the Requirement for Subgenomic Promoter Sequences by RNA Recombination of Brome Mosaic Virus In Vivo: Evidence for Functional Separation of Transcription and Recombination
Authors: Wierzchoslawski Rafal , Dzianott Aleksandra , Bujarski Jozef 
Published: July 2004

ABSTRACTPreviously, we and others mapped an increased homologous recombination activity within the subgenomic promoter (sgp) region in brome mosaic virus (BMV) RNA3 (A. Bruyere et al., J. Virol. 74:4214-4219, 2000; R. Wierzchoslawski et al., J. Virol. 77:6769-6776, 2003). In order to correlate sgp-mediated recombination and transcription, in the present work we used BMV RNA3 constructs that carried altered sgp repeats. We observed that the removal or extension of the poly(U) tract reduced or increased recombination, respectively. Deletion of the sgp core hairpin or its replacement by a different stem-loop structure inhibited recombination activity. Nucleotide substitutions at the +1 or +2 transcription initiation position reduced recombination. The sgp core alone supported only basal recombination activity. The sites of crossovers mapped to the poly(U) region and to the core hairpin. The observed effects on recombination did not parallel those observed for transcription. To explain how both activities operate within the sgp sequence, we propose a dual mechanism whereby recombination is primed at the poly(U) tract by the predetached nascent plus strand, whereas transcription is initiated de novo at the sgp core.
Pulmonary Collectins Modulate Strain-Specific Influenza A Virus Infection and Host Responses
Authors: Hawgood Samuel , Brown Cynthia , Edmondson Jess , Stumbaugh Amber , Allen Lennell , Goerke Jon , Clark Howard , Poulain Francis 
Published: July 2004

ABSTRACT

Collectins are secreted collagen-like lectins that bind, agglutinate, and neutralize influenza A virus (IAV) in vitro. Surfactant proteins A and D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and could have a role in the regulation of IAV infection in vivo. Previous studies have shown that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1 domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1 domain is dependent on the glycosylation of the carbohydrate recognition domain of SP-A. Here, using SP-A and SP-D gene-targeted mice on a common C57BL6 background, we report that viral replication and the host response as measured by weight loss, neutrophil influx into the lung, and local cytokine release are regulated by SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1 domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at N165. With the exception of a small difference on day 2 after infection with X-79, we did not find any significant difference in viral load in SP-A
−/−
mice with either IAV strain, although small differences in the cytokine responses to IAV were detected in SP-A
−/−
mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SP-D may play a role in protection from IAV infection.
Evidence that Stable Retroviral Transduction and Cell Survival following DNA Integration Depend on Components of the Nonhomologous End Joining Repair Pathway
Authors: Daniel René , Greger James G. , Katz Richard A. , Taganov Konstantin D. , Wu Xiaoyun , Kappes John C. , Skalka Anna Marie 
Published: July 2004

ABSTRACT

We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PK
CS
) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PK
CS
-deficient
scid
fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in
scid
cells that are complemented with a functional DNA-PK
CS
gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.
Herpes Simplex Virus 1 Induces Cytoplasmic Accumulation of TIA-1/TIAR and both Synthesis and Cytoplasmic Accumulation of Tristetraprolin, Two Cellular Proteins That Bind and Destabilize AU-Rich RNAs
Authors: Esclatine Audrey , Taddeo Brunella , Roizman Bernard 
Published: July 2004

ABSTRACTHerpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the UL41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking UL41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking UL41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.
Role of JC Virus Agnoprotein in DNA Repair
Authors: Darbinyan Armine , Siddiqui Khwaja M. , Slonina Dorota , Darbinian Nune , Amini Shohreh , White Martyn K. , Khalili Kamel 
Published: July 2004

ABSTRACTThe late region of human neurotropic JC virus encodes a small 71-amino-acid agnoprotein that is also found in the polyomaviruses simian virus 40 and BK virus. Several functions of agnoprotein have been identified, including roles in regulating viral transcription and virion maturation. Earlier studies showed that agnoprotein expressed alone induced p21/WAF-1 expression and caused cells to accumulate in the G2/M stage of the cell cycle. Here we report that agnoprotein expression sensitized cells to the cytotoxic effects of the DNA-damaging agent cisplatin. Agnoprotein reduced the viability of cisplatin-treated cells and increased chromosome fragmentation and micronucleus formation. Whereas cisplatin-treated control cells accumulated in S phase, cells expressing agnoprotein did not, instead becoming aneuploid. Agnoprotein expression correlated with impaired double-strand-break repair activity in cellular extracts and reduced expression of the Ku70 and Ku80 DNA repair proteins. After agnoprotein expression, much of the Ku70 protein was located in the perinuclear space, where agnoprotein was also found. Results from binding studies showed an interaction of agnoprotein with Ku70 which was mediated by the N terminus. The ability of agnoprotein to inhibit double-strand break repair activity when it was added to cellular extracts was also mediated by the N terminus. We conclude that agnoprotein inhibits DNA repair after DNA damage and interferes with DNA damage-induced cell cycle regulation. Since Ku70 is a subunit of the DNA-dependent protein kinase that is responsible both for double-strand break repair and for signaling damage-induced cell cycle arrest, the modulation of Ku70 and/or Ku80 by agnoprotein may represent an important event in the polyomavirus life cycle and in cell transformation.