bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Severe Acute Respiratory Syndrome Virus Replication by Small Interfering RNAs in Mammalian Cells
Authors: Wang Zhi , Ren Lili , Zhao Xingang , Hung Tao , Meng Anming , Wang Jianwei , Chen Ye-Guang 
Published: June 2004

ABSTRACT
Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.
CC Chemokine Receptor 7 Expression by Effector/Memory CD4+T Cells Depends on Antigen Specificity and Tissue Localization during Influenza A Virus Infection
Authors: Debes Gudrun F. , Bonhagen Kerstin , Wolff Thorsten , Kretschmer Ute , Krautwald Stefan , Kamradt Thomas , Hamann Alf 
Published: June 2004

ABSTRACTThe lung is an important entry site for respiratory pathogens such as influenza A virus. In order to combat such invading infectious agents, effector/memory T cells home to the lung and other peripheral tissues as well as lymphoid organs. In this process, chemokines and their receptors fulfill important roles in the guidance of T cells into such organs and specialized microenvironments within tissues. In this study, we determined if CD4+T cells residing in different lung compartments and draining lymph nodes of influenza A virus-infected and naïve mice express receptors allowing their recirculation into secondary lymphoid tissues. We found high levels ofl-selectin and CC chemokine receptor 7 (CCR7) expression in lung-derived CD4+T cells, similar to that detected on T cells in secondary lymphoid organs. Upon influenza A virus infection, the bulk of gamma interferon-positive (IFN-γ+) and IFN-γ−CD4+T cells recovered from lung parenchyma retained functional CCR7, whereas virus-specific IFN-γ-producing T cells were CCR7−. In contrast, a majority of virus-specific IFN-γ+T cells in the lung draining lymph node were CCR7+. Independent of infection, CD4+T cells obtained from the lung airways exhibited the lowest expression level ofl-selectin and CCR7, indicating that T cells at this anatomical site represent the most differentiated effector cell type, lacking the ability to recirculate. Our results suggest that effector/memory T cells that enter inflammatory sites retain functional CCR7 expression, which is lost only upon response to viral antigen and after localization to the final effector site.
Gain of Virulence Caused by Loss of a Gene in Murine Cytomegalovirus
Authors: Bubić Ivan , Wagner Markus , Krmpotić Astrid , Saulig Tanja , Kim Sungjin , Yokoyama Wayne M. , Jonjić Stipan , Koszinowski Ulrich H. 
Published: June 2004

ABSTRACTMouse strains are either resistant or susceptible to murine cytomegalovirus (MCMV). Resistance is determined by theCmv1r(Ly49h) gene, which encodes the Ly49H NK cell activation receptor. The protein encoded by them157gene of MCMV has been defined as a ligand for Ly49H. To find out whether the m157 protein is the only Ly49H ligand encoded by MCMV, we constructed them157deletion mutant and a revertant virus. Viruses were tested for susceptibility to NK cell control in Ly49H+and Ly49H−mouse strains. Deletion of them157gene abolished the viral activation of Ly49H+NK cells, resulting in higher virus virulence in vivo. Thus, in the absence ofm157, Ly49H+mice react like susceptible strains. 129/SvJ mice lack the Ly49H activation NK cell receptor but express the inhibitory Ly49I NK cell receptor that binds to the m157 protein. The Δm157inhibitory phenotype was weak because MCMV encodes a number of proteins that mediate NK inhibition, whose contribution could be shown by another mutant.
Transmitted Human Immunodeficiency Virus Type 1 Carrying the D67N or K219Q/E Mutation Evolves Rapidly to Zidovudine Resistance In Vitro and Shows a High Replicative Fitness in the Presence of Zidovudine
Authors: García-Lerma J. Gerardo , MacInnes Hamish , Bennett Diane , Weinstock Hillard , Heneine Walid 
Published: June 2004

ABSTRACT

Drug-naive patients infected with drug-resistant human immunodeficiency virus type 1 (HIV-1) who initiate antiretroviral therapy show a shorter time to virologic failure than patients infected with wild-type (WT) viruses. Resistance-related HIV genotypes not commonly seen in treated patients, which likely result from reversion or loss of primary resistance mutations, have also been recognized in drug-naive persons. Little work has been done to characterize the patterns of mutations in these viruses and the frequency of occurrence, their association with phenotypic resistance, and their effect on fitness and evolution of resistance. Through the analysis of resistance mutations in 1082 newly diagnosed antiretroviral-naive persons from the United States, we found that 35 of 48 (72.9%) persons infected with HIV-1 containing thymidine analog mutations (TAMs) had viruses that lacked a primary mutation (T215Y/F, K70R, or Q151M). Of these viruses, 9 (25.7%) had only secondary TAMs (D67N, K219Q, M41L, or F77L), and all were found to be sensitive to zidovudine (AZT) and other drugs. To assess the impact of secondary TAMs on the evolution of AZT resistance, we generated recombinant viruses from cloned plasma-derived reverse transcriptase sequences. Two viruses had D67N, three had D67N and K219Q/E, and three were WT. Four site-directed mutants with D67N, K219Q, K219E, and D67N/K219Q were also made in HIV-1
HXB2
. In vitro selection of AZT resistance showed that viruses with D67N and/or K219Q/E acquired AZT resistance mutations more rapidly than WT viruses (36 days compared to 54 days;
P
= 0.003). To investigate the factors associated with the rapid selection of AZT mutations in these viruses, we evaluated fitness differences among HXB2
WT
and HXB2
D67N
or HXB2
D67N/K219Q
in the presence of AZT. Both HXB2
D67N/K219Q
and HXB2
D67N
were more fit than HXB2
WT
in the presence of either low or high AZT concentrations, likely reflecting low-level resistance to AZT that is not detectable by phenotypic testing. In the absence of AZT, the fitness cost conferred by D67N or K219Q was modest. Our results demonstrate that viruses with unique patterns of TAMs, including D67N and/or K219Q/E, are commonly found among newly diagnosed persons and illustrate the expanding diversity of revertant viruses in this population. The modest fitness cost conferred by D67N and K219Q supports persistence of these mutants in the untreated population and highlights the potential for secondary transmission. The faster evolution of these mutants toward AZT resistance is consistent with the higher viral fitness in the presence of AZT and shows that these viruses are phenotypically different from WT HIV-1. Our study emphasizes the need for clinical studies to better define the impact of these mutants on treatment responses and evolution of resistance.
Cellular Distribution of Lysyl-tRNA Synthetase and Its Interaction with Gag during Human Immunodeficiency Virus Type 1 Assembly
Authors: Halwani Rabih , Cen Shan , Javanbakht Hassan , Saadatmand Jenan , Kim Sunghoon , Shiba Kiyotaka , Kleiman Lawrence 
Published: June 2004

ABSTRACT

Lysyl-tRNA synthetase (LysRS) is packaged into human immunodeficiency virus type 1 (HIV-1) via its interaction with Gag, and this enzyme facilitates the selective packaging of tRNA
3
Lys
, the primer for initiating reverse transcription, into HIV-1. The Gag/LysRS interaction is detected at detergent-resistant membrane but not in membrane-free cell compartments that contain Gag and LysRS. LysRS is found (i) in the nucleus, (ii) in a cytoplasmic high-molecular-weight aminoacyl-tRNA synthetase complex (HMW aaRS complex), (iii) in mitochondria, and (iv) associated with plasma membrane. The cytoplasmic form of LysRS lacking the mitochondrial import signal was previously shown to be efficiently packaged into virions, and in this report we also show that LysRS compartments in nuclei, in the HMW aaRS complex, and at the membrane are also not required as a primary source for viral LysRS. Exogenous mutant LysRS species unable to either enter the nucleus or bind to the cell membrane are still incorporated into virions. Many HMW aaRS components are not packaged into the virion along with LysRS, and the interaction of LysRS with p38, a protein that binds tightly to LysRS in the HMW aaRS complex, is not required for the incorporation of LysRS into virions. These data indicate that newly synthesized LysRS may interact rapidly with Gag before the enzyme has the opportunity to move to the above-mentioned cellular compartments. In confirmation of this idea, we found that newly synthesized LysRS is associated with Gag after a 10-min pulse with [
35
S]cysteine/methionine. This observation is also supported by previous work indicating that the incorporation of LysRS into HIV-1 is very sensitive to the inhibition of new synthesis of LysRS.
Novel Hepatitis B Virus Genotype A Subtyping Assay That Distinguishes Subtype Aa from Ae and Its Application in Epidemiological Studies
Authors: Hasegawa Izumi , Tanaka Yasuhito , Kramvis Anna , Kato Takanobu , Sugauchi Fuminaka , Acharya Subrat K. , Orito Etsuro , Ueda Ryuzo , Kew Michael C. , Mizokami Masashi 
Published: June 2004

ABSTRACTThe eight genotypes of hepatitis B virus (HBV) have different geographical distributions, virological characteristics, and clinical manifestations. A unique subtype of HBV genotype A (HBV/A) was reported in sub-Saharan Africa, raising the possibility that patients infected with this subtype (HBV/Aa [“a” for African and Asian]) may have different clinical outcomes than other HBV/A isolates (HBV/Ae [“e” for European]). Comparison between 30 HBV/Aa and 30 HBV/Ae isolates indicated that almost all HBV/Ae isolates had G at nucleotide (nt) 1809 and C at nt 1812, whereas HBV/Aa isolates had T1809/T1812. Taking advantage of these two single nucleotide polymorphisms (SNPs), a novel subtype-specific PCR assay in the X/precore/core region was developed. This assay was combined with a restriction fragment length polymorphism assay using BglII in a different region (nt 1984 to 1989), which has a SNP distinguishing HBV/Aa from HBV/Ae, resulting in 100% specificity for the combined assay. Application of the subtyping assay using sera from 109 paid donors in the United States indicated significantly different distributions of HBV/A subtypes among races; African-Americans, Caucasians, and Hispanics had HBV/Ae, whereas Asians had mainly HBV/Aa, suggesting that the HBV/Aa isolates may have been imported by recent immigration from Asia. In conclusion, the specificity and sensitivity of the combined subtyping assay were confirmed, and its usefulness was demonstrated in a practical context.
Determinants of Human Immunodeficiency Virus Type 1 Baseline Susceptibility to the Fusion Inhibitors Enfuvirtide and T-649 Reside outside the Peptide Interaction Site
Authors: Heil Marintha L. , Decker Julie M. , Sfakianos Jeffrey N. , Shaw George M. , Hunter Eric , Derdeyn Cynthia A. 
Published: June 2004

ABSTRACT

The peptide fusion inhibitor (PFI) enfuvirtide is the first of a new class of entry inhibitors to receive FDA approval. We previously determined the susceptibility of 55 PFI-naïve-patient isolates to enfuvirtide and a second peptide inhibitor, T-649. Seven of the 55 viral isolates were insusceptible to enfuvirtide, T-649, or both inhibitors in the absence of prior exposure. To determine the molecular basis of the insusceptible phenotypes, we PCR amplified and cloned five PFI-insusceptible and one PFI-susceptible, full-length, biologically functional
env
genes and characterized viruses pseudotyped with the Env proteins in a single-round drug sensitivity assay. Overall, the mean 50% inhibitory concentrations of enfuvirtide and T-649 for the PFI-insusceptible Env pseudotypes were 1.4 to 1.7 log
10
and 1.2 to 1.8 log
10
greater, respectively, than those for a PFI-susceptible lab strain, NLHX; however, all of the PFI-insusceptible Env proteins conserved the sequence of a critical enfuvirtide interaction site (residues 36 to 38 of gp41, GIV) in HR-1. In contrast, multiple amino acid changes were observed C-terminal to HR-1, many of which were located in regions of HR-2 corresponding to the PFI. Nevertheless, peptides based on patient-derived HR-2 sequences were not more potent inhibitors than enfuvirtide or T-649, arguing that the basis of PFI susceptibility is not a higher-affinity, competitive HR-1/HR-2 interaction. These results demonstrate that regions of Env outside the enfuvirtide interaction site can significantly impact the PFI susceptibility of patient-derived Env, even prior to drug exposure. We hypothesize that both gp120 gene- and gp41 gene-encoded determinants that minimize the window of opportunity for PFI to bind provide a growth advantage and possibly a predisposition to resistance to this new class of drugs in vivo.
Identification and Characterization of
cis
-Acting Elements Residing in the Walleye Dermal Sarcoma Virus Promoter
Authors: Hronek Brett W. , Meagher Ashley , Rovnak Joel , Quackenbush Sandra L. 
Published: June 2004

ABSTRACT
Walleye dermal sarcoma virus (WDSV) is a complex retrovirus found associated with tumors that appear and regress on a seasonal basis. There are quantitative and qualitative differences in the amount of virus expression between developing and regressing tumors. To understand the role of host cell factors in WDSV expression, DNase I footprint analysis, electrophoretic mobility shift assays (EMSA), and reporter gene assays were employed. DNase I footprint analysis of the U3 region of the WDSV long terminal repeat with nuclear extract prepared from a walleye cell line revealed protection of an Oct1, AP1, Whn, and two E4BP4 sites. Additionally, three regions that contained no putative transcription factor binding sites were protected. EMSA confirmed the specific binding of the protected sites and revealed three additional sites, NF1, AP3, and LVa, not protected in DNase I footprint analysis. Site-directed mutagenesis of the individual sites, in the context of a luciferase reporter plasmid, revealed that the NF1, Oct1, AP1, E4BP4#2, AP3, and LVa sites contributed to transcription activation driven by the WDSV U3 region. Mutation of Novel#2 resulted in an increase in luciferase activity, suggesting the Novel#2 site may function to bind a negative regulator of transcription. Anti-Jun and anti-Fos antiserum specifically inhibited protein-DNA complex formation, indicating the presence of c-Jun and c-Fos in the walleye cell nuclear extracts and their participation in binding to the AP1 site. Interestingly, degenerative 15-bp repeats found in the U3 region are differentially protected in DNase I footprint analysis by the walleye cell line nuclear extract and regressing-tumor nuclear extract. EMSA utilizing the 15-bp repeat probe revealed that there are similarities of binding with W12 cell and developing-tumor nuclear extracts and that the binding differs from that observed with regressing-tumor nuclear extract.
PSC-RANTES Blocks R5 Human Immunodeficiency Virus Infection of Langerhans Cells Isolated from Individuals with a Variety ofCCR5Diplotypes
Authors: Kawamura Tatsuyoshi , Bruce Shannon E. , Abraha Awet , Sugaya Makoto , Hartley Oliver , Offord Robin E. , Arts Eric J. , Zimmerman Peter A. , Blauvelt Andrew 
Published: June 2004

ABSTRACTTopical microbicides that effectively block interactions between CCR5+immature Langerhans cells (LC) residing within genital epithelia and R5 human immunodeficiency virus (HIV) may decrease sexual transmission of HIV. Here, we investigated the ability of synthetic RANTES analogues (AOP-, NNY-, and PSC-RANTES) to block R5 HIV infection of human immature LC by using a skin explant model. In initial experiments using activated peripheral blood mononuclear cells, each analogue compound demonstrated marked antiviral activity against two R5 HIV isolates. Next, we found that 20-min preincubation of skin explants with each RANTES analogue blocked R5 HIV infection of LC in a dose-dependent manner (1 to 100 nM) and that PSC-RANTES was the most potent of these compounds. Similarly, preincubation of LC with each analogue was able to block LC-mediated infection of cocultured CD4+T cells. Competition experiments between primary R5 and X4 HIV isolates showed blocking of R5 HIV by PSC-RANTES and no evidence of increased propagation of X4 HIV, data that are consistent with the specificity of PSC-RANTES for CCR5 and the CCR5+CXCR4−phenotype of immature LC. Finally, whenCCR5genetic polymorphism data were integrated with results from the in vitro LC infection studies, PSC-RANTES was found to be equally effective in inhibiting R5 HIV in LC isolated from individuals withCCR5diplotypes known to be associated with low, intermediate, and high cell surface levels of CCR5. In summary, PSC-RANTES is a potent inhibitor of R5 HIV infection in immature LC, suggesting that it may be useful as a topical microbicide to block sexual transmission of HIV.
Immunostimulant Patch Enhances Immune Responses to Influenza Virus Vaccine in Aged Mice
Authors: Guebre-Xabier Mimi , Hammond Scott A. , Ellingsworth Larry R. , Glenn Gregory M. 
Published: June 2004

ABSTRACTImprovement in the immune response to influenza virus vaccination in the elderly represents the primary unmet need in influenza virus vaccination. We have shown that topical application of immunostimulating (IS) patches containing heat-labile enterotoxin ofEscherichia coli(LT) enhances immune responses to injected vaccines. We extend these findings and show that LT-IS patch application enhances the antibody responses to influenza virus vaccination in both young and aged mice. LT-IS patches markedly increased influenza virus-specific immunoglobulin G (IgG), hemagglutination inhibition antibody, mucosal antibody, and T-cell responses. The magnitude of the immune responses in aged mice receiving an LT-IS patch was equivalent to or greater than that of the immune responses in young mice given vaccine alone. These results suggest that addition of an LT-IS patch may compensate for the deficient immune function seen in the aged in response to influenza virus vaccination. Therefore, use of an LT-IS patch could be a new, safe, and simple immunization strategy that may significantly improve the outcome of influenza virus vaccination in the elderly.
Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon
Authors: Zhang Guohua , Zhang Jiuchun , Simon Anne E. 
Published: June 2004

ABSTRACT

Plus-strand viral RNAs contain sequences and structural elements that allow cognate RNA-dependent RNA polymerases (RdRp) to correctly initiate and transcribe asymmetric levels of plus and minus strands during RNA replication.
cis-
acting sequences involved in minus-strand synthesis, including promoters, enhancers, and, recently, transcriptional repressors (J. Pogany, M. R. Fabian, K. A. White, and P. D. Nagy, EMBO J.
22:
5602-5611, 2003), have been identified for many viruses. A second example of a transcriptional repressor has been discovered in satC, a replicon associated with turnip crinkle virus. satC hairpin 5 (H5), located proximal to the core hairpin promoter, contains a large symmetrical internal loop (LSL) with sequence complementary to 3′-terminal bases. Deletion of satC 3′-terminal bases or alteration of the putative interacting bases enhanced transcription in vitro, while compensatory exchanges between the LSL and 3′ end restored near-normal transcription. Solution structure analysis indicated that substantial alteration of the satC H5 region occurs when the three 3′-terminal cytidylates are deleted. These results indicate that H5 functions to suppress synthesis of minus strands by sequestering the 3′ terminus from the RdRp. Alteration of a second sequence strongly repressed transcription in vitro and accumulation in vivo, suggesting that this sequence may function as a derepressor to free the 3′ end from interaction with H5. Hairpins with similar sequence and/or structural features that contain sequence complementary to 3′-terminal bases, as well as sequences that could function as derepressors, are located in similar regions in other carmoviruses, suggesting a general mechanism for controlling minus-strand synthesis in the genus.
Human Immunodeficiency Virus Type 1 Evades T-Helper Responses by Exploiting Antibodies That Suppress Antigen Processing
Authors: Chien Peter C. , Cohen Sandra , Tuen Michael , Arthos James , Chen Pei-de , Patel Sukeshi , Hioe Catarina E. 
Published: June 2004

ABSTRACTT-helper responses are important for controlling chronic viral infections, yet T-helper responses specific to human immunodeficiency virus type 1 (HIV-1), particularly to envelope glycoproteins, are lacking in the vast majority of HIV-infected individuals. It was previously shown that the presence of antibodies to the CD4-binding domain (CD4bd) of HIV-1 glycoprotein 120 (gp120) prevents T-helper responses to gp120, but their suppressive mechanisms were undefined (C. E. Hioe et al., J. Virol. 75:10950-10957, 2001). The present study demonstrates that gp120, when complexed to anti-CD4bd antibodies, becomes more resistant to proteolysis by lysosomal enzymes from antigen-presenting cells such that peptide epitopes are not released and presented efficiently by major histocompatibility complex class II molecules to gp120-specific CD4 T cells. Antibodies to other gp120 regions do not confer this effect. Thus, HIV may evade anti-viral T-helper responses by inducing and exploiting antibodies that conceal the virus envelope antigens from T cells.
Replication of Herpes Simplex Virus 1 Depends on the γ
1
34.5 Functions That Facilitate Virus Response to Interferon and Egress in the Different Stages of Productive Infection
Authors: Jing Xianghong , Cerveny Melissa , Yang Kui , He Bin 
Published: June 2004

ABSTRACT

The ability of the γ
1
34.5 protein to suppress the PKR response plays a crucial role in herpes simplex virus pathogenesis. In this process, the γ
1
34.5 protein associates with protein phosphatase 1 to form a large complex that dephosphorylates eIF-2α and thereby prevents translation shutoff mediated by PKR. Accordingly, γ
1
34.5 null mutants are virulent in PKR-knockout mice but not in wild-type mice. However, γ
1
34.5 deletion mutants, with an extragenic compensatory mutation, inhibit PKR activity but remain avirulent, suggesting that the γ
1
34.5 protein has additional functions. Here, we show that a substitution of the γ
1
34.5 gene with the NS1 gene from influenza A virus renders viral resistance to interferon involving PKR. The virus replicates as efficiently as wild-type virus in SK-N-SH and CV-1 cells. However, in mouse 3T6 cells, the virus expressing the NS1 protein grows at an intermediate level between the wild-type virus and the γ
1
34.5 deletion mutant. This decrease in growth, compared to that of the wild-type virus, is due not to an inhibition of viral protein synthesis but rather to a block in virus release or egress. Virus particles are predominantly present in the nucleus and cytoplasm. Notably, deletions in the amino terminus of the γ
1
34.5 protein lead to a significant decrease in virus growth in mouse 3T6 cells, which is independent of eIF-2α dephosphorylation. In correlation, a series of deletions in the amino-terminal domain impair nuclear as well as cytoplasmic egress. These results indicate that efficient viral replication depends on the γ
1
34.5 functions required to prevent the PKR response and to facilitate virus egress in the different stages during virus infection.