bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Amino Acid Residues of Sendai Virus C Protein That Are Critically Involved in Its Interferon Antagonism and RNA Synthesis Down-Regulation
Authors: Kato Atsushi , Cortese-Grogan Case , Moyer Sue A. , Sugahara Fumihiro , Sakaguchi Takemasa , Kubota Toru , Otsuki Noriyuki , Kohase Masayoshi , Tashiro Masato , Nagai Yoshiyuki
Published: June 2004

Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or translationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C′, C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions (A. Kato et al., J. Virol.
75:
3802-3810, 2001, and A. Kato et al., J. Virol.
76:
7114-7124, 2002). Here, we further generated HeLa cell lines expressing the mutated C (Cm) proteins with charged amino acids substituted for alanine residues at either positions 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; or 173, 175, and 176. We found that only the mutations at positions 151, 153, and 154 abolished IFN antagonism. All the Cm proteins lost the ability to bind with STAT1 under our assay conditions, regardless of their ability to inhibit IFN signaling. On the other hand, the Cm proteins that altered the tyrosine phosphorylation and dephosphorylation of STAT1 and STAT2 always retained IFN antagonism. Thus, the abnormality of phosphorylation or dephosphorylation appeared to be a cause of the IFN antagonism by SeV C. Regarding viral RNA synthesis inhibition, all mutants but the mutant with replacements at positions 114 and 115 greatly reduced the inhibitory activity, indicating that anti-RNA synthesis by the C protein is governed by amino acids scattered across its C-terminal half. Thus, amino acid sequence requirements differ greatly between IFN antagonism and RNA synthesis inhibition. In addition, we confirmed that another SeV accessory protein, V, does not antagonize IFN.
Short Interfering RNA Accumulation Correlates with Host Recovery in DNA Virus-Infected Hosts, and Gene Silencing Targets Specific Viral Sequences
Authors: Chellappan Padmanabhan , Vanitharani Ramachandran , Fauquet Claude M.
Published: June 2004

Viruses are both inducers and targets of posttranscriptional gene silencing (PTGS), a natural defense mechanism in plants. Here we report molecular evidence of the ability of single-stranded DNA (ssDNA) viruses to induce PTGS in infected plants irrespective of the severity of or recovery from the symptoms. Our results reveal that five distinct species of cassava-infecting geminiviruses were capable of triggering PTGS by producing two classes of virus-specific short interfering RNAs (siRNAs) of 21 to 26 nucleotides in two plant hosts, tobacco (
Nicotiana benthamiana
) and cassava (
Manihot esculenta
, Crantz). However, the efficacy of virus-induced PTGS varied depending on the intrinsic features of the virus and its interaction with the plant host. We found that symptom recovery over time in plants infected with the isolates of
African cassava mosaic virus
(ACMV-[CM]) or
Sri Lankan cassava mosaic virus
was associated with a much higher level of virus-derived siRNA accumulation compared to plants infected with viruses that do not show symptom recovery. Furthermore, we determined that the C terminus of
AC1
that overlaps with the N terminus of
AC2
early viral genes involved in virus replication were the primary targets for ACMV-[CM]-induced PTGS, whereas the C terminus of BC1 was targeted for the
East African cassava mosaic Cameroon virus
. In addition, our results reveal the possibility for double-stranded RNA formation during transcription in ssDNA viruses, which explains in part how these viruses can trigger PTGS in plants.
Gene Profiling of Cottontail Rabbit Papillomavirus-Induced Carcinomas Identifies Upregulated Genes Directly Involved in Stroma Invasion as Shown by Small Interfering RNA-Mediated Gene Silencing
Authors: Huber Evamaria , Vlasny Daniela , Jeckel Sonja , Stubenrauch Frank , Iftner Thomas
Published: June 2004

To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 ζ and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin β, which are involved in cell motility and tissue invasion, as well as casein kinase 1 α, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin β in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.
Heterologous Envelope Immunogens Contribute to AIDS Vaccine Protection in Rhesus Monkeys
Authors: Letvin Norman L. , Huang Yue , Chakrabarti Bimal K. , Xu Ling , Seaman Michael S. , Beaudry Kristin , Korioth-Schmitz Birgit , Yu Faye , Rohne Daniela , Martin Kristi L. , Miura Ayako , Kong Wing-Pui , Yang Zhi-Yong , Gelman Rebecca S. , Golubeva Olga G. , Montefiori David C. , Mascola John R. , Nabel Gary J.
Published: June 2004

Because a strategy to elicit broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies has not yet been found, the role of an Env immunogen in HIV-1 vaccine candidates remains undefined. We sought to determine whether an HIV-1 Env immunogen genetically disparate from the Env of the challenge virus can contribute to protective immunity. We vaccinated Indian-origin rhesus monkeys with Gag-Pol-Nef immunogens, alone or in combination with Env immunogens that were either matched or mismatched with the challenge virus. These animals were then challenged with a pathogenic simian-human immunodeficiency virus. The vaccine regimen included a plasmid DNA prime and replication-defective adenoviral vector boost. Vaccine regimens that included the matched or mismatched Env immunogens conferred better protection against CD4
+
T-lymphocyte loss than that seen with comparable regimens that did not include Env immunogens. This increment in protective immunity was associated with anamnestic Env-specific cellular immunity that developed in the early days following viral challenge. These data suggest that T-lymphocyte immunity to Env can broaden the protective cellular immune response to HIV despite significant sequence diversity of the strains of the Env immunogens and can contribute to immune protection in this AIDS vaccine model.
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

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

The 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

Mouse 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

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

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

The 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

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

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

Topical 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

Improvement 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

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

T-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

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.
Human Herpesvirus 8 K14 Protein Mimics CD200 in Down-Regulating Macrophage Activation through CD200 Receptor
Authors: Foster-Cuevas Mildred , Wright Gavin J. , Puklavec Michael J. , Brown Marion H. , Barclay A. Neil
Published: June 2004

Many viral proteins limit host immune defenses, and their genes often originate from their hosts. CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) that is implicated in locally preventing macrophage activation. Distant, but recognizable, homologues of CD200 have been identified in many herpesviruses and poxviruses. Here, we show that the product of the K14 open reading frame from human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) interacts with human CD200R and is expressed at the surfaces of infected cells solely during the lytic cycle. Despite sharing only 40% primary sequence identity, K14 and CD200 interacted with CD200R with an almost identical and low affinity (KD= 0.5 μM), in contrast to other characterized viral homologue interactions. Cells expressing CD200 or K14 on the cell surface were able to inhibit secretion by activated macrophages of proinflammatory cytokines such as tumor necrosis factor alpha, an effect that could be specifically relieved by addition of monoclonal antibodies and soluble monomeric CD200 protein. We conclude that CD200 delivers local down-modulatory signals to myeloid cells through direct cell-cell contact and that the K14 viral homologue closely mimics this.
E1B-55-Kilodalton Protein Is Not Required To Block p53-Induced Transcription during Adenovirus Infection
Authors: Hobom Urs , Dobbelstein Matthias
Published: June 2004

The adenovirus E1B-55-kDa protein binds and inactivates the tumor suppressor protein p53. However, the role of this interaction during infection is still poorly understood and was therefore examined here. Infection with a virus carrying the E1B-55-kDa mutation R239A, preventing the interaction with p53, led to the accumulation of p53. However, p53 target genes were not activated in the infected cells, although p53 phosphorylation did occur and the p53 antagonists Mdm2 and ΔNp73 did not accumulate. Deletion of E4orf6, alone or in combination with E1B-55-kDa, did not allow the induction of p53-responsive genes either. In transient reporter assays, the viral E1A-13S protein antagonized p53 activity; mutational analysis suggested that this depends partially on p300 binding, but it depends even more strongly on the interaction of E1A with the p400/TRRAP protein complex. However, viruses expressing E1A mutants lacking these binding activities, in combination with E1B-55-kDa R239A, still abolished p53 activity. In contrast, when the mutation of E1B-55-kDa at R239A was combined with a deletion of the apoptosis inhibitor E1B-19-kDa, infected cells showed more extensive apoptosis than after infection with single mutants, suggesting that accumulated p53, albeit transcriptionally inactive, might nonetheless enhance apoptosis. Despite extensive apoptosis of the infected cells, the deletion of E1B-19-kDa, in combination with the E1B-55-kDa mutation or in the presence of the constitutively active p53 mutant p53mt24-28, reduced virus replication less than fivefold. In conclusion, adenovirus does not need direct binding of E1B-55-kDa to inactivate p53, and forced p53 activity with consecutive apoptosis does not severely impair virus replication.
Structural Basis for Competitive Inhibition of eIF4G-Mnk1 Interaction by the Adenovirus 100-Kilodalton Protein
Authors: Cuesta Rafael , Xi Qiaoran , Schneider Robert J.
Published: June 2004

Translation of most cellular mRNAs involves cap binding by the translation initiation complex. Among this complex of proteins are cap-binding protein eIF4E and the eIF4E kinase Mnk1. Cap-dependent mRNA translation generally correlates with Mnk1 phosphorylation of eIF4E when both are bound to eIF4G. During the late phase of adenovirus (Ad) infection translation of cellular mRNA is inhibited, which correlates with displacement of Mnk1 from eIF4G by the viral 100-kDa (100K) protein and dephosphorylation of eIF4E. Here we describe the molecular mechanism for 100K protein displacement of Mnk1 from eIF4G and elucidate a structural basis for eIF4G interaction with Mnk1 and 100K proteins and Ad inhibition of cellular protein synthesis. The eIF4G-binding site is located in an N-terminal 66-amino-acid peptide of 100K which is sufficient to bind eIF4G, displace Mnk1, block eIF4E phosphorylation, and inhibit eIF4F (cap)-dependent cellular mRNA translation. Ad 100K and Mnk1 proteins possess a common eIF4G-binding motif, but 100K protein binds more strongly to eIF4G than does Mnk1. Unlike Mnk1, for which binding to eIF4G is RNA dependent, competitive binding by 100K protein is RNA independent. These data support a model whereby 100K protein blocks cellular protein synthesis by coopting eIF4G and cap-initiation complexes regardless of their association with mRNA and displacing or blocking binding by Mnk1, which occurs only on preassembled complexes, resulting in dephosphorylation of eIF4E.
Human Cytomegalovirus Carries a Cell-Derived Phospholipase A2 Required for Infectivity
Authors: Allal Cuider , Buisson-Brenac Claire , Marion Vincent , Claudel-Renard Clotilde , Faraut Thomas , Dal Monte Paola , Streblow Daniel , Record Michel , Davignon Jean-Luc
Published: June 2004

Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2α, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity.
Crystal Structure of Species D Adenovirus Fiber Knobs and Their Sialic Acid Binding Sites
Authors: Burmeister Wim P. , Guilligay Delphine , Cusack Stephen , Wadell Göran , Arnberg Niklas
Published: June 2004

Adenovirus serotype 37 (Ad37) belongs to species D and can cause epidemic keratoconjunctivitis, whereas the closely related Ad19p does not. Primary cell attachment by adenoviruses is mediated through receptor binding of the knob domain of the fiber protein. The knobs of Ad37 and Ad19p differ at only two positions, Lys240Glu and Asn340Asp. We report the high-resolution crystal structures of the Ad37 and Ad19p knobs, both native and in complex with sialic acid, which has been proposed as a receptor for Ad37. Overall, the Ad37 and Ad19p knobs are very similar to previously reported knob structures, especially to that of Ad5, which binds the coxsackievirus-adenovirus receptor (CAR). Ad37 and Ad19p knobs are structurally identical with the exception of the changed side chains and are structurally most similar to CAR-binding knobs (e.g., that of Ad5) rather than non-CAR-binding knobs (e.g., that of Ad3). The two mutations in Ad19p result in a partial loss of the exceptionally high positive surface charge of the Ad37 knob but do not affect sialic acid binding. This site is located on the top of the trimer and binds both α(2,3) and α(2,6)-linked sialyl-lactose, although only the sialic acid residue makes direct contact. Amino acid alignment suggests that the sialic acid binding site is conserved in several species D serotypes. Our results show that the altered viral tropism and cell binding of Ad19p relative to those of Ad37 are not explained by a different binding ability toward sialyl-lactose.
The Host Response to West Nile Virus Infection Limits Viral Spread through the Activation of the Interferon Regulatory Factor 3 Pathway
Authors: Fredericksen Brenda L. , Smith Maria , Katze Michael G. , Shi Pei-Yong , Gale Michael
Published: June 2004

Recent outbreaks of West Nile Virus (WNV) have been associated with an increase in morbidity and mortality in humans, birds, and many other species. We have initiated studies to define the molecular mechanisms by which a recent pathogenic isolate of WNV evades the host cell innate antiviral response. Biochemical and microarray analyses demonstrated that WNV induced the expression of beta interferon (IFN-β) and several IFN-stimulated genes late in infection of cultured human cells. The late expression of these antiviral genes was due to the delayed activation of the transcription factor IFN regulatory factor 3 (IRF-3). Despite this host response, WNV was still able to replicate efficiently. The effect of the IRF-3 pathway on WNV replication was assessed by examining virus replication and spread in cultures of wild-type or IRF-3-null mouse embryo fibroblasts. The absence of IRF-3 was marked by a significant increase in plaque size and a sustained production of infectious particles. Although the activation of the IRF-3 pathway was not sufficient to block virus replication, our results suggest that IRF-3 target genes function to constrain WNV infection and limit cell-to-cell virus spread.
New Simian Immunodeficiency Virus Infecting De Brazza's Monkeys (
Cercopithecus neglectus
): Evidence for a
Cercopithecus
Monkey Virus Clade
Authors: Bibollet-Ruche Frederic , Bailes Elizabeth , Gao Feng , Pourrut Xavier , Barlow Katrina L. , Clewley Jonathan P. , Mwenda Jason M. , Langat Daudi K. , Chege Gerald K. , McClure Harold M. , Mpoudi-Ngole Eitel , Delaporte Eric , Peeters Martine , Shaw George M. , Sharp Paul M. , Hahn Beatrice H.
Published: June 2004

Nearly complete sequences of simian immunodeficiency viruses (SIVs) infecting 18 different nonhuman primate species in sub-Saharan Africa have now been reported; yet, our understanding of the origins, evolutionary history, and geographic distribution of these viruses still remains fragmentary. Here, we report the molecular characterization of a lentivirus (SIVdeb) naturally infecting De Brazza's monkeys (
Cercopithecus neglectus
). Complete SIVdeb genomes (9,158 and 9,227 bp in length) were amplified from uncultured blood mononuclear cell DNA of two wild-caught De Brazza's monkeys from Cameroon. In addition, partial
pol
sequences (650 bp) were amplified from four offspring of De Brazza's monkeys originally caught in the wild in Uganda. Full-length (9,068 bp) and partial
pol
(650 bp) SIVsyk sequences were also amplified from Sykes's monkeys (
Cercopithecus albogularis
) from Kenya. Analysis of these sequences identified a new SIV clade (SIVdeb), which differed from previously characterized SIVs at 40 to 50% of sites in Pol protein sequences. The viruses most closely related to SIVdeb were SIVsyk and members of the SIVgsn/SIVmus/SIVmon group of viruses infecting greater spot-nosed monkeys (
Cercopithecus nictitans
), mustached monkeys (
Cercopithecus cephus
), and mona monkeys (
Cercopithecus mona
), respectively. In phylogenetic trees of concatenated protein sequences, SIVdeb, SIVsyk, and SIVgsn/SIVmus/SIVmon clustered together, and this relationship was highly significant in all major coding regions. Members of this virus group also shared the same number of cysteine residues in their extracellular envelope glycoprotein and a high-affinity AIP1 binding site (YPD/SL) in their p6 Gag protein, as well as a unique transactivation response element in their viral long terminal repeat; however, SIVdeb and SIVsyk, unlike SIVgsn, SIVmon, and SIVmus, did not encode a
vpu
gene. These data indicate that De Brazza's monkeys are naturally infected with SIVdeb, that this infection is prevalent in different areas of the species' habitat, and that geographically diverse SIVdeb strains cluster in a single virus group. The consistent clustering of SIVdeb with SIVsyk and the SIVmon/SIVmus/SIVgsn group also suggests that these viruses have evolved from a common ancestor that likely infected a
Cercopithecus
host in the distant past. The
vpu
gene appears to have been acquired by a subset of these
Cercopithecus
viruses after the divergence of SIVdeb and SIVsyk.
Parvovirus B19-Induced Apoptosis of Hepatocytes
Authors: Poole Brian D. , Karetnyi Yuory V. , Naides Stanley J.
Published: June 2004

Parvovirus B19 (B19 virus) can persist in multiple tissues and has been implicated in a variety of diseases, including acute fulminant liver failure. The mechanism by which B19 virus induces liver failure remains unknown. Hepatocytes are nonpermissive for B19 virus replication. We previously reported that acute fulminant liver failure associated with B19 virus infection was characterized by hepatocellular dropout. We inoculated both primary hepatocytes and the hepatocellular carcinoma cell line Hep G2 with B19 virus and assayed for apoptosis by using annexin V staining. Reverse transcriptase PCR analysis and immunofluorescence demonstrated that B19 virus was able to infect the cells and produce its nonstructural protein but little or no structural capsid protein. Infection with B19 virus induced means of 28% of Hep G2 cells and 10% of primary hepatocytes to undergo apoptosis, which were four- and threefold increases, respectively, over background levels. Analysis of caspase involvement showed that B19 virus-inoculated cultures had a significant increase in the number of cells with active caspase 3. Inhibition studies demonstrated that caspases 3 and 9, but not caspase 8, are required for B19 virus-induced apoptosis.
Rotavirus Serotype G9 Strains Belonging to VP7 Gene Phylogenetic Sequence Lineage 1 May Be More Suitable for Serotype G9 Vaccine Candidates than Those Belonging to Lineage 2 or 3
Authors: Hoshino Yasutaka , Jones Ronald W. , Ross Jerri , Honma Shinjiro , Santos Norma , Gentsch Jon R. , Kapikian Albert Z.
Published: June 2004

A safe and effective group A rotavirus vaccine that could prevent severe diarrhea or ameliorate its symptoms in infants and young children is urgently needed in both developing and developed countries. Rotavirus VP7 serotypes G1, G2, G3, and G4 have been well established to be of epidemiologic importance worldwide. Recently, serotype G9 has emerged as the fifth globally common type of rotavirus of clinical importance. Sequence analysis of the VP7 gene of various G9 isolates has demonstrated the existence of at least three phylogenetic lineages. The goal of our study was to determine the relationship of the phylogenetic lineages to the neutralization specificity of various G9 strains. We generated eight single VP7 gene substitution reassortants, each of which bore a single VP7 gene encoding G9 specificity of one of the eight G9 strains (two lineage 1, one lineage 2 and five lineage 3 strains) and the remaining 10 genes of bovine rotavirus strain UK, and two hyperimmune guinea pig antisera to each reassortant, and we then analyzed VP7 neutralization characteristics of the eight G9 strains as well as an additional G9 strain belonging to lineage 1; the nine strains were isolated in five countries. Antisera to lineage 1 viruses neutralized lineage 2 and 3 strains to at least within eightfold of the homotypic lineage viruses. Antisera to lineage 2 virus neutralized lineage 3 viruses to at least twofold of the homotypic lineage 2 virus; however, neutralization of lineage 1 viruses was fourfold (F45 and AU32) to 16- to 64-fold (WI61) less efficient. Antisera to lineage 3 viruses neutralized the lineage 2 strain 16- to 64-fold less efficiently, the lineage 1 strains F45 and AU32 8- to 128-fold less efficiently, and WI61 (prototype G9 strain) 128- to 1,024-fold less efficiently than the homotypic lineage 3 viruses. These findings may have important implications for the development of G9 rotavirus vaccine candidates, as the strain with the broadest reactivity (i.e., a prime strain) would certainly be the ideal strain for inclusion in a vaccine.