bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three-Dimensional Localization of the Smallest Capsid Protein in the Human Cytomegalovirus Capsid
Authors: Yu Xuekui , Shah Sanket , Atanasov Ivo , Lo Pierrette , Liu Fenyong , Britt William J. , Zhou Z. Hong 
Published: December 2004

ABSTRACT
The smallest capsid proteins (SCPs) of the human herpesviruses differ substantially in size and sequence and are thought to impart some unique aspects of infection to their respective viruses. We used electron cryomicroscopy and antibody labeling to show that the 8-kDa SCP of human cytomegalovirus is attached only to major capsid protein subunits of the hexons, not the pentons. Thus, the SCPs of different herpesviruses illustrate that a protein can evolve significantly in sequence, structure, and function, while preserving its role in the architecture of the virus by binding to a specific partner in a specific oligomeric state.
Virus-Like Particles as Carriers for T-Cell Epitopes: Limited Inhibition of T-Cell Priming by Carrier-Specific Antibodies
Authors: Ruedl Christiane , Schwarz Katrin , Jegerlehner Andrea , Storni Tazio , Manolova Vania , Bachmann Martin F. 
Published: December 2004

ABSTRACTVirus-like particles (VLPs) are able to induce cytotoxic T-cell responses in the absence of infection or replication. This makes VLPs promising candidates for the development of recombinant vaccines. However, VLPs are also potent inducers of B-cell responses, and it is generally assumed that such VLP-specific antibodies interfere with the induction of protective immune responses, a phenomenon summarized as carrier suppression. In this study, we investigated the impact of preexisting VLP-specific antibodies on the induction of specific cytotoxic T-cell and Th-cell responses in mice. The data show that VLP-specific antibodies did not measurably reduce antigen presentation in vitro or in vivo. Nevertheless, T-cell priming was slightly reduced by antigen-specific antibodies; however, the overall reduction was limited and vaccination with VLPs in the presence of VLP-specific antibodies still resulted in protective T-cell responses. Thus, carrier suppression is unlikely to be a limiting factor for VLP-based T-cell vaccines.
Integrating Reptilian Herpesviruses into the Family
Herpesviridae
Authors: McGeoch Duncan J. , Gatherer Derek 
Published: December 2004

ABSTRACT

The phylogeny of reptilian herpesviruses (HVs) relative to mammalian and avian HVs was investigated by using available gene sequences and by alignment of encoded amino acid sequences and derivation of trees by maximum-likelihood and Bayesian methods. Phylogenetic loci were obtained for green turtle HV (GTHV) primarily on the basis of DNA polymerase (POL) and DNA binding protein sequences, and for lung-eye-trachea disease-associated HV (LETV) primarily from its glycoprotein B sequence; both have nodes on the branch leading to recognized species in the
Alphaherpesvirinae
subfamily and should be regarded as new members of that subfamily. A similar but less well defined locus was obtained for an iguanid HV based on a partial POL sequence. On the basis of short POL sequences (around 60 amino acid residues), it appeared likely that GTHV and LETV belong to a private clade and that three HVs of gerrhosaurs (plated lizards) are associated with the iguanid HV. Based on phylogenetic branching patterns for mammalian HV lineages that mirror those of host lineages, we estimated a date for the HV tree's root of around 400 million years ago. Estimated dates for branching events in the development of reptilian, avian, and mammalian
Alphaherpesvirinae
lineages could plausibly be accounted for in part but not completely by ancient coevolution of these virus lines with reptilian lineages and with the development of birds and mammals from reptilian progenitors.
Involvement of Influenza Virus PA Subunit in Assembly of Functional RNA Polymerase Complexes
Authors: Kawaguchi Atsushi , Naito Tadasuke , Nagata Kyosuke 
Published: December 2004

ABSTRACT

The RNA-dependent RNA polymerase of influenza virus consists of three subunits, PB1, PB2, and PA, and synthesizes three kinds of viral RNAs, vRNA, cRNA, and mRNA. PB1 is a catalytic subunit; PB2 recognizes the cap structure for generation of the primer for transcription; and PA is thought to be involved in viral RNA replication. However, the process of polymerase complex assembly and the exact nature of polymerase complexes involved in synthesis of the three different RNA species are not yet clear.
ts
53 virus is a temperature-sensitive (
ts
) mutant derived from A/WSN/33 (A. Sugiura, M. Ueda, K. Tobita, and C. Enomoto, Virology
65:
363-373, 1975). We confirmed that the mRNA synthesis level of
ts
53 remains unaffected at the nonpermissive temperature, whereas vRNA synthesis is largely reduced. Sequencing of the gene encoding
ts
53 PA and recombinant virus rescue experiments revealed that an amino acid change from Leu to Pro at amino acid position 226 is causative of temperature sensitivity. By glycerol density gradient analyses of nuclear extracts prepared from wild-type virus-infected cells, we found that polymerase proteins sediment in three fractions: one (H fraction) consists of RNP complexes, another (M fraction) contains active polymerases but not viral RNA, and the other (L fraction) contains inactive forms of polymerases. Pulse-chase experiments showed that polymerases in the L fraction are converted to those in the M fraction. In
ts
53-infected cells, polymerases accumulated in the L fraction. These results strongly suggest that PA is involved in the assembly of functional viral RNA polymerase complexes from their inactive intermediates.
Functional Analysis of Vif Protein Shows Less Restriction of Human Immunodeficiency Virus Type 2 by APOBEC3G
Authors: Ribeiro Ana Clara , Maia e Silva Alexandra , Santa-Marta Mariana , Pombo Ana , Moniz-Pereira José , Goncalves Joao , Barahona Isabel 
Published: December 2004

ABSTRACT

Viral infectivity factor (Vif) is one of the human immunodeficiency virus (HIV) accessory proteins and is conserved in the primate lentivirus group. This protein is essential for viral replication in vivo and for productive infection of nonpermissive cells, such as peripheral blood mononuclear cells (PBMC). Vif counteracts an antiretroviral cellular factor in nonpermissive cells named CEM15/APOBEC3G. Although HIV type 1 (HIV-1) Vif protein (Vif1) can be functionally replaced by HIV-2 Vif protein (Vif2), its identity is very small. Most of the functional studies have been carried out with Vif1. Characterization of functional domains of Vif2 may elucidate its function, as well as differences between HIV-1 and HIV-2 infectivity. Our aim was to identify the permissivity of different cell lines for HIV-2
vif
-minus viruses. By mutagenesis specific conserved motifs of HIV-2 Vif protein were analyzed, as well as in conserved motifs between Vif1 and Vif2 proteins. Vif2 mutants were examined for their stability, expression, and cellular localization in order to characterize essential domains of Vif2 proteins. Viral replication in various target cells (PBMC and H9, A3.01, U38, and Jurkat cells) and infectivity in single cycle assays in the presence of APOBEC3G were also analyzed. Our results of viral replication show that only PBMC have a nonpermissive phenotype in the absence of Vif2. Moreover, the HIV-1
vif
-minus nonpermissive cell line H9 does not show a similar phenotype for
vif
-negative HIV-2. We also report a limited effect of APOBEC3G in a single-cycle infectivity assay, where only conserved domains between HIV-1 and HIV-2 Vif proteins influence viral infectivity. Taken together, these results allow us to speculate that viral inhibition by APOBEC3G is not the sole and most important determinant of antiviral activity against HIV-2.
Directed Evolution of Retrovirus Envelope Protein Cytoplasmic Tails Guided by Functional Incorporation into Lentivirus Particles
Authors: Merten Christoph A. , Stitz Jörn , Braun Gundula , Poeschla Eric M. , Cichutek Klaus , Buchholz Christian J. 
Published: December 2004

ABSTRACTIn contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.
Epstein-Barr Virus gp42 Is Posttranslationally Modified To Produce Soluble gp42 That Mediates HLA Class II Immune Evasion
Authors: Ressing Maaike E. , van Leeuwen Daphne , Verreck Frank A. W. , Keating Sinéad , Gomez Raquel , Franken Kees L. M. C. , Ottenhoff Tom H. M. , Spriggs Melanie , Schumacher Ton N. , Hutt-Fletcher Lindsey M. , Rowe Martin , Wiertz Emmanuel J. H. J. 
Published: December 2004

ABSTRACTEpstein-Barr virus (EBV) resides as a persistent infection in human leukocyte antigen (HLA) class II+B lymphocytes and is associated with a number of malignancies. The EBV lytic-phase protein gp42 serves at least two functions: gp42 acts as the coreceptor for viral entry into B cells and hampers T-cell recognition via HLA class II molecules through steric hindrance of T-cell receptor-class II-peptide interactions. Here, we show that gp42 associates with class II molecules at their various stages of maturation, including immature αβIi heterotrimers and mature αβ-peptide complexes. When analyzing the biosynthesis and maturation of gp42 in cells stably expressing the viral protein, we found that gp42 occurs in two forms: a full-length type II membrane protein and a truncated soluble form. Soluble gp42 is generated by proteolytic cleavage in the endoplasmic reticulum and is secreted. Soluble gp42 is sufficient to inhibit HLA class II-restricted antigen presentation to T cells. In an almost pure population of Burkitt's lymphoma cells in the EBV lytic cycle, both transmembrane and soluble forms of gp42 are detected. These results imply that soluble gp42 is generated during EBV lytic infection and could contribute to undetected virus production by mediating evasion from T-cell immunity.
Selection for Virulent Dengue Viruses Occurs in Humans and Mosquitoes
Authors: Cologna Raymond , Armstrong Philip M. , Rico-Hesse Rebeca 
Published: December 2004

ABSTRACT

Dengue is the most common mosquito-borne viral disease in humans. The spread of both mosquito vectors and viruses has led to the resurgence of epidemic dengue fever (a self-limited flu-like syndrome) and the emergence of dengue hemorrhagic fever (severe dengue with bleeding abnormalities) in urban centers of the tropics. There are no animal or laboratory models of dengue disease; indirect evidence suggests that dengue viruses differ in virulence, including their pathogenicities for humans and epidemic potential. We developed two assay systems (using human dendritic cells and
Aedes aegypti
mosquitoes) for measuring differences in virus replication that correlate with the potential to cause hemorrhagic dengue and increased virus transmission. Infection and growth experiments showed that dengue serotype 2 viruses causing dengue hemorrhagic fever epidemics (Southeast Asian genotype) can outcompete viruses that cause dengue fever only (American genotype). This fact implies that Southeast Asian genotype viruses will continue to displace other viruses, causing more hemorrhagic dengue epidemics.
Human Immunodeficiency Virus Type 1 Clade B Superinfection: Evidence for Differential Immune Containment of Distinct Clade B Strains
Authors: Yang Otto O. , Daar Eric S. , Jamieson Beth D. , Balamurugan Arumugam , Smith Davey M. , Pitt Jacqueline A. , Petropoulos Christos J. , Richman Douglas D. , Little Susan J. , Brown Andrew J. Leigh 
Published: December 2004

ABSTRACT

Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development. Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain. Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting. A low set point of viremia (∼1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (∼40,000 HIV-1 copies/ml) manifested about 5 months after infection. Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of
pol
genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B). Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by
pol
, and
env
sequences indicated that the two strains had the same R5 coreceptor phenotype. Delineation of CD8
+
-T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes. An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B. In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development.
Structural Requirements for Recognition of the Human Immunodeficiency Virus Type 1 Core during Host Restriction in Owl Monkey Cells
Authors: Forshey Brett M. , Shi Jiong , Aiken Christopher 
Published: December 2004

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) infection of simian cells is restricted at an early postentry step by host factors whose mechanism of action is unclear. These factors target the viral capsid protein (CA) and attenuate reverse transcription, suggesting that they bind to the HIV-1 core and interfere with its uncoating. To identify the relevant binding determinants in the capsid, we tested the capacity of viruses containing Gag cleavage site mutations and amino acid substitutions in CA to inhibit restriction of a wild type HIV-1 reporter virus in owl monkey cells. The results demonstrated that a stable, polymeric capsid and a correctly folded amino-terminal CA subunit interface are essential for saturation of host restriction in target cells by HIV-1 cores. We conclude that the owl monkey cellular restriction machinery recognizes a polymeric array of CA molecules, most likely via direct engagement of the HIV-1 capsid in target cells prior to uncoating.
CpG Methylation Directly Regulates Transcriptional Activity of the Human Endogenous Retrovirus Family HERV-K(HML-2)
Authors: Lavie Laurence , Kitova Milena , Maldener Esther , Meese Eckart , Mayer Jens 
Published: December 2004

ABSTRACT
A significant proportion of the human genome consists of stably inherited retroviral sequences. Most human endogenous retroviruses (HERVs) became defective over time. The HERV-K(HML-2) family is exceptional because of its coding capacity and the possible involvement in germ cell tumor (GCT) development. HERV-K(HML-2) transcription is strongly upregulated in GCTs. However, regulation of HERV-K(HML-2) transcription remains poorly understood. We investigated in detail the role of CpG methylation on the transcriptional activity of HERV-K(HML-2) long terminal repeats (LTRs). We find that CpG sites in various HERV-K(HML-2) proviral 5′ LTRs are methylated at different levels in the cell line Tera-1. Methylation levels correlate with previously observed transcriptional activities of these proviruses. CpG-mediated silencing of HERV-K(HML-2) LTRs is further corroborated by transcriptional inactivity of in vitro-methylated 5′ LTR reporter plasmids. However, CpG methylation levels do not solely regulate HERV-K(HML-2) 5′ LTR activity, as evidenced by different LTR activities in the cell line T47D. A significant number of mutated CpG sites in evolutionary old HERV-K(HML-2) 5′ LTRs suggests that CpG methylation had already silenced HERV-K(HML-2) proviruses millions of years ago. Direct silencing of HERV-K(HML-2) expression by CpG methylation enlightens upregulated HERV-K(HML-2) expression in usually hypomethylated GCT tissue.
Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with Pneumonia
Authors: Woo Patrick C. Y. , Lau Susanna K. P. , Chu Chung-ming , Chan Kwok-hung , Tsoi Hoi-wah , Huang Yi , Wong Beatrice H. L. , Poon Rosana W. S. , Cai James J. , Luk Wei-kwang , Poon Leo L. M. , Wong Samson S. Y. , Guan Yi , Peiris J. S. Malik , Yuen Kwok-yung 
Published: December 2004

ABSTRACT

Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 × 10
6
copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 × 10
6
copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.
Efficient Rescue of Hepatitis C Virus RNA Replication bytrans-Complementation with Nonstructural Protein 5A
Authors: Appel Nicole , Herian Ulrike , Bartenschlager Ralf 
Published: December 2004

ABSTRACTStudies ofHepatitis C virus(HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued bytrans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for theBovine viral diarrhea virus(C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented intrans; (ii) replication of the helper RNA is not necessary to allow efficienttrans-complementation; and (iii) the minimal sequence required fortrans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.
Covalent Modification of Human Immunodeficiency Virus Type 1 p6 by SUMO-1
Authors: Gurer Cagan , Berthoux Lionel , Luban Jeremy 
Published: December 2004

ABSTRACTThe p6 domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein mediates virion budding from infected cells via protein-protein contacts with the class E vacuolar protein sorting factors, Tsg101 and AIP1/ALIX. Interaction with Tsg101 is strengthened by covalent attachment of monovalent ubiquitin to HIV-1 p6. To identify additional host factors that bind to HIV-1 p6, a human cDNA library was screened in the yeast two-hybrid system. HIV-1 p6 was found to interact with small ubiquitin-like modifier 1 (SUMO-1) as well as the E2 SUMO-1 transfer enzyme, Ubc9. Interaction with p6 was also detected with Daxx, a cellular protein to which SUMO-1 is sometimes covalently attached. SUMO-1 was incorporated into HIV-1 virions where it was protected within the virion membrane from digestion by exogenous protease. Of the two lysine residues in p6, lysine 27 uniquely served as a site of covalent SUMO-1 attachment. As previously reported, though, HIV-1 bearing the p6-K27R mutation replicated just like the wild type. Overproduction of SUMO-1 in HIV-1 producer cells had no apparent effect on virion release or on virion protein or RNA content. Infectivity of the resulting virions, though, was decreased, with the defect occurring after membrane fusion, at the time of viral cDNA synthesis. HIV-1 bearing the p6-K27R mutation was insensitive to SUMO-1 overexpression, suggesting that covalent attachment of SUMO-1 to p6 is detrimental to HIV-1 replication.