bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the Flavivirus Helicase: Implications for Catalytic Activity, Protein Interactions, and Proteolytic Processing
Authors: Wu Jinhua , Bera Aloke Kumar , Kuhn Richard J. , Smith Janet L.
Published: July 2005

Yellow fever virus (YFV), a member of the
Flavivirus
genus, has a plus-sense RNA genome encoding a single polyprotein. Viral protein NS3 includes a protease and a helicase that are essential to virus replication and to RNA capping. The 1.8-Å crystal structure of the helicase region of the YFV NS3 protein includes residues 187 to 623. Two familiar helicase domains bind nucleotide in a triphosphate pocket without base recognition, providing a site for nonspecific hydrolysis of nucleoside triphosphates and RNA triphosphate. The third, C-terminal domain has a unique structure and is proposed to function in RNA and protein recognition. The organization of the three domains indicates that cleavage of the viral polyprotein NS3-NS4A junction occurs in
trans
.
Molecular Determinants of Infectious Pancreatic Necrosis Virus Virulence and Cell Culture Adaptation
Authors: Song Haichen , Santi Nina , Evensen Øystein , Vakharia Vikram N.
Published: July 2005

Infectious pancreatic necrosis viruses (IPNVs) exhibit a wide range of virulence in salmonid species. In previous studies, we have shown that the amino acid residues at positions 217 and 221 in VP2 are implicated in virulence. To pinpoint the molecular determinants of virulence in IPNV, we generated recombinant IPNV strains using the cRNA-based reverse-genetics system. In two virulent strains, residues at positions 217 and 247 were replaced by the corresponding amino acids of a low-virulence strain. The growth characteristics of the recovered chimeric strains in cell culture were similar to the low-virulence strains, and these viruses induced significantly lower mortality in Atlantic salmon fry than the parent strains did in in vivo challenge studies. Furthermore, the virulent strain was serially passaged in CHSE-214 cells 10 times and was completely characterized by nucleotide sequencing. Deduced amino acid sequence analyses revealed a single amino acid substitution of Ala to Thr at position 221 in VP2 of this virus, which became highly attenuated and induced 15% cumulative mortality in Atlantic salmon fry, compared to 68% mortality induced by the virulent parent strain. The attenuated strain grows to higher titers in CHSE cells and can be distinguished antigenically from the wild-type virus by use of a monoclonal antibody. However, the virulent strain passaged 10 times in RTG-2 cells was stable, and it retained its antigenicity and virulence. Our results indicate that residues Thr at position 217 (Thr217) and Ala221 of VP2 are the major determinants of virulence in IPNV of the Sp serotype. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221; and strains containing Thr221 are almost avirulent, irrespective of the residue at position 217.
Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase
Authors: Lu Richard , Ghory Hina Z. , Engelman Alan
Published: July 2005

Results of in vitro assays identified residues in the C-terminal domain (CTD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) important for IN-IN and IN-DNA interactions, but the potential roles of these residues in virus replication were mostly unknown. Sixteen CTD residues were targeted here, generating 24 mutant viruses. Replication-defective mutants were typed as class I (blocked at integration) or class II (additional reverse transcription and/or assembly defects). Most defective viruses (15 of 17) displayed reverse transcription defects. In contrast, replication-defective HIV-1
E246K
synthesized near-normal cDNA levels but processing of Pr55

g

ag

was largely inhibited in virus-producing cells. Because single-round HIV-1
E246K.Luc(R-)
transduced cells at approximately 8% of the wild-type level, we concluded that the late-stage processing defect contributed significantly to the overall replication defect of HIV-1
E246K
. Results of complementation assays revealed that the CTD could function in
trans
to the catalytic core domain (CCD) in in vitro assays, and we since determined that certain class I and class II mutants defined a novel genetic complementation group that functioned in cells independently of IN domain boundaries. Seven of eight novel Vpr-IN mutant proteins efficiently
trans
-complemented class I active-site mutant virus, demonstrating catalytically active CTD mutant proteins during infection. Because most of these mutants inefficiently complemented a class II CCD mutant virus, the majority of CTD mutants were likely more defective for interactions with cellular and/or viral components that affected reverse transcription and/or preintegration trafficking than the catalytic activity of the IN enzyme.
Vaccinia Virus Tropism for Primary Hematolymphoid Cells Is Determined by Restricted Expression of a Unique Virus Receptor
Authors: Chahroudi Ann , Chavan Rahul , Koyzr Natalia , Waller Edmund K. , Silvestri Guido , Feinberg Mark B.
Published: July 2005

The presumed broad tropism of poxviruses has stymied attempts to identify both the cellular receptor(s) and the viral determinant(s) for binding. Detailed studies of poxvirus binding to and infection of primary human cells have not been conducted. In particular, the determinants of target cell infection and the consequences of infection for cells involved in the generation of antiviral immune responses are incompletely understood. In this report, we show that vaccinia virus (VV) exhibits a more restricted tropism for primary hematolymphoid human cells than has been previously recognized. We demonstrate that vaccinia virus preferentially infects antigen-presenting cells (dendritic cells, monocytes/macrophages, and B cells) and activated T cells, but not resting T cells. The infection of activated T cells is permissive, with active viral replication and production of infectious progeny. Susceptibility to infection is determined by restricted expression of a cellular receptor that is induced de novo upon T-cell activation and can be removed from the cell surface by either trypsin or pronase treatment. The VV receptor expressed on activated T cells displays unique characteristics that distinguish it from the receptor used to infect cell lines in culture. The observed restricted tropism of VV may have significant consequences for the understanding of natural poxvirus infection and immunity and for poxvirus-based vaccine development.
Efficient cDNA-Based Rescue of La Crosse Bunyaviruses Expressing or Lacking the Nonstructural Protein NSs
Authors: Blakqori Gjon , Weber Friedemann
Published: July 2005

La Crosse virus (LACV) belongs to the
Bunyaviridae
family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology
330:
493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3′ ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10
8
infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.
Regulation of the Interaction between Glycogen Synthase Kinase 3 and the Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen
Authors: Fujimuro Masahiro , Liu Jianyong , Zhu Jian , Yokosawa Hideyoshi , Hayward S. Diane
Published: July 2005

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein stabilizes β-catenin by the novel mechanism of binding to the negative regulator, glycogen synthase kinase 3 (GSK-3), and depleting cytoplasmic GSK-3 levels. The two domains of LANA required for interaction with GSK-3 were further characterized. Evidence for similarity between the C-terminal LANA interaction domain and the axin GSK-3 interaction domain was obtained using GSK-3 and LANA mutants. GSK-3(F291L), which does not interact with axin, also failed to bind to LANA, and a mutation in the axin homology domain of LANA, L1132P, destroyed binding to GSK-3. The N-terminal LANA interaction domain was found to mediate interaction by acting as a substrate for GSK-3. GSK-3(R96A), a priming pocket mutant, did not bind to LANA, suggesting that LANA was a primed GSK-3 substrate. Phosphorylation of endogenous LANA precipitated from primary effusion lymphoma cells was inhibited by the GSK-3 inhibitor LiCl. GST-LANA(1-340) was phosphorylated by GSK-3, and mitogen-activated protein kinase (MAPK) and casein kinase I functioned as priming kinases in vitro. Mutation of consensus GSK-3 sites revealed that sites between LANA amino acids 219 and 268 were important for GSK-3 phosphorylation. Immunoprecipitation assays revealed that loss of GSK-3 phosphorylation of this N-terminal domain correlated with loss of GSK-3 interaction. Although LANA-associated GSK-3 actively phosphorylated LANA, GSK-3 coprecipitated with LANA was unable to phosphorylate an exogenous peptide substrate. LANA sequestration of GSK-3 may explain the ability of KSHV-infected cells to tolerate increased levels of nuclear GSK-3.
Mutagenesis-Induced, Large Fitness Variations with an Invariant Arenavirus Consensus Genomic Nucleotide Sequence
Authors: Grande-Pérez Ana , Gómez-Mariano Gema , Lowenstein Pedro R. , Domingo Esteban
Published: July 2005

Enhanced mutagenesis may result in RNA virus extinction, but the molecular events underlying this process are not well understood. Here we show that 5-fluorouracil (FU)-induced mutagenesis of the arenavirus lymphocytic choriomeningitis virus (LCMV) resulted in preextinction populations whose consensus genomic nucleotide sequence remained unaltered. Furthermore, fitness recovery passages in the absence of FU, or alternate virus passages in the presence and absence of FU, led to profound differences in the capacity of LCMV to produce progeny, without modification of the consensus genomic sequence. Molecular genetic analysis failed to produce evidence of hypermutated LCMV genomes. The results suggest that low-level mutagenesis to enrich the viral population with defector, interfering genomes harboring limited numbers of mutations may mediate the loss of infectivity that accompanies viral extinction.
Role of the Antigenic Loop of the Hepatitis B Virus Envelope Proteins in Infectivity of Hepatitis Delta Virus
Authors: Jaoudé Georges Abou , Sureau Camille
Published: July 2005

The infectious particles of hepatitis B virus (HBV) and hepatitis delta virus (HDV) are coated with the large, middle, and small envelope proteins encoded by HBV. While it is clear that the N-terminal pre-S1 domain of the large protein, which is exposed at the virion surface, is implicated in binding to a cellular receptor at viral entry, the role in infectivity of the envelope protein antigenic loop, also exposed to the virion surface and accessible to neutralizing antibodies, remains to be established. In the present study, mutations were created in the antigenic loop of the three envelope proteins, and the resulting mutants were evaluated for their capacity to assist in the maturation and infectivity of HDV. We observed that short internal combined deletions and insertions, affecting residues 109 to 133 in the antigenic loop, were tolerated for secretion of both subviral HBV particles and HDV virions. However, when assayed for infectivity on primary cultures of human hepatocytes or on the recently described HepaRG cell line, virions carrying deletions between residues 118 and 129 were defective. Single amino acid substitutions in this region revealed that Gly-119, Pro-120, Cys-121, Arg-122, and Cys-124 were instrumental in viral entry. These results demonstrate that in addition to a receptor-binding site previously identified in the pre-S1 domain of the L protein, a determinant of infectivity resides in the antigenic loop of HBV envelope proteins.
A Chimeric Respiratory Syncytial Virus Fusion Protein Functionally Replaces the F and HN Glycoproteins in Recombinant Sendai Virus
Authors: Zimmer Gert , Bossow Sascha , Kolesnikova Larissa , Hinz Matthias , Neubert Wolfgang J. , Herrler Georg
Published: July 2005

Entry of most paramyxoviruses is accomplished by separate attachment and fusion proteins that function in a cooperative manner. Because of this close interdependence, it was not possible with most paramyxoviruses to replace either of the two protagonists by envelope glycoproteins from related paramyxoviruses. By using reverse genetics of Sendai virus (SeV), we demonstrate that chimeric respiratory syncytial virus (RSV) fusion proteins containing either the cytoplasmic domain of the SeV fusion protein or in addition the transmembrane domain were efficiently incorporated into SeV particles provided the homotypic SeV-F was deleted. In the presence of SeV-F, the chimeric glycoproteins were incorporated with significantly lower efficiency, indicating that determinants in the SeV-F ectodomain exist that contribute to glycoprotein uptake. Recombinant SeV in which the homotypic fusion protein was replaced with chimeric RSV fusion protein replicated in a trypsin-independent manner and was neutralized by antibodies directed to RSV-F. However, replication of this virus also relied on the hemagglutinin-neuraminidase (HN) as pretreatment of cells with neuraminidase significantly reduced the infection rate. Finally, recombinant SeV was generated with chimeric RSV-F as the only envelope glycoprotein. This virus was not neutralized by antibodies to SeV and did not use sialic acids for attachment. It replicated more slowly than hybrid virus containing HN and produced lower virus titers. Thus, on the one hand RSV-F can mediate infection in an autonomous way while on the other hand it accepts support by a heterologous attachment protein.
An Exonic Splicing Silencer Downstream of the 3′ Splice Site A2 Is Required for Efficient Human Immunodeficiency Virus Type 1 Replication
Authors: Madsen Joshua M. , Stoltzfus C. Martin
Published: July 2005

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1-infected cell. Regulation of splicing at HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of splicing occurs through binding of cellular factors tocis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A2, which producesvprmRNA and promotes inclusion of HIV-1 exon 3, is repressed by the hnRNP A/B-dependent exonic splicing silencer ESSV. Here we show that ESSV activity downstream of 3′ss A2 is localized to a 16-nucleotide element within HIV-1 exon 3. HIV-1 replication was reduced by 95% when ESSV was inactivated by mutagenesis. Reduced replication was concomitant with increased inclusion of exon 3 within spliced viral mRNA and decreased accumulation of unspliced viral mRNA, resulting in decreased cell-associated p55 Gag. Prolonged culture of ESSV mutant viruses resulted in two independent second-site reversions disrupting the splice sites that define exon 3, 3′ss A2 and 5′ splice site D3. Either of these changes restored both HIV-1 replication and regulated viral splicing. Therefore, inhibition of HIV-1 3′ss A2 splicing is necessary for HIV-1 replication.
Herpes Simplex Virus Type 2 UL24 Gene Is a Virulence Determinant in Murine and Guinea Pig Disease Models
Authors: Blakeney Susan , Kowalski Jacek , Tummolo Donna , DeStefano Joanne , Cooper David , Guo Min , Gangolli Seema , Long Deborah , Zamb Timothy , Natuk Robert J. , Visalli Robert J.
Published: July 2005

A herpes simplex virus type 2 (HSV-2) UL24 β-glucuronidase (UL24-βgluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-βgluc yielded cytopathic effect with syncytium formation. UL24-βgluc replicated to wild-type viral titers in three different cell lines. UL24-βgluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD
50
) of the parental virus. Furthermore, few UL24-βgluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-βgluc at a dose equivalent to the LD
50
of parental virus (≈5 × 10
3
PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-βgluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.
Tyrosine Phosphorylation of the Tio Oncoprotein Is Essential for Transformation of Primary Human T Cells
Authors: Albrecht Jens-Christian , Müller-Fleckenstein Ingrid , Schmidt Monika , Fleckenstein Bernhard , Biesinger Brigitte
Published: July 2005

Human T cells are transformed to antigen-independent permanent growth in vitro upon infection with herpesvirus saimiri subgroup C strains. The viral oncoproteins required for this process, StpC and Tip, could be replaced by Tio, the oncoprotein of herpesvirus ateles. Here we demonstrate that proliferation of lymphocytes transformed with Tio-recombinant herpesvirus saimiri required the activity of Src family kinases. Src kinases had previously been identified as interaction partners of Tio. This interaction was now shown to be independent of any of the four tyrosine residues of Tio but to be dependent on an SH3-binding motif. Mutations within this motif abrogated the transforming capabilities of Tio-recombinant herpesvirus saimiri. Furthermore, kinase interaction resulted in the phosphorylation of Tio on a single tyrosine residue at position 136. Mutation of this residue in the viral context revealed that this phosphorylation site, but none of the other tyrosine residues, was required for T-cell transformation. These data indicate that the interaction of Tio with a Src kinase is essential for both the initiation and the maintenance of T-cell transformation by recombinant herpesvirus saimiri. The requirement for the tyrosine phosphorylation site at position 136 suggests a role for Tio beyond simple deregulation of the kinase.
Primary Sooty Mangabey Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 2nefAlleles Modulate Cell Surface Expression of Various Human Receptors and Enhance Viral Infectivity and Replication
Authors: Münch Jan , Schindler Michael , Wildum Steffen , Rücker Elke , Bailer Nicola , Knoop Volker , Novembre Francis J. , Kirchhoff Frank
Published: July 2005

Thenefgene of the pathogenic simian immunodeficiency virus (SIV) mac239 clone has been well characterized. Little is known, however, about the function ofnefalleles derived from naturally SIVsm-infected sooty mangabeys (Cercocebus atys) and from human immunodeficiency virus type 2 (HIV-2)-infected individuals. Addressing this, we demonstrate that, similarly to the SIVmac239nef, primary SIVsm and HIV-2nefalleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II major histocompatibility complex (MHC-I or MHC-II, respectively) molecules, up-regulate surface expression of the invariant chain (Ii) associated with immature MHC-II, inhibit early T-cell activation events, and enhance virion infectivity. Both also stimulate viral replication, although HIV-2nefalleles were less active in this assay than SIVsmnefalleles. Mutational analysis showed that a dileucine-based sorting motif in the C-proximal loop of SIV or HIV-2 Nef is critical for its effects on CD4, CD28, and Ii but dispensable for down-regulation of CD3, MHC-I, and MHC-II. The C terminus of SIV and HIV-2 Nef was exclusively required for down-modulation of MHC-I, further demonstrating that analogous functions are mediated by different domains in Nef proteins derived from different groups of primate lentiviruses. Our results demonstrate that none of the eight Nef functions investigated had been newly acquired after cross-species transmission of SIVsm from naturally infected mangabeys to humans or macaques. Notably, HIV-2 and SIVsmnefalleles efficiently down-modulate CD3 and C28 surface expression and inhibit T-cell activation more efficiently than HIV-1nefalleles. These differences in Nef function might contribute to the relatively low levels of immune activation observed in HIV-2-infected human individuals.
Capsid Processing Requirements for Abrogation of Fv1 and Ref1 Restriction
Authors: Dodding Mark P. , Bock Michael , Yap Melvyn W. , Stoye Jonathan P.
Published: July 2005

Murine leukemia virus is restricted in mouse cells lines by a host factor known as Fv1 and in human cell lines by Ref1. Genetic evidence indicates that these restriction factors target the virus capsid (CA) protein. Restriction can be overcome by adding virus at a high multiplicity of infection, indicating that the restriction factors can be saturated. Cells preexposed to restricted virus will allow infection by a second virus which would normally be restricted. This phenomenon is known as abrogation; it provides us with a tool with which to study the interaction of virus with restriction factors. We tested the abilities of several Gag processing mutants to abrogate restriction. Our results show that CA must be cleaved from both p12 and nucleocapsid in order for the incoming virion to interact with the restriction factor. Endogenous expression of properly processed CA, however, failed to abrogate restriction. These results suggest that as well as being processed, CA must also be properly assembled in the form of a condensed viral core in order to interact with Fv1 and Ref1. This polymeric structure may contain restriction factor binding sites not present in monomeric CA.
Role of Inflammatory Cytokine-Induced Cycloxygenase 2 in the Ocular Immunopathologic Disease Herpetic Stromal Keratitis
Authors: Biswas Partha S. , Banerjee Kaustuv , Kim Bumseok , Kinchington Paul R. , Rouse Barry T.
Published: July 2005

Ocular infection with herpes simplex virus (HSV) results in a blinding immunoinflammatory stromal keratitis (SK) lesion. Early preclinical events include polymorphonuclear neutrophil (PMN) infiltration and neovascularization in the corneal stroma. We demonstrate here that HSV infection of the cornea results in the upregulation of the cycloxygenase 2 (COX-2) enzyme. Early after infection, COX-2 was produced from uninfected stromal fibroblasts as an indirect effect of virus infection. Subsequently, COX-2 may also be produced from other inflammatory cells that infiltrate the cornea. The induction of COX-2 is a critical event, since inhibition of COX-2 with a selective inhibitor was shown to reduce corneal angiogenesis and SK severity. The administration of a COX-2 inhibitor resulted in compromised PMN infiltration into the cornea, as well as diminished corneal vascular endothelial growth factor levels, likely accounting for the reduced angiogenic response. COX-2 stimulation by HSV infection represents a critical early event accessible for therapy and the control of SK severity.
Ordered Processing of the Human Immunodeficiency Virus Type 1 GagPol Precursor Is Influenced by the Context of the Embedded Viral Protease
Authors: Pettit Steven C. , Clemente Jose C. , Jeung Jennifer A. , Dunn Ben M. , Kaplan Andrew H.
Published: July 2005

Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in
cis
, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.
CD8+T-Cell Dysfunction due to Cytolytic Granule Deficiency in Persistent Friend Retrovirus Infection
Authors: Zelinskyy Gennadiy , Robertson Shelly J. , Schimmer Simone , Messer Ronald J. , Hasenkrug Kim J. , Dittmer Ulf
Published: July 2005

Virus-specific CD8+T cells are critical for the control of acute Friend virus (FV) infections, but are rendered impotent by CD4+regulatory T cells during the chronic phase of infection. The current study examines this CD8+T-cell dysfunction by analyzing the production and release of cytolytic molecules by CD8+T cells. CD8+T cells with an activated phenotype (CD43+) from acutely infected mice produced all three key components of lytic granules: perforin, granzyme A, and granzyme B. Furthermore, they displayed evidence of recent degranulation and in vivo cytotoxicity. In contrast, activated CD8+T cells from chronically infected mice were deficient in cytolytic molecules and showed little evidence of recent degranulation and poor in vivo cytotoxicity. Evidence from tetramer-positive CD8+T cells with known virus specificity confirmed the findings from the activated subset of CD8+T cells. Interestingly, perforin and granzyme A mRNA levels were not significantly reduced during chronic infection, indicating control at a posttranscriptional level. Granzyme B deficiency was associated with a significant decrease in mRNA levels, but posttranscriptional control also appeared to contribute to deficiency. These results demonstrate a broad impairment of cytotoxic CD8+T-cell effector function during chronic retroviral infection and explain the inability of virus-specific CD8+T cells to eliminate persistent virus.
Evolution of Human Immunodeficiency Virus Type 1 Protease Genotypes and Phenotypes In Vivo under Selective Pressure of the Protease Inhibitor Ritonavir
Authors: Resch Wolfgang , Parkin Neil , Watkins Terri , Harris Janera , Swanstrom Ronald
Published: July 2005

We examined the population dynamics of human immunodeficiency virus type 1
pro
variants during the evolution of resistance to the protease inhibitor ritonavir (RTV) in vivo.
pro
variants were followed in subjects who had added RTV to their previously failed reverse transcriptase inhibitor therapy using a heteroduplex tracking assay designed to detect common resistance-associated mutations. In most cases the initial variant appeared rapidly within 2 to 3 months followed by one or more subsequent population turnovers. Some of the subsequent transitions between variants were rapid, and some were prolonged with the coexistence of multiple variants. In several cases variants without resistance mutations persisted despite the emergence of new variants with an increasing number of resistance-associated mutations. Based on the rate of turnover of
pro
variants in the RTV-treated subjects we estimated that the mean fitness of newly emerging variants was increased 1.2-fold (range, 1.02 to 1.8) relative to their predecessors. A subset of
pro
genes was introduced into infectious molecular clones. The corresponding viruses displayed impaired replication capacity and reduced susceptibility to RTV. A subset of these clones also showed increased susceptibility to two nonnucleoside reverse transcriptase inhibitors and the protease inhibitor saquinavir. Finally, a significant correlation between the reduced replication capacity and reduced processing at the
gag
NC-p1 processing site was noted. Our results reveal a complexity of patterns in the evolution of resistance to a protease inhibitor. In addition, these results suggest that selection for resistance to one protease inhibitor can have pleiotropic effects that can affect fitness and susceptibility to other drugs.
Nef Proteins from Diverse Groups of Primate Lentiviruses Downmodulate CXCR4 To Inhibit Migration to the Chemokine Stromal Derived Factor 1
Authors: Hrecka Kasia , Swigut Tomek , Schindler Michael , Kirchhoff Frank , Skowronski Jacek
Published: July 2005

Nef proteins of primate lentiviruses promote viral replication, virion infectivity, and evasion of antiviral immune responses by modulating signal transduction pathways and downregulating expression of receptors at the cell surface that are important for efficient antigen-specific responses, such as CD4, CD28, T-cell antigen receptor, and class I and class II major histocompatibility complex. Here we show that Nef proteins from diverse groups of primate lentiviruses which do not require the chemokine receptor CXCR4 for entry into target cells strongly downmodulate the cell surface expression of CXCR4. In contrast, all human immunodeficiency virus type 1 (HIV-1) and the majority of HIV-2 Nef proteins tested did not have such strong effects. SIVmac239 Nef strongly inhibited lymphocyte migration to CXCR4 ligand, the chemokine stromal derived factor 1 (SDF-1). SIVmac239 Nef downregulated CXCR4 by accelerating the rate of its endocytosis. Downmodulation of CXCR4 was abolished by mutations that disrupt the constitutively strong AP-2 clathrin adaptor binding element located in the N-terminal region of the Nef molecule, suggesting that Nef accelerates CXCR4 endocytosis via an AP-2-dependent pathway. Together, these results point to CXCR4 as playing an important role in simian immunodeficiency virus and possibly also HIV-2 persistence in vivo that is unrelated to viral entry into target cells. We speculate that Nef targets CXCR4 to disrupt ordered trafficking of infected leukocytes between local microenvironments in order to facilitate their dissemination and/or impair the antiviral immune response.
An S101P Substitution in the Putative Cleavage Motif of the Human Metapneumovirus Fusion Protein Is a Major Determinant for Trypsin-Independent Growth in Vero Cells and Does Not Alter Tissue Tropism in Hamsters
Authors: Schickli Jeanne H. , Kaur Jasmine , Ulbrandt Nancy , Spaete Richard R. , Tang Roderick S.
Published: July 2005

Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F
0
) into F
1
and F
2
exposes a fusion peptide at the N terminus of the F
1
fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.
High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations
Authors: Ceccherini-Silberstein Francesca , Gago Federico , Santoro Maria , Gori Caterina , Svicher Valentina , Rodríguez-Barrios Fátima , d'Arrigo Roberta , Ciccozzi Massimo , Bertoli Ada , Monforte Antonella d'Arminio , Balzarini Jan , Antinori Andrea , Perno Carlo-Federico
Published: July 2005

To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs.
A Begomovirus DNAβ-Encoded Protein Binds DNA, Functions as a Suppressor of RNA Silencing, and Targets the Cell Nucleus
Authors: Cui Xiaofeng , Li Guixin , Wang Daowen , Hu Dongwei , Zhou Xueping
Published: July 2005

Our previous results demonstrated that the DNAβ satellite (Y10β) associated with
Tomato yellow leaf curl China virus
Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its
βC1
gene in
Nicotiana
plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the βC1 proteins of Y10β and DNAβ (Y35β) found in the
Tobacco curly shoot virus
Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in
Escherichia coli
. Electrophoretic mobility shift assays and UV cross-linking experiments revealed that βC1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing
Nicotiana benthamiana
plants coinoculated by TYLCCNV-Y10 plus Y10β or by TbCSV-Y35 plus Y35β. In a patch agroinfiltration assay, the transiently expressed
βC1
gene of Y10β or Y35β was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic
N. benthamiana
plants. The βC1 protein of Y10β accumulated primarily in the nuclei of plant and insect cells when fused with β-glucuronidase or GFP and immunogold labeling showed that the βC1 protein is present in the nuclei of infected
N. benthamiana
plants. A mutant version of Y10β carrying the mutations within the putative nuclear localization sequence of the Y10 βC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the βC1 protein is a key requirement for symptom induction and silencing suppression.
Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus
Authors: Beer Christiane , Andersen Ditte S. , Rojek Aleksandra , Pedersen Lene
Published: July 2005

Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (
t
≈5 h), which is dependent on plasma membrane cholesterol but independent of NH
4
Cl treatment of cells; NH
4
Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.
Interactions between Rubella Virus Capsid and Host Protein p32 Are Important for Virus Replication
Authors: Beatch Martin D. , Everitt Jason C. , Law LokMan J. , Hobman Tom C.
Published: July 2005

The distribution and morphology of mitochondria are dramatically affected during infection with rubella virus (RV). Expression of the capsid, in the absence of other viral proteins, was found to induce both perinuclear clustering of mitochondria and the formation of electron-dense intermitochondrial plaques, both hallmarks of RV-infected cells. We previously identified p32, a host cell mitochondrial matrix protein, as a capsid-binding protein. Here, we show that two clusters of arginine residues within capsid are required for stable binding to p32. Mutagenic ablation of the p32-binding site in capsid resulted in decreased mitochondrial clustering, indicating that interactions with this cellular protein are required for capsid-dependent reorganization of mitochondria. Recombinant viruses encoding arginine-to-alanine mutations in the p32-binding region of capsid exhibited altered plaque morphology and replicated to lower titers. Further analysis indicated that disruption of stable interactions between capsid and p32 was associated with decreased production of subgenomic RNA and, consequently, infected cells produced significantly lower amounts of viral structural proteins under these conditions. Together, these results suggest that capsid-p32 interactions are important for nonstructural functions of capsid that include regulation of virus RNA replication and reorganization of mitochondria during infection.
Bovine Viral Diarrhea Virus Entry Is Dependent on Clathrin-Mediated Endocytosis
Authors: Lecot Steve , Belouzard Sandrine , Dubuisson Jean , Rouillé Yves
Published: July 2005

Cellular mechanisms of bovine viral diarrhea virus (BVDV) entry in MDBK cells were investigated. Chloroquine, bafilomycin A1, or ammonium chloride inhibited BVDV infection, indicating that an acidic endosomal pH is required for BVDV entry. The tyrosine kinase inhibitor genistein partially inhibited BVDV infection at a postentry step, whereas BVDV entry was strongly inhibited by chlorpromazine or by the overexpression of a dominant-negative form of EPS15, a protein essential for the formation of clathrin-coated vesicles at the plasma membrane. Together, these data indicate that BVDV infection requires an active clathrin-dependent endocytic pathway.
BOUND STATES IN THE DYNAMICS OF A DIPOLE IN THE PRESENCE OF A CONICAL DEFECT
Authors: DE LIMA RIBEIRO C. A. , FURTADO CLAUDIO , MORAES FERNANDO
Published: July 2005

In this work we investigate the quantum dynamics of an electric dipole in a (2+1)-dimensional conical spacetime. For specific conditions, the Schrödinger equation is solved and bound states are found with the energy spectrum and eigenfunctions determined. We find that the bound states spectrum extends from minus infinity to zero with a point of accumulation at zero. This unphysical result is fixed when a finite radius for the defect is introduced.
RECENT RESULTS FROM THE KLOE EXPERIMENT AT DAΦNE
Authors:   (Representing the KLOE Collaboration: F. Ambrosino, A. Antonelli, M. Antonelli, C. Bacci, P. Beltrame, G. Bencivenni, S. Bertolucci, C. Bini, C. Bloise, V. Bocci, F. Bossi, D. Bowring, P. Branchini, R. Caloi, P. Campana, G. Capon, T. Capussela, F. Ceradini, S. Chi, G. Chiefari, P. Ciambrone, S. Conetti, E. De Lucia, A. De Santis, P. De Simone, G. De Zorzi, S. Dell'Agnello, A. Denig, A. Di Domenico, C. Di Donato, S. Di Falco, B. Di Micco, A. Doria, M. Dreucci, G. Felici, A. Ferrari, M. L. Ferrer, G. Finocch, PALUTAN MATTEO
Published: July 2005

The KLOE experiment at DAΦNE has collected data for an integrated luminosity of ~ 1.3 fb-1 from e+e- collisions at center-of-mass energy around the ϕ-meson peak, W ~1.02 GeV . A selection of results obtained from a sample of 450 pb-1 of data acquired during year 2001 and 2002 is presented. Studying tagged KS and KL-mesons produced in the reaction e+e-→ϕ→KS KL we measured the branching ratio of the decay KS→πeν, the upper limit on the decay KS→π0π0π0, and the main KL decay branching ratios. Exploiting the initial state radiation in the reaction e+e-→π+π-γ, we extracted the hadronic cross-section as a function of the squared center-of-mass energy sπ of the π π system, in the range 0.35 < sπ < 0.95 GeV 2.
PECULIARITIES OF THE CANONICAL ANALYSIS OF THE TWO-DIMENSIONAL FIRST-ORDER EINSTEIN–HILBERT ACTION IN TERMS OF THE METRIC TENSOR OR THE METRIC DENSITY
Authors: KIRIUSHCHEVA N. , KUZMIN S. V. , MCKEON D. G. C.
Published: July 2005

The peculiarities of doing a canonical analysis of the first-order formulation of the Einstein–Hilbert action in terms of either the metric tensor gαβ or the metric density [Formula: see text] along with the affine connection are discussed. It is shown that the difference between using gαβ as opposed to hαβ appears only in two spacetime dimensions. Despite there being a different number of constraints in these two approaches, both formulations result in there being a local Poisson brackets algebra of constraints with field independent structure constants, closed off-shell generators of gauge transformations and off-shell invariance of the action. The formulation in terms of the metric tensor is analyzed in detail and compared with earlier results obtained using the metric density. The gauge transformations, obtained from the full set of first-class constraints, are different from a diffeomorphism transformation in both cases.
IMPLIED KERNEL MODELS
Authors: WEIGEL PETER
Published: July 2005

We develop a class of models within the pricing kernel framework, i.e., we model the pricing kernel directly, and not a particular interest rate or a set of rates. The construction of the kernel is explicitly linked to the calibrating set of instruments. Thus, once the kernel is constructed it will price correctly the chosen set of instruments, and have a low-dimensional Markov structure. We test our model on yield, at-the-money cap, caplet implied volatility surface, and swaption data. The quality of fit is very good.
CONTINUOUS TIME RANDOM WALK AND TIME FRACTIONAL DIFFUSION: A NUMERICAL COMPARISON BETWEEN THE FUNDAMENTAL SOLUTIONS
Authors: MAINARDI FRANCESCO , VIVOLI ALESSANDRO , GORENFLO RUDOLF
Published: July 2005

We consider the basic models for anomalous transport provided by the integral equation for continuous time random walk (CTRW) and by the time fractional diffusion equation to which the previous equation is known to reduce in the diffusion limit. We compare the corresponding fundamental solutions of these equations, in order to investigate numerically the increasing quality of approximation with advancing time.
Reconstitution of local Ca2+ signaling between cardiac L-type Ca2+ channels and ryanodine receptors: insights into regulation by FKBP12.6
Authors: Goonasekera Sanjeewa A. , Chen S. R. Wayne , Dirksen Robert T.
Published: July 2005

Ca+-induced Ca2+ release (CICR) in the heart involves local Ca2+ signaling between sarcolemmal L-type Ca2+ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) α1-subunit (α1CYM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing α1CYM and RyR2 were vesiculated and exhibited spontaneous Ca2+ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with α1CYM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca2+ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca2+ transients that required extracellular Ca2+. Depolarization-induced Ca2+ release in α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing α1CYM alone (maximal fluorescence change was 2.10 ± 0.39 and 0.54 ± 0.07, respectively), despite similar Ca2+ current densities. In addition, the gain of CICR in α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local α1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca2+ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca2+ release and the incidence of spontaneous Ca2+ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6.