bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirements for the Assembly and Release of Newcastle Disease Virus-Like Particles
Authors: Pantua Homer D. , McGinnes Lori W. , Peeples Mark E. , Morrison Trudy G.
Published: October 2006

Paramyxoviruses, such as Newcastle disease virus (NDV), assemble in and bud from plasma membranes of infected cells. To explore the role of each of the NDV structural proteins in virion assembly and release, virus-like particles (VLPs) released from avian cells expressing all possible combinations of the nucleoprotein (NP), membrane or matrix protein (M), an uncleaved fusion protein (F-K115Q), and hemagglutinin-neuraminidase (HN) protein were characterized for densities, protein content, and efficiencies of release. Coexpression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm
3
and 83.8% ± 1.1%, respectively) similar to those of authentic virions. Expression of M protein alone, but not NP, F-K115Q, or HN protein individually, resulted in efficient VLP release, and expression of all different combinations of proteins in the absence of M protein did not result in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F, and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by coimmunoprecipitation. The colocalization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M-HN and M-NP interactions are responsible for incorporation of HN and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein.
Vaccination of Pigs against Swine Influenza Viruses by Using an NS1-Truncated Modified Live-Virus Vaccine
Authors: Richt Jürgen A. , Lekcharoensuk Porntippa , Lager Kelly M. , Vincent Amy L. , Loiacono Christina M. , Janke Bruce H. , Wu Wai-Hong , Yoon Kyoung-Jin , Webby Richard J. , Solórzano Alicia , García-Sastre Adolfo
Published: October 2006

Swine influenza viruses (SIV) naturally infect pigs and can be transmitted to humans. In the pig, genetic reassortment to create novel influenza subtypes by mixing avian, human, and swine influenza viruses is possible. An SIV vaccine inducing cross-protective immunity between different subtypes and strains circulating in pigs is highly desirable. Previously, we have shown that an H3N2 SIV (A/swine/Texas/4199-2/98 [TX98]) containing a deleted NS1 gene expressing a truncated NS1 protein of 126 amino acids, NS1▴126, was attenuated in swine. In this study, 4-week-old pigs were vaccinated with the TX98 NS1▴126 modified live virus (MLV). Ten days after boosting, pigs were challenged with wild-type homologous H3N2 or heterosubtypic H1N1 SIV and sacrificed 5 days later. The MLV was highly attenuated and completely protected against challenge with the homologous virus. Vaccinated pigs challenged with the heterosubtypic H1N1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated H1N1 control pigs. Remarkably, vaccinated pigs challenged with the H1N1 SIV had significantly lower microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, H1N1-challenged pigs. All vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin A antibodies against H3N2 SIV antigens. Vaccinated pigs were seronegative for NS1, indicating the potential use of the TX98 NS1▴126 MLV as a vaccine to differentiate infected from vaccinated animals.
Kaposi's Sarcoma-Associated Herpesvirus Virions Inhibit Interferon Responses Induced by Envelope Glycoprotein gpK8.1
Authors: Perry Stuart T. , Compton Teresa
Published: October 2006

The Kaposi's sarcoma-associated herpesvirus (KSHV) envelope glycoprotein gpK8.1 contributes to cellular attachment through binding cell surface heparan sulfate proteoglycans. By using a soluble recombinant form of gpK8.1, we discovered that a consequence of gpK8.1 interaction with human fibroblasts is the induction of an antiviral response, as characterized by the activation of interferon regulatory factor 3 (IRF-3), production of interferon beta (IFN-β), and expression of interferon-stimulated antiviral genes. In contrast, neither IFN-β expression nor a functional antiviral response is observed in cells treated with KSHV virions. The interferon response induced by soluble gpK8.1 can be inhibited by simultaneous treatment with UV-inactivated virions, while the induction of an indicator inflammatory cytokine, interleukin-6, was readily evident in the response to both gpK8.1 and KSHV. In addition, KSHV virions abrogate gpK8.1-mediated activation of IRF-3, an early transcriptional regulator for cellular antiviral responses. Although innate immune responses are initiated during contact between gpK8.1 and cellular receptor(s), these results suggest that the virion contains one or more structural elements that selectively repress an effective antiviral response while allowing cellular responses favorable to the KSHV life cycle.
Cellular Immune Responses Associated with Occult Hepatitis C Virus Infection of the Liver
Authors: Quiroga Juan A. , Llorente Silvia , Castillo Inmaculada , Rodríguez-Iñigo Elena , Pardo Margarita , Carreño Vicente
Published: October 2006

Occult hepatitis C virus (HCV) infection is a type of recently identified chronic infection that is evidenced only by detection of HCV RNA in liver; patients consistently test negative for antibodies to HCV and HCV RNA in serum. Using ex vivo and in vitro measures of T-cell responses, we have identified functional virus-specific memory CD4+and CD8+T cells in the peripheral blood of patients with occult HCV infection. The features of the virus-specific T cells were consistent with immune surveillance functions, supporting previous exposure to HCV. In addition, the magnitudes of CD4+and CD8+T-cell responses were in parallel and correlated inversely with the extent of liver HCV infection. The detection of HCV-specific T cells in individuals in whom HCV RNA can persist in the liver despite the absence of viremia and antibodies indicates that HCV replication is prolonged in the face of virus-specific CD4+and CD8+T-cell responses. These findings demonstrate that HCV-specific cellular immune responses are markers not only of previous exposure to and recovery from HCV but also of ongoing occult HCV infection.
Epstein-Barr Virus EBNA-3C Is Targeted to and Regulates Expression from the Bidirectional LMP-1/2B Promoter
Authors: Jiménez-Ramírez Carmilia , Brooks Andrew J. , Forshell Linus Plym , Yakimchuk Konstantin , Zhao Bo , Fulgham Tacha Zi , Sample Clare E.
Published: October 2006

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is essential for EBV-mediated immortalization of human B lymphocytes and regulates both the cell cycle and transcription. Transient reporter gene assays have implicated a pivotal role for EBNA-3C in the regulation of transcription of the majority of latency-associated genes expressed during the EBV growth program, including the viral oncoprotein LMP-1. To examine the regulation of latency gene expression by EBNA-3C, we generated an EBV-positive cell line that inducibly expresses EBNA-3C. This cell line allowed us to examine expression from the endogenous latency gene promoters in the context of an actual latent infection and the presence of other EBNA proteins, in particular EBNA-2, which is presumed to coregulate transcription with EBNA-3C. EBNA-3C induced the expression of both LMP-1 and LMP-2B mRNAs from the bidirectional LMP-1/LMP-2B promoter. In contrast, no effect was seen on expression from the common EBNA promoter Cp, which is responsive to EBNA-3C in reporter assays. Activation of LMP expression was not the consequence of increases in EBNA-2, PU.1 or Spi-B transcription factors, all of which are believed to be critical for activation of LMP-1. Chromatin immunoprecipitation assays furthermore indicated that EBNA-3C is present at the bidirectional LMP-1/LMP-2B promoter. These results indicate that EBNA-3C directly activates the expression of LMP-1 and LMP-2B but is unlikely to significantly regulate EBNA expression via Cp under normal growth conditions.
Porcine Endogenous Retrovirus Integration Sites in the Human Genome: Features in Common with Those of Murine Leukemia Virus
Authors: Moalic Yann , Blanchard Yannick , Félix Hélène , Jestin André
Published: October 2006

Porcine endogenous retroviruses (PERV) are a major concern when porcine tissues and organs are used for xenotransplantation. PERV has been shown to infect human cells in vitro, highlighting a potential zoonotic risk. No pathology is associated with PERV in its natural host, but the pathogenic potential might differ in the case of cross-species transmission and can only be inferred from knowledge of related gammaretroviruses. We therefore investigated the integration features of the PERV DNA in the human genome in vitro in order to further characterize the risk associated with PERV transmission. In this study, we characterized 189 PERV integration site sequences from human HEK-293 cells. Data showed that PERV integration was strongly enhanced at transcriptional start sites and CpG islands and that the frequencies of integration events increased with the expression levels of the genes, except for the genes with the highest levels of expression, which were disfavored for integration. Finally, we extracted genomic sequences directly flanking the integration sites and found an original 8-base statistical palindromic consensus sequence [TG(int)GTACCAGC]. All these results show similarities between PERV and murine leukemia virus integration site selection, suggesting that gammaretroviruses have a common pattern of integration and that the mechanisms of target site selection within a retrovirus genus might be similar.
Evidence of Viral Adaptation to HLA Class I-Restricted Immune Pressure in Chronic Hepatitis C Virus Infection
Authors: Gaudieri Silvana , Rauch Andri , Park Lawrence P. , Freitas Elizabeth , Herrmann Susan , Jeffrey Gary , Cheng Wendy , Pfafferott Katja , Naidoo Kiloshni , Chapman Russell , Battegay Manuel , Weber Rainer , Telenti Amalio , Furrer Hansjakob , James Ian , Lucas Michaela , Mallal Simon A.
Published: October 2006

Cellular immune responses are an important correlate of hepatitis C virus (HCV) infection outcome. These responses are governed by the host's human leukocyte antigen (HLA) type, and HLA-restricted viral escape mutants are a critical aspect of this host-virus interaction. We examined the driving forces of HCV evolution by characterizing the in vivo selective pressure(s) exerted on single amino acid residues within nonstructural protein 3 (NS3) by the HLA types present in two host populations. Associations between polymorphisms within NS3 and HLA class I alleles were assessed in 118 individuals from Western Australia and Switzerland with chronic hepatitis C infection, of whom 82 (69%) were coinfected with human immunodeficiency virus. The levels and locations of amino acid polymorphisms exhibited within NS3 were remarkably similar between the two cohorts and revealed regions under functional constraint and selective pressures. We identified specific HCV mutations within and flanking published epitopes with the correct HLA restriction and predicted escaped amino acid. Additional HLA-restricted mutations were identified that mark putative epitopes targeted by cell-mediated immune responses. This analysis of host-virus interaction reveals evidence of HCV adaptation to HLA class I-restricted immune pressure and identifies in vivo targets of cellular immune responses at the population level.
DNA Helicase Activity Is Associated with the Replication Initiator Protein Rep of Tomato Yellow Leaf Curl Geminivirus
Authors: Clérot Danielle , Bernardi Françoise
Published: October 2006

The Rep protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a single-stranded DNA virus of plants, is the replication initiator essential for virus replication. TYLCSV Rep has been classified among ATPases associated with various cellular activities (AAA+ ATPases), in superfamily 3 of small DNA and RNA virus replication initiators whose paradigmatic member is simian virus 40 large T antigen. Members of this family are DNA- or RNA-dependent ATPases with helicase activity necessary for viral replication. Another distinctive feature of AAA+ ATPases is their quaternary structure, often composed of hexameric rings. TYLCSV Rep has ATPase activity, but the helicase activity, which is instrumental in further characterization of the mechanism of rolling-circle replication used by geminiviruses, has been a longstanding question. We present results showing that TYLCSV Rep lacking the 121 N-terminal amino acids has helicase activity comparable to that of the other helicases: requirements for a 3′ overhang and 3′-to-5′ polarity of unwinding, with some distinct features and with a minimal AAA+ ATPase domain. We also show that the helicase activity is dependent on the oligomeric state of the protein.
Cooperative Involvement of the S1 and S2 Subunits of the Murine Coronavirus Spike Protein in Receptor Binding and Extended Host Range
Authors: de Haan Cornelis A. M. , te Lintelo Eddie , Li Zhen , Raaben Matthijs , Wurdinger Tom , Bosch Berend Jan , Rottier Peter J. M.
Published: October 2006

To study the process of spike (S)-receptor interaction during coronavirus entry, we evaluated the contributions of mutations in different regions of the murine hepatitis virus (MHV) S protein to natural receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (CEACAM1a) dependence and to the acquisition of extended host range. Extended-host-range variants of MHV strain A59 were previously obtained from persistently infected cells (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9504, 1997). These variant viruses contain several mutations in the S protein that confer to the viruses the ability to enter cells in a heparan sulfate-dependent manner (C. A. de Haan, Z. Li, E. te Lintelo, B. J. Bosch, B. J. Haijema, and P. J. M. Rottier, J. Virol. 79:14451-14456, 2005). While the parental MHV-A59 is fully dependent on murine CEACAM1a for its entry, viruses carrying the variant mutations in the amino-terminal part of their S protein had become dependent on both CEACAM1a and heparan sulfate. Substitutions in a restricted, downstream part of the S protein encompassing heptad repeat region 1 (HR1) and putative fusion peptide (FP) did not alter the CEACAM1a dependence. However, when the mutations in both parts of the S protein were combined, the resulting viruses became independent of CEACAM1a and acquired the extended host range. In addition, these viruses showed a decreased binding to and inhibition by soluble CEACAM1a. The observations suggest that the amino-terminal region of the S protein, including the receptor-binding domain, and a region in the central part of the S protein containing HR1 and FP, i.e., regions far apart in the linear sequence, communicate and may even interact physically in the higher-order structure of the spike.
E6/E7 Expression of Human Papillomavirus Type 20 (HPV-20) and HPV-27 Influences Proliferation and Differentiation of the Skin in UV-Irradiated SKH-hr1 Transgenic Mice
Authors: Michel Angelika , Kopp-Schneider Annette , Zentgraf Hanswalter , Gruber Achim D. , de Villiers Ethel-Michele
Published: October 2006

The functional role of UV irradiation, in combination with the E6 and E7 proteins of the cutaneous human papillomavirus (HPV) types in the malignant conversion of benign papillomatous lesions, has not been elucidated. Transgenic SKH-hr1 hairless mice expressing HPV-20 and HPV-27 E6 and E7 proteins in the suprabasal compartment were generated and exposed to chronic UV irradiation. Histological and immunohistochemical examination of skin samples revealed enhanced proliferation of the epidermal layers and papilloma formation in both transgenic strains in comparison to what was observed with nontransgenic mice. Squamous cell carcinoma developed in the HPV-20 E6/E7 transgenic line as well as in the HPV-27 E6/E7 transgenic line. Several weeks after cessation of UV-B exposure, enhanced proliferation, as measured by BrdU incorporation, was maintained only in HPV-20 transgenic skin. Keratin 6 expression was increased in the transgenic mice throughout all cell layers. Expression of the differentiation markers involucrin and loricrin was reduced and disturbed. p63α expression was differentially regulated with high levels of cytoplasmic expression in clusters of cells in the granular layer of the skin in the transgenic lines 20 weeks after cessation of UV-B exposure, in contrast to uninterrupted staining in the nontransgenic lines. p53 was expressed in clusters of cells in nontransgenic and HPV-27 transgenic mice, in contrast to an even distribution in a higher number of cells in HPV-20 transgenic animals.
The NS1 Gene Contributes to the Virulence of H5N1 Avian Influenza Viruses
Authors: Li Zejun , Jiang Yongping , Jiao Peirong , Wang Aiqin , Zhao Fengju , Tian Guobin , Wang Xijun , Yu Kangzhen , Bu Zhigao , Chen Hualan
Published: October 2006

In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.
Recombination and Selection in the Evolution of Picornaviruses and Other Mammalian Positive-Stranded RNA Viruses
Authors: Simmonds Peter
Published: October 2006

Picornaviridae
are a large virus family causing widespread, often pathogenic infections in humans and other mammals. Picornaviruses are genetically and antigenically highly diverse, with evidence for complex evolutionary histories in which recombination plays a major part. To investigate the nature of recombination and selection processes underlying the evolution of serotypes within different picornavirus genera, large-scale analysis of recombination frequencies and sites, segregation by serotype within each genus, and sequence selection and composition was performed, and results were compared with those for other nonenveloped positive-stranded viruses (astroviruses and human noroviruses) and with flavivirus and alphavirus control groups. Enteroviruses, aphthoviruses, and teschoviruses showed phylogenetic segregation by serotype only in the structural region; lack of segregation elsewhere was attributable to extensive interserotype recombination. Nonsegregating viruses also showed several characteristic sequence divergence and composition differences between genome regions that were absent from segregating virus control groups, such as much greater amino acid sequence divergence in the structural region, markedly elevated ratios of nonsynonymous-to-synonymous substitutions, and differences in codon usage. These properties were shared with other picornavirus genera, such as the parechoviruses and erboviruses. The nonenveloped astroviruses and noroviruses similarly showed high frequencies of recombination, evidence for positive selection, and differential codon use in the capsid region, implying similar underlying evolutionary mechanisms and pressures driving serotype differentiation. This process was distinct from more-recent sequence evolution generating diversity within picornavirus serotypes, in which neutral or purifying selection was prominent. Overall, this study identifies common themes in the diversification process generating picornavirus serotypes that contribute to understanding of their evolution and pathogenicity.
Antibody Binding Is a Dominant Determinant of the Efficiency of Human Immunodeficiency Virus Type 1 Neutralization
Authors: Yang Xinzhen , Lipchina Inna , Cocklin Simon , Chaiken Irwin , Sodroski Joseph
Published: October 2006

Primary and laboratory-adapted variants of human immunodeficiency virus type 1 (HIV-1) exhibit a wide range of sensitivities to neutralization by antibodies directed against the viral envelope glycoproteins. An antibody directed against an artificial FLAG epitope inserted into the envelope glycoproteins of three HIV-1 isolates with vastly different neutralization sensitivities inhibited all three viruses equivalently. Thus, naturally occurring HIV-1 isolates that are neutralization resistant are not necessarily more impervious to the inhibitory consequences of bound antibody. Moreover, the binding affinity of the anti-FLAG antibody correlated with neutralizing potency, underscoring the dominant impact on neutralization of antibody binding to the envelope glycoproteins.
Herpes Simplex Virus Capsids Are Transported in Neuronal Axons without an Envelope Containing the Viral Glycoproteins
Authors: Snyder Aleksandra , Wisner Todd W. , Johnson David C.
Published: October 2006

Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini.
Differential Biophysical Properties of Infectious Intracellular and Secreted Hepatitis C Virus Particles
Authors: Gastaminza Pablo , Kapadia Sharookh B. , Chisari Francis V.
Published: October 2006

The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (∼65 to 70 nm) but different in buoyant density (∼1.15 to 1.20 g/ml) from extracellular particles (∼1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.
Acid Sphingomyelinase Deficiency Increases Susceptibility to Fatal Alphavirus Encephalomyelitis
Authors: Ng Ching G. , Griffin Diane E.
Published: October 2006

Sindbis virus (SV), an enveloped virus with a single-stranded, plus-sense RNA genome, is the prototype alphavirus in theTogaviridaefamily. In mice, SV infects neurons and can cause apoptosis of immature neurons. Sphingomyelin (SM) is the most prevalent cellular sphingolipid, is particularly abundant in the nervous systems of mammals, and is required for alphavirus fusion and entry. The level of SM is tightly regulated by sphingomyelinases. A defect in acid sphingomyelinase (ASMase) results in SM storage and subsequent intracellular accumulation of SM. To better understand the role of the SM pathway in SV pathogenesis, we have characterized SV infection of transgenic mice deficient in the ASMase gene. ASMase knockout (ASM-KO) mice were more susceptible to SV infection than wild-type (WT) or heterozygous (Het) animals. Titers of SV were higher in the brains of ASM-KO mice than in the brains of WT mice. More SV RNA was detected by in situ hybridization, more SV protein was detected by immunohistochemistry, and more terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were present in the cortex and hippocampus of ASM-KO mice than in those of WT or Het mice. Interleukin-6 (IL-6), but not IL-1β or tumor necrosis factor alpha, was elevated in infected ASM-KO mice compared to levels in WT or Het mice, but studies with IL-6-KO mice and recombinant SV expressing IL-6 showed no role for IL-6 in fatal disease. Together these data indicate that the increase in susceptibility of ASM-KO mice to SV infection was the result of more-rapid replication and spread of SV in the nervous system and increased neuronal death.
PD-1 Expression in Acute Hepatitis C Virus (HCV) Infection Is Associated with HCV-Specific CD8 Exhaustion
Authors: Urbani Simona , Amadei Barbara , Tola Daniela , Massari Marco , Schivazappa Simona , Missale Gabriele , Ferrari Carlo
Published: October 2006

Hepatitis C virus (HCV)-specific CD8 cell exhaustion may represent a mechanism of HCV persistence. The inhibitory receptor PD-1 has been reported to be up-regulated in exhausted CD8 cells. Therefore, we studied PD-1 expression longitudinally during acute HCV infection. Most HCV-specific CD8 cells expressed PD-1 at the time of acute illness, irrespective of the final outcome. PD-1 expression declined with the acquisition of a memory phenotype and recovery of an efficient CD8 cell function in resolving HCV infections, whereas high levels were maintained when HCV persisted and HCV-specific CD8 cells remained dysfunctional. Blocking PD-1/PDL-1 interaction with an anti-PDL-1 antibody improved the capacity of expansion of virus-specific CD8 cells.
Functional Role of the Cytoplasmic Tail Domain of the Major Envelope Fusion Protein of Group II Baculoviruses
Authors: Long Gang , Pan Xiaoyu , Westenberg Marcel , Vlak Just M.
Published: October 2006

F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD), ranging from 48 (
Spodoptera litura
multicapsid NPV [MNPV]) to 78 (
Adoxophyes honmai
NPV) amino acid (aa) residues, with a nonassigned function. This CTD is much longer than the CTD of GP64-like envelope fusion proteins (7 aa), which appear to be nonessential for BV infectivity. Here we have investigated the functional role of the CTD of
Helicoverpa armigera
single-capsid NPV (HearNPV), a group II NPV. We combined a newly constructed HearNPV
f
-null bacmid knockout-repair system and an
Autographa californica
MNPV (AcMNPV)
gp64
-null bacmid knockout-pseudotype system with mutation and rescue experiments to study the functional role of the baculovirus F protein CTD. We show that except for the 16 C-terminal aa, the HearNPV F CTD is essential for virus spread from cell to cell. In addition, the CTD of HearNPV F is involved in BV production in a length-dependent manner and is essential for BV infectivity. The tyrosine residue Y658, located 16 aa from the C terminus, seems to be critical. However, HearNPV F without a CTD still rescues the infectivity of
gp64
-null AcMNPV BV, indicating that the CTD is not involved in processing and fusogenicity. Altogether, our results indicate that the F protein is essential for baculovirus BV infectivity and that the CTD is important for F protein incorporation into BV.
Oligomerization of Hepatitis C Virus Core Protein Is Crucial for Interaction with the Cytoplasmic Domain of E1 Envelope Protein
Authors: Nakai Kousuke , Okamoto Toru , Kimura-Someya Tomomi , Ishii Koji , Lim Chang Kweng , Tani Hideki , Matsuo Eiko , Abe Takayuki , Mori Yoshio , Suzuki Tetsuro , Miyamura Tatsuo , Nunberg Jack H. , Moriishi Kohji , Matsuura Yoshiharu
Published: October 2006

Hepatitis C virus (HCV) contains two membrane-associated envelope glycoproteins, E1 and E2, which assemble as a heterodimer in the endoplasmic reticulum (ER). In this study, predictive algorithms and genetic analyses of deletion mutants and glycosylation site variants of the E1 glycoprotein were used to suggest that the glycoprotein can adopt two topologies in the ER membrane: the conventional type I membrane topology and a polytopic topology in which the protein spans the ER membrane twice with an intervening cytoplasmic loop (amino acid residues 288 to 360). We also demonstrate that the E1 glycoprotein is able to associate with the HCV core protein, but only upon oligomerization of the core protein in the presence of tRNA to form capsid-like structures. Yeast two-hybrid and immunoprecipitation analyses reveal that oligomerization of the core protein is promoted by amino acid residues 72 to 91 in the core. Furthermore, the association between the E1 glycoprotein and the assembled core can be recapitulated using a fusion protein containing the putative cytoplasmic loop of the E1 glycoprotein. This fusion protein is also able to compete with the intact E1 glycoprotein for binding to the core. Mutagenesis of the cytoplasmic loop of E1 was used to define a region of four amino acids (residues 312 to 315) that is important for interaction with the assembled HCV core. Taken together, our studies suggest that interaction between the self-oligomerized HCV core and the E1 glycoprotein is mediated through the cytoplasmic loop present in a polytopic form of the E1 glycoprotein.
Molecular Epidemiology of the Foot-and-Mouth Disease Virus Outbreak in the United Kingdom in 2001
Authors: Cottam Eleanor M. , Haydon Daniel T. , Paton David J. , Gloster John , Wilesmith John W. , Ferris Nigel P. , Hutchings Geoff H. , King Donald P.
Published: October 2006

The objective of this study was to quantify the extent to which the genetic diversity of foot-and-mouth disease virus (FMDV) arising over the course of infection of an individual animal becomes fixed, is transmitted to other animals, and thereby accumulates over the course of an outbreak. Complete consensus sequences of 23 genomes (each of 8,200 nucleotides) of FMDV were recovered directly from epithelium tissue acquired from 21 farms infected over a nearly 7-month period during the 2001 FMDV outbreak in the United Kingdom. An analysis of these consensus sequences revealed very few apparently ambiguous sites but clear evidence of 197 nucleotide substitutions at 191 different sites. We estimated the rate of nucleotide substitution to be 2.26 × 10
−5
per site per day (95% confidence interval [CI], 1.75 × 10
−5
to 2.80 × 10
−5
) and nucleotide substitutions to accrue in the consensus sequence at an average rate of 1.5 substitutions per farm infection. This is a sufficiently high rate showing that detailed histories of the transmission pathways can be reliably reconstructed. Coalescent methods indicated that the date at which FMDV first infected livestock in the United Kingdom was 7 February 2001 (95% CI, 20 January to 19 February 2001), which was identical to estimates obtained on the basis of purely clinical evidence. Nucleotide changes appeared to have occurred evenly across the genome, and within the open reading frame, the ratio of nonsynonymous-to-synonymous change was 0.09. The ability to recover particular transmission pathways of acutely acting RNA pathogens from genetic data will help resolve uncertainties about how virus is spread and could help in the control of future epidemics.
Evidence that the Herpes Simplex Virus Type 1 ICP0 Protein Does Not Initiate Reactivation from Latency In Vivo
Authors: Thompson R. L. , Sawtell N. M.
Published: October 2006

The stress-induced host cell factors initiating the expression of the herpes simplex virus lytic cycle from the latent viral genome are not known. Previous studies have focused on the effect of specific viral proteins on reactivation, i.e., the production of detectable infectious virus. However, identification of the viral protein(s) through which host cell factors transduce entry into the lytic cycle and analysis of the promoter(s) of this (these) first protein(s) will provide clues to the identity of the stress-induced host cell factors important for reactivation. In this report, we present the first strategy developed for this type of analysis and use this strategy to test the established hypothesis that the herpes simplex virus ICP0 protein initiates reactivation from the latent state. To this end, ICP0 null and promoter mutants were analyzed for the abilities (i) to exit latency and produce lytic-phase viral proteins (initiate reactivation) and (ii) to produce infectious viral progeny (reactivate) in explant and in vivo. Infection conditions were manipulated so that approximately equal numbers of latent infections were established by the parental strains, the mutants, and their genomically restored counterparts, eliminating disparate latent pool sizes as a complicating factor. Following hyperthermic stress (HS), which induces reactivation in vivo, equivalent numbers of neurons exited latency (as evidenced by the expression of lytic-phase viral proteins) in ganglia latently infected with either the ICP0 null mutant dl1403 or the parental strain. In contrast, infectious virus was detected in the ganglia of mice latently infected with the parental strain but not with ICP0 null mutant dl1403 or FXE. These data demonstrate that the role of ICP0 in the process of reactivation is not as a component of the switch from latency to lytic-phase gene expression; rather, ICP0 is required after entry into the lytic cycle has occurred. Similar analyses were carried out with the ΔTfi mutant, which contains a 350-bp deletion in the ICP0 promoter, and the genomically restored isolate, ΔTfiR. The numbers of latently infected neurons exiting latency were not different for ΔTfi and ΔTfiR. However, ΔTfi did not reactivate in vivo, whereas ΔTfiR reactivated in ∼38% of the mice. In addition, ICP0 was detected in ΔTfiR-infected neurons exiting latency but was not detected in those neurons exiting latency infected with ΔTfi. We conclude that while ICP0 is important and perhaps essential for infectious virus production during reactivation in vivo, this protein is not required and appears to play no major role in the initiation of reactivation in vivo.
Single Amino Acid Changes Can Influence Titer, Heparin Binding, and Tissue Tropism in Different Adeno-Associated Virus Serotypes
Authors: Wu Zhijian , Asokan Aravind , Grieger Joshua C. , Govindasamy Lakshmanan , Agbandje-McKenna Mavis , Samulski R. Jude
Published: October 2006

Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors.
Papillomavirus Particles Assembled in 293TT Cells Are Infectious In Vivo
Authors: Culp Timothy D. , Cladel Nancy M. , Balogh Karla K. , Budgeon Lynn R. , Mejia Andres F. , Christensen Neil D.
Published: October 2006

Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article, we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal. Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected 293TT cells.
Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Recruits Uracil DNA Glycosylase 2 at the Terminal Repeats and Is Important for Latent Persistence of the Virus
Authors: Verma Subhash C. , Bajaj Bharat G. , Cai Qiliang , Si Huaxin , Seelhammer Todd , Robertson Erle S.
Published: October 2006

Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with LANA. One of the identified proteins was uracil DNA glycosylase 2 (UNG2). UNG2 is important for removing uracil residues yielded after either misincorporation of dUTP during replication or deamination of cytosine. The specificity of the ′LANA-UNG2 interaction was confirmed by using a scrambled DNA sequence affinity column. Interaction of LANA and UNG2 was further confirmed by in vitro binding and coimmunoprecipitation assays. Colocalization of these proteins was also detected in primary effusion lymphoma (PEL) cells, as well as in a cotransfected KSHV-negative cell line. UNG2 binds to the carboxyl terminus of LANA and retains its enzymatic activity in the complex. However, no major effect on TR-mediated DNA replication was observed when a UNG2-deficient (UNG
−/−
) cell line was used. Infection of UNG
−/−
and wild-type mouse embryonic fibroblasts with KSHV did not reveal any difference; however, UNG
−/−
cells produced a significantly reduced number of virion particles after induction. Interestingly, depletion of UNG2 in PEL cells with short hairpin RNA reduced the number of viral genome copies and produced infection-deficient virus.
Fusion-Induced Apoptosis Contributes to Thymocyte Depletion by a Pathogenic Human Immunodeficiency Virus Type 1 Envelope in the Human Thymus
Authors: Meissner Eric G. , Zhang Liguo , Jiang S. , Su Lishan
Published: October 2006

The mechanisms of CD4+T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection remain incompletely characterized. Of particular importance is how CD4+T cells are depleted within the lymphoid organs, including the lymph nodes and thymus. Herein we characterize the pathogenic mechanisms of an envelope from a rapid progressor (R3A Env) in the NL4-3 backbone (NL4-R3A) which is able to efficiently replicate and deplete CD4+thymocytes in the human fetal-thymus organ culture (HF-TOC). We demonstrate that uninterrupted replication is required for continual thymocyte depletion. During depletion, NL4-R3A induces an increase in thymocytes which uptake 7AAD, a marker of cell death, and which express active caspase-3, a marker of apoptosis. While 7AAD uptake is observed predominantly in uninfected thymocytes (p24−), active caspase-3 is expressed in both infected (p24+) and uninfected thymocytes (p24−). When added to HF-TOC with ongoing infection, the protease inhibitor saquinavir efficiently suppresses NL4-R3A replication. In contrast, the fusion inhibitors T20 and C34 allow for sustained HIV-1 production. Interestingly, T20 and C34 effectively prevent thymocyte depletion in spite of this sustained replication. Apoptosis of both p24−and p24+thymocytes appears to be envelope fusion dependent, as T20, but not saquinavir, is capable of reducing thymocyte apoptosis. Together, our data support a model whereby pathogenic envelope-dependent fusion contributes to thymocyte depletion in HIV-1-infected thymus, correlated with induction of apoptosis in both p24+and p24−thymocytes.
Two Modes of Herpesvirus Trafficking in Neurons: Membrane Acquisition Directs Motion
Authors: Antinone Sarah E. , Smith Gregory A.
Published: October 2006

Alphaherpesvirus infection of the mammalian nervous system is dependent upon the long-distance intracellular transport of viral particles in axons. How viral particles are effectively trafficked in axons to either sensory ganglia following initial infection or back out to peripheral sites of innervation following reactivation remains unknown. The mechanism of axonal transport has, in part, been obscured by contradictory findings regarding whether capsids are transported in axons in the absence of membrane components or as enveloped virions. By imaging actively translocated viral structural components in living peripheral neurons, we demonstrate that herpesviruses use two distinct pathways to move in axons. Following entry into cells, exposure of the capsid to the cytosol resulted in efficient retrograde transport to the neuronal cell body. In contrast, progeny virus particles moved in the anterograde direction following acquisition of virion envelope proteins and membrane lipids. Retrograde transport was effectively shut down in this membrane-bound state, allowing for efficient delivery of progeny viral particles to the distal axon. Notably, progeny viral particles that lacked a membrane were misdirected back to the cell body. These findings show that cytosolic capsids are trafficked to the neuronal cell body and that viral egress in axons occurs after capsids are enshrouded in a membrane envelope.
The α Isoform of Protein Kinase CKI Is Responsible for Hepatitis C Virus NS5A Hyperphosphorylation
Authors: Quintavalle Manuela , Sambucini Sonia , Di Pietro Chiara , De Francesco Raffaele , Neddermann Petra
Published: October 2006

Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspects of the virus life cycle are still a mystery. The HCV nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulating HCV replication. Using kinase inhibitors that specifically inhibit the formation of NS5A-p58 in cells, we identified the CKI kinase family as a target. NS5A-p58 increased upon overexpression of CKI-α, CKI-δ, and CKI-ε, whereas the RNA interference of only CKI-α reduced NS5A hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by CKI-α overexpression, and we demonstrated that the CKI-α isoform is targeted by NS5A hyperphosphorylation inhibitors in living cells. Finally, we showed that down-regulation of CKI-α attenuates HCV RNA replication.
Expression of Nef Downregulates CXCR4, the Major Coreceptor of Human Immunodeficiency Virus, from the Surfaces of Target Cells and Thereby Enhances Resistance to Superinfection
Authors: Venzke Stephanie , Michel Nico , Allespach Ina , Fackler Oliver T. , Keppler Oliver T.
Published: October 2006

Lentiviral Nef proteins are key factors for pathogenesis and are known to downregulate functionally important molecules, including CD4 and major histocompatibility complex class I (MHC-I), from the surfaces of infected cells. Recently, we demonstrated that Nef reduces cell surface levels of the human immunodeficiency virus type 1 (HIV-1) entry coreceptor CCR5 (N. Michel, I. Allespach, S. Venzke, O. T. Fackler, and O. T. Keppler, Curr. Biol. 15:714-723, 2005). Here, we report that Nef downregulates the second major HIV-1 coreceptor, CXCR4, from the surfaces of HIV-infected primary CD4 T lymphocytes with efficiencies comparable to those of the natural CXCR4 ligand, stromal cell-derived factor-1 alpha. Analysis of a panel of mutants of HIV-1
SF2
Nef revealed that the viral protein utilized the same signature motifs for downmodulation of CXCR4 and MHC-I, including the proline-rich motif P
73
P
76
P
79
P
82
and the acidic cluster motif E
66
E
67
E
68
E
69.
Expression of wild-type Nef, but not of specific Nef mutants, resulted in a perinuclear accumulation of the coreceptor. Remarkably, the carboxy terminus of CXCR4, which harbors the classical motifs critical for basal and ligand-induced receptor endocytosis, was dispensable for the Nef-mediated reduction of surface exposure. Functionally, the ability of Nef to simultaneously downmodulate CXCR4 and CD4 correlated with maximum-level protection of Nef-expressing target cells from fusion with cells exposing X4 HIV-1 envelopes. Furthermore, the Nef-mediated downregulation of CXCR4 alone on target T lymphocytes was sufficient to diminish cells' susceptibility to X4 HIV-1 virions at the entry step. The downregulation of chemokine coreceptors is a conserved activity of Nef to modulate infected cells, an important functional consequence of which is an enhanced resistance to HIV superinfection.