bluuebunny/crossref_metadata_2025 huggingface dataset:
Retroviral Recombination In Vivo: Viral Replication Patterns and Genetic Structure of Simian Immunodeficiency Virus (SIV) Populations in Rhesus Macaques after Simultaneous or Sequential Intravaginal Inoculation with SIVmac239Δ
vpx/
Δ
vpr
and SIVmac239Δ
nef
Authors: Kim Eun-Young , Busch Marc , Abel Kristina , Fritts Linda , Bustamante Patty , Stanton Jenny , Lu Ding , Wu Samuel , Glowczwskie Jenny , Rourke Tracy , Bogdan Derek , Piatak Mike , Lifson Jeffrey D. , Desrosiers Ronald C. , Wolinsky Steven , Miller Christopher J.
Published: March 2005

To characterize the occurrence, frequency, and kinetics of retroviral recombination in vivo, we intravaginally inoculated rhesus macaques, either simultaneously or sequentially, with attenuated simian immunodeficiency virus (SIV) strains having complementary deletions in their accessory genes and various degrees of replication impairment. In monkeys inoculated simultaneously with SIVmac239Δ
vpx/
Δ
vpr
and SIVmac239Δ
nef
, recombinant wild-type (wt) virus and wild-type levels of plasma viral RNA (vRNA) were detected in blood by 2 weeks postinoculation. In monkeys inoculated first with SIVmac239Δ
vpx/
Δ
vpr
and then with SIVmac239Δ
nef
, recombination occurred but was associated with lower plasma vRNA levels than plasma vRNA levels seen for monkeys inoculated intravaginally with wt SIVmac239. In one monkey, recombination occurred 6 weeks after the challenge with SIVmac239Δ
nef
when plasma SIVmac239Δ
vpx/
Δ
vpr
RNA levels were undetectable. In monkeys inoculated first with the more highly replicating strain, SIVmac239Δ
nef
, and then with SIVmac239Δ
vpx/
Δ
vpr
, wild-type recombinant virus was not detected in blood or tissues. Instead, a virus that had repaired the deletion in the
nef
gene by a compensatory mutation was found in one animal. Overall, recombinant SIV was eventually found in four of six animals intravaginally inoculated with the two SIVmac239 deletion mutants. These findings show that recombination can occur readily in vivo after mucosal SIV exposure and thus contributes to the generation of viral genetic diversity and enhancement of viral fitness.
CD4+T-Cell Responses to Epstein-Barr Virus (EBV) Latent-Cycle Antigens and the Recognition of EBV-Transformed Lymphoblastoid Cell Lines
Authors: Long H. M. , Haigh T. A. , Gudgeon N. H. , Leen A. M. , Tsang C.-W. , Brooks J. , Landais E. , Houssaint E. , Lee S. P. , Rickinson A. B. , Taylor G. S.
Published: March 2005

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.
Human Immunodeficiency Virus (HIV)-Specific Gamma Interferon Secretion Directed against All Expressed HIV Genes: Relationship to Rate of CD4 Decline
Authors: Peretz Yoav , Alter Galit , Boisvert Marie-Pierre , Hatzakis George , Tsoukas Christos M. , Bernard Nicole F.
Published: March 2005

Immune responses to human immunodeficiency virus (HIV) are detected at all stages of infection and are believed to be responsible for controlling viremia. This study seeks to determine whether gamma interferon (IFN-γ)-secreting HIV-specific T-cell responses influence disease progression as defined by the rate of CD4 decline. The study population consisted of 31 subjects naïve to antiretroviral therapy. All were monitored clinically for a median of 24 months after the time they were tested for HIV-specific responses. The rate of CD4
+
-T-cell loss was calculated for all participants from monthly CD4 counts. Within this population, 17 subjects were classified as typical progressors, 6 subjects were classified as fast progressors, and 8 subjects were classified as slow progressors. Peripheral blood mononuclear cells were screened for HIV-specific IFN-γ responses to all expressed HIV genes. Among the detected immune responses, 48% of the recognized peptides were encoded by Gag and 19% were encoded by Nef gene products. Neither the breadth nor the magnitude of HIV-specific responses correlated with the viral load or rate of CD4 decline. The breadth and magnitude of HIV-specific responses did not differ significantly among typical, fast, and slow progressors. These results support the conclusion that although diverse HIV-specific IFN-γ-secreting responses are mounted during the asymptomatic phase, these responses do not seem to modulate disease progression rates.
Induction of the Human Papillomavirus Type 31 Late Promoter Requires Differentiation but Not DNA Amplification
Authors: Spink Kathryn M. , Laimins Laimonis A.
Published: March 2005

The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine β-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the
cis
-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.
Induction of Humoral Immune Responses following Vaccination with Envelope-Containing, Formaldehyde-Treated, Thermally Inactivated Human Immunodeficiency Virus Type 1
Authors: Poon B. , Safrit J. T. , McClure H. , Kitchen C. , Hsu J. F. , Gudeman V. , Petropoulos C. , Wrin T. , Chen I. S. Y. , Grovit-Ferbas K.
Published: March 2005

The lack of success of subunit human immunodeficiency virus type 1 (HIV-1) vaccines to date suggests that multiple components or a complex virion structure may be required. We previously demonstrated retention of the major conformational epitopes of HIV-1 envelope following thermal treatment of virions. Moreover, antibody binding to some of these epitopes was significantly enhanced following thermal treatment. These included the neutralizing epitopes identified by monoclonal antibodies 1b12, 2G12, and 17b, some of which have been postulated to be partially occluded or cryptic in native virions. Based upon this finding, we hypothesized that a killed HIV vaccine could be derived to elicit protective humoral immune responses. Shedding of HIV-1 envelope has been described for some strains of HIV-1 and has been cited as one of the major impediments to developing an inactivated HIV-1 vaccine. In the present study, we demonstrate that treatment of virions with low-dose formaldehyde prior to thermal inactivation retains the association of viral envelope with virions. Moreover, mice and nonhuman primates vaccinated with formaldehyde-treated, thermally inactivated virions produce antibodies capable of neutralizing heterologous strains of HIV in peripheral blood mononuclear cell-, MAGI cell-, and U87-based infectivity assays. These data indicate that it is possible to create an immunogen by using formaldehyde-treated, thermally inactivated HIV-1 virions to induce neutralizing antibodies. These findings have broad implications for vaccine development.
Latent Membrane Protein 1 Regulates STAT1 through NF-κB-Dependent Interferon Secretion in Epstein-Barr Virus-Immortalized B Cells
Authors: Najjar Imen , Baran-Marszak Fanny , Le Clorennec Christophe , Laguillier Christelle , Schischmanoff Olivier , Youlyouz-Marfak Ibtissam , Schlee Martin , Bornkamm Georg W. , Raphaël Martine , Feuillard Jean , Fagard Remi
Published: March 2005

Constitutive activation of signal transducer and activator of transcription 1 (STAT1) is a distinctive feature of Epstein-Barr virus (EBV)-immortalized B cells (lymphoblastoid cell lines [LCLs]). The expression of STAT1 in these cells is modulated by the latent membrane protein 1 (LMP1), but the mechanism of STAT1 activation has remained unclear. We demonstrate that the tyrosine phosphorylation of STAT1 in LCLs results from an indirect pathway encompassing an NF-κB-dependent secretion of interferons (IFNs). The cell culture supernatant of LCLs induced tyrosine phosphorylation of STAT1 in cells with no constitutively activated STAT1. Moreover, removal of supernatant from LCLs was sufficient to decrease the phosphorylation of STAT1. Inhibition of NF-κB activity by different pharmacological inhibitors (i.e., parthenolide, MG132 and BAY 11-7082) and by overexpressed mutated IκBα prevented the activation of STAT1. To identify the factors involved, we performed macroarray cDNA profiling with or without inhibition of NF-κB. The expression of several cytokines was NF-κB dependent among those alpha and gamma IFNs (IFN-α and IFN-γ), known activators of STAT1. By real-time PCR and enzyme-linked immunosorbent assay we show that IFN-α and IFN-γ are expressed and released by LCLs in an NF-κB-dependent manner. Finally, the blocking of the IFN-α and IFN-γ by neutralizing antibodies led to the complete inhibition of tyrosine phosphorylation of STAT1. Taken together, our results clearly show that LMP1-induced tyrosine phosphorylation of STAT1 is almost exclusively due to the NF-κB-dependent secretion of IFNs. Whether this response, which is usually considered to be antiviral, is in fact required for the persistence of the virus remains to be elucidated.
CD4-Independent Entry and Replication of Simian Immunodeficiency Virus in Primary Rhesus Macaque Astrocytes Are Regulated by the Transmembrane Protein
Authors: Overholser Emily D. , Babas Tahar , Zink M. Christine , Barber Sheila A. , Clements Janice E.
Published: March 2005

Previous studies have demonstrated that the genetic determinants of simian immunodeficiency virus (SIV) neurovirulence map to the
env
and
nef
genes. Recent studies from our laboratory demonstrated that SIV replication in primary rhesus macaque astrocyte cultures is dependent upon the
nef
gene. Here, we demonstrate that macrophage tropism is not sufficient for replication in astrocytes and that specific amino acids in the transmembrane (TM) portion of Env are also important for optimal SIV replication in astrocytes. Specifically, a Gly at amino acid position 751 and truncation of the cytoplasmic tail of TM are required for efficient replication in these cells. Studies using soluble CD4 demonstrated that these changes within the TM protein regulate CD4-independent, CCR5-dependent entry of virus into astrocytes. In addition, we observed that two distinct CD4-independent, neuroinvasive strains of SIV/DeltaB670 also replicated efficiently in astrocytes, further supporting the role of CD4 independence as an important determinant of SIV infection of astrocytes in vitro and in vivo.
Host and Viral Proteins in the Virion of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Bechtel Jill T. , Winant Richard C. , Ganem Don
Published: March 2005

Infection of cultured cells with Kaposi's sarcoma associated herpesvirus (KSHV) typically establishes a latent infection, in which only a few viral genes are expressed. Recently, it has been reported that a subset of lytic genes are transiently expressed very early after viral entry but that this burst of abortive lytic gene expression is terminated with the supervention of latency (H. H. Krishnan, P. P. Naranatt, M. S. Smith, L. Zeng, C. Bloomer, and B. Chandran, J. Virol.
78:
3601-3620, 2004). To identify molecules imported into cells by KSHV that might influence this gene expression program, we have examined the protein composition of the KSHV particle. Immunoblotting of virus particles demonstrated that RTA, the lytic switch protein, and RAP, a viral protein that is a transcriptional and cell cycle modulator, were both incorporated into virus particles. In a second approach, polypeptides isolated from purified virions were identified by mass-spectrometric analysis of their constituent tryptic peptides. With this approach we were able to identify 18 major virion proteins, including structural, regulatory, and signaling proteins of both viral and cellular origin.
Preferential Feline Immunodeficiency Virus (FIV) Infection of CD4+CD25+T-Regulatory Cells Correlates both with Surface Expression of CXCR4 and Activation of FIV Long Terminal Repeat Binding Cellular Transcriptional Factors
Authors: Joshi Anjali , Garg Himanshu , Tompkins Mary B. , Tompkins Wayne A.
Published: March 2005

Previously, we have characterized feline CD4+CD25+T-regulatory (Treg) cells with regard to their immune regulatory properties and ability to support feline immunodeficiency virus (FIV) replication in vitro and in vivo. Our studies showed that while CD4+CD25+cells were capable of replicating FIV in the presence of interleukin-2 (IL-2) alone, CD4+CD25−cells harbored a latent infection that required a strong mitogenic stimulus to activate virus replication. In the present study, we investigated the mechanisms governing the preferential replication of FIV in highly purified CD4+CD25+Treg cells compared to their CD4+CD25−counterparts. Studies aimed at elucidating mechanisms regulating infection of these cells revealed that CD4+CD25−cells were less susceptible to FIV binding and entry than CD4+CD25+cells, which correlated with increased surface expression of FIV coreceptor CXCR4. In addition, the number of CD4+CD25+cells that expressed the primary receptor CD134 was greater than for CD4+CD25−cells. Although increased permissiveness to FIV infection of CD4+CD25−cells following mitogenic stimulation correlated strongly with upregulation of surface CXCR4, it did not correlate with CD134 expression. Further, study of intracellular factors regulating FIV replication revealed that CD4+CD25+but not CD4+CD25−T cells showed constitutive and IL-2-responsive transactivation of activating transcription factor, CAAT enhancer binding protein, and activating protein 1 transcription factors that are important for FIV replication. These factors were upregulated in CD4+CD25−T cells following ConA stimulation, which correlated with FIV replication. This is the first report elucidating the mechanisms that allow for productive lentiviral infection of CD4+CD25+Treg cells.
Evidence of Recombination in the Norovirus Capsid Gene
Authors: Rohayem Jacques , Münch Julia , Rethwilm Axel
Published: March 2005

Noroviruses are single-stranded RNA viruses with high genomic variability. They have emerged in the last decade as a major cause of acute gastroenteritis. It remains so far unclear whether norovirus evolution is driven by sequence mutation and/or recombination. In this study, we have assessed the occurrence of recombination in the norovirus capsid gene. For this purpose, 69 complete capsid sequences of norovirus strains accessible in GenBank as well as 25 complete capsid sequences generated from norovirus-positive clinical samples were examined. Unreported recombination was detected in about 8% of norovirus strains belonging to genetic clusters I/1 (n= 1), II/1 (n= 1), II/3 (n= 1), II/4 (n= 3), and II/5 (n= 1). Recombination breakpoints were mainly located at the interface of the putative P1-1 and P2 domains of the capsid protein and/or within the P2 domain. The recombination region displayed features such as length, sequence composition (upstream and downstream GC- and AU-rich sequences, respectively), and predicted RNA secondary structure that are characteristic of homologous recombination activators. Our results suggest that recombination in the norovirus capsid gene may naturally occur, involving capsid domains presumably exposed to immunological pressure.
Enfuvirtide Resistance Mutations: Impact on Human Immunodeficiency Virus Envelope Function, Entry Inhibitor Sensitivity, and Virus Neutralization
Authors: Reeves Jacqueline D. , Lee Fang-Hua , Miamidian John L. , Jabara Cassandra B. , Juntilla Marisa M. , Doms Robert W.
Published: March 2005

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.
The Majority of Currently Circulating Human Immunodeficiency Virus Type 1 Clade B Viruses Fail To Prime Cytotoxic T-Lymphocyte Responses against an Otherwise Immunodominant HLA-A2-Restricted Epitope: Implications for Vaccine Design
Authors: Altfeld Marcus , Allen Todd M. , Kalife Elizabeth T. , Frahm Nicole , Addo Marylyn M. , Mothe Bianca R. , Rathod Almas , Reyor Laura L. , Harlow Jason , Yu Xu G. , Perkins Beth , Robinson Loren K. , Sidney John , Alter Galit , Lichterfeld Mathias , Sette Alessandro , Rosenberg Eric S. , Goulder Philip J. R. , Brander Christian , Walker Bruce D.
Published: March 2005

Human immunodeficiency virus type 1 (HIV-1) mutates to escape immune selection pressure, but there is little evidence of selection mediated through HLA-A2, the dominant class I allele in persons infected with clade B virus. Moreover, HLA-A2-restricted responses are largely absent in the acute phase of infection as the viral load is being reduced, suggesting that circulating viruses may lack immunodominant epitopes targeted through HLA-A2. Here we demonstrate an A2-restricted epitope within Vpr (Vpr
59-67
) that is targeted by acute-phase HIV-1-specific CD8
+
T cells, but only in a subset of persons expressing HLA-A2. Individuals in the acute stage of infection with viruses containing the most common current sequence within this epitope (consensus sequence) were unable to mount epitope-specific T-cell responses, whereas subjects infected with the less frequent I
60
L variant all developed these responses. The I
60
L variant epitope was a stronger binder to HLA-A2 and was recognized by epitope-specific T cells at lower peptide concentrations than the consensus sequence epitope. These data demonstrate that HLA-A2 is capable of contributing to the acute-phase cytotoxic T-lymphocyte response in infected subjects, but that most currently circulating viruses lack a dominant immunogenic epitope presented by this allele, and suggest that immunodominant epitopes restricted by common HLA alleles may be lost as the epidemic matures.
Hepatitis C Virus NS5A-Mediated Activation of Phosphoinositide 3-Kinase Results in Stabilization of Cellular β-Catenin and Stimulation of β-Catenin-Responsive Transcription
Authors: Street Andrew , Macdonald Andrew , McCormick Christopher , Harris Mark
Published: March 2005

The hepatitis C virus (HCV) nonstructural NS5A protein has been shown to bind to and activate phosphoinositide 3-kinase (PI3K), resulting in activation of the downstream effector serine/threonine kinase Akt/protein kinase B. Here we present data pertaining to the effects of NS5A-mediated Akt activation on its downstream targets. Using a recombinant baculovirus to deliver the complete HCV polyprotein to human hepatoma cells in a tetracycline-regulable fashion, we confirm that expression of the complete HCV polyprotein also activates PI3K and Akt. We further show that this results in the inhibition of the Akt substrate Forkhead transcription factor and the stimulation of phosphorylation of a second key Akt substrate, glycogen synthase kinase-3β (GSK-3β). Phosphorylation of GSK-3β results in its inactivation; consistent with this, we show that expression of the HCV polyprotein results in the accumulation of β-catenin. Finally, we show that levels of β-catenin-dependent transcription are also elevated in the presence of the HCV polyprotein. Given the prevalence of β-catenin mutations in many human tumors, especially colon and hepatocellular carcinomas, these data implicate NS5A-mediated PI3K activation as a contributory factor in the increasingly common association between HCV infection and the development of hepatocellular carcinoma.
Penetration of Enveloped Double-Stranded RNA Bacteriophages φ13 and φ6 intoPseudomonas syringaeCells
Authors: Daugelavičius Rimantas , Cvirkaitė Virginija , Gaidelytė Aušra , Bakienė Elena , Gabrėnaitė-Verkhovskaya Rasa , Bamford Dennis H.
Published: March 2005

Bacteriophages φ6 and φ13 are related enveloped double-stranded RNA viruses that infect gram-negativePseudomonas syringaecells. φ6 uses a pilus as a receptor, and φ13 attaches to the host lipopolysaccharide. We compared the entry-related events of these two viruses, including receptor binding, envelope fusion, peptidoglycan penetration, and passage through the plasma membrane. The infection-related events are dependent on the multiplicity of infection in the case of φ13 but not with φ6. A temporal increase of host outer membrane permeability to lipophilic ions was observed from 1.5 to 4 min postinfection in both virus infections. This enhanced permeability period coincided with the fast dilution of octadecyl rhodamine B-labeled virus-associated lipid molecules. This result is in agreement with membrane fusion, and the presence of temporal virus-derived membrane patches on the outer membrane. Similar to φ6, φ13 contains a thermosensitive lytic enzyme involved in peptidoglycan penetration. The phage entry also caused a limited depolarization of the plasma membrane. Inhibition of host respiration considerably decreased the efficiency of irreversible virus binding and membrane fusion. An active role of cell energy metabolism in restoring the infection-induced defects in the cell envelope was also observed.
Characterization of the Elements and Proteins Responsible for Interferon-Stimulated Gene Induction by Human Cytomegalovirus
Authors: Yang Shaojun , Netterwald James , Wang Weijia , Zhu Hua
Published: March 2005

Human cytomegalovirus (HCMV) infection of human fibroblast cells activates a large number of interferon-stimulated genes (ISGs) in a viral envelope-cell membrane fusion-dependent mechanism. In this study, we identified two interferon response elements, the interferon-stimulated response element (ISRE) and the gamma interferon-activated site (GAS), which act as HCMV response sites (VRS). Gel mobility shift assays showed that cellular proteins form specific and identical complexes with ISRE and GAS elements, and the binding of these complexes to ISRE and GAS is stimulated by HCMV infection. Point mutations in the consensus sequences of ISRE and GAS completely abolished their activities in response to HCMV-mediated transactivation, as well as their abilities to interact with HCMV-activated VRS-binding proteins. Interferon regulatory factor 3 does not appear to be present in the VRS-binding complexes or to be involved directly in HCMV-mediated ISG activation. Using ProteinChip technology, four potential proteins were identified, ranging from 20 to 42 kDa, in the VRS-binding complexes. The data suggest that HCMV infection activates VRS-binding proteins, which then bind to the VRS and stimulate ISG expression.
Two Gamma Interferon-Activated Site-Like Elements in the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer Are Important for Viral Replication
Authors: Netterwald James , Yang Shaojun , Wang Weijia , Ghanny Salena , Cody Michael , Soteropoulos Patricia , Tian Bin , Dunn Walter , Liu Fenyong , Zhu Hua
Published: March 2005

Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.
Structure of Adeno-Associated Virus Type 4
Authors: Padron Eric , Bowman Valorie , Kaludov Nikola , Govindasamy Lakshmanan , Levy Hazel , Nick Phillip , McKenna Robert , Muzyczka Nicholas , Chiorini John A. , Baker Timothy S. , Agbandje-McKenna Mavis
Published: March 2005

Adeno-associated virus
(AAV) is a member of the
Parvoviridae
, belonging to the
Dependovirus
genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-Å resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.
The Murine Gammaherpesvirus 68 ORF27 Gene Product Contributes to Intercellular Viral Spread
Authors: May Janet S. , Walker Jennifer , Colaco Susanna , Stevenson Philip G.
Published: March 2005

Herpesviruses remain predominantly cell associated within their hosts, implying that they spread between cells by a mechanism distinct from free virion release. We previously identified the efficient release of murine gammaherpesvirus 68 (MHV-68) virions as a function of the viral gp150 protein. Here we show that the MHV-68 ORF27 gene product, gp48, contributes to the direct spread of viruses from lytically infected to uninfected cells. Monoclonal antibodies to gp48 identified it on infected cell surfaces and in virions. gp48-deficient viruses showed no obvious deficit in virion cell binding, single-cycle replication, or virion release but had reduced lytic propagation between cells. After intranasal infection of mice, ORF27-deficient viruses were impaired predominantly in lytic replication in the lungs. There was a small deficit in latency establishment, but long-term latency appeared normal. Since ORF27 has homologs in both Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, it is likely part of a conserved mechanism employed by gammaherpesviruses to disseminate lytically in their hosts.
Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency
Authors: Cohen Jeffrey I. , Krogmann Tammy , Bontems Sebastien , Sadzot-Delvaux Catherine , Pesnicak Lesley
Published: March 2005

Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is one of the most abundant transcripts expressed during VZV latency in humans, and ORF63 protein has been detected in human ganglia by several laboratories. Deletion of over 90% of the ORF63 gene showed that the protein is required for efficient establishment of latency in rodents. We have constructed viruses with a series of mutations in ORF63. While prior experiments showed that transfection of cells with a plasmid expressing ORF63 but lacking the putative nuclear localization signal of the protein resulted in increased expression of the protein in the cytoplasm, we found that ORF63 protein remained in the nucleus in cells infected with a VZV ORF63 nuclear localization signal deletion mutant. This mutant was not impaired for growth in cell culture or for latency in rodents. Replacement of five serine or threonine phosphorylation sites in ORF63 with alanines resulted in a virus that was impaired for replication in vitro and for latency. A series of ORF63 carboxy-terminal mutants showed that the last 70 amino acids do not affect replication in vitro or latency in rodents; however, the last 108 amino acids are important for replication and latency. Thus, regions of ORF63 that are important for replication in vitro are also required for efficient establishment of latency.
ND10 Components Relocate to Sites Associated with Herpes Simplex Virus Type 1 Nucleoprotein Complexes during Virus Infection
Authors: Everett Roger D. , Murray Jill
Published: March 2005

Infections with DNA viruses commonly result in the association of viral genomes and replication compartments with cellular nuclear substructures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. While there is evidence that viral genomes can associate with preexisting ND10, we demonstrate in this study by live-cell microscopy that structures resembling ND10 form de novo and in association with viral genome complexes during the initial stages of herpes simplex virus type 1 (HSV-1) infection. Consistent with previous studies, we found that the major ND10 proteins PML, Sp100, and hDaxx are exchanged very rapidly between ND10 foci and the surrounding nucleoplasm in live cells. The dynamic nature of the individual protein molecule components of ND10 provides a mechanism by which ND10 proteins can be recruited to novel sites during virus infection. These observations explain why the genomes and replication compartments of DNA viruses that replicate in the cell nucleus are so commonly found in association with ND10. These findings are discussed with reference to the nature, location, and potential number of HSV-1 prereplication compartments and to the dynamic aspects of HSV-1 genomes and viral products during the early stages of lytic infection.
Development and Assessment of Human Adenovirus Type 11 as a Gene Transfer Vector
Authors: Stone Daniel , Ni Shaoheng , Li Zong-Yi , Gaggar Anuj , DiPaolo Nelson , Feng Qinghua , Sandig Volker , Lieber André
Published: March 2005

Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low. E1-deleted Ad11 vector genomes were generated by homologous recombination in 293 cells expressing the Ad11-E1B55K protein or by recombination in
Escherichia coli
. E1-deleted Ad11 genomes did not display transforming activity in rodent cells. Transduction of primary human CD34
+
hematopoietic progenitor cells and immature dendritic cells was more efficient with Ad11 vectors than with Ad5 vectors. Thirty minutes after intravenous injection into mice that express one of the Ad11 receptors (CD46), we found, in a pattern and at a level comparable to what is found in humans, Ad11 vector genomes in all analyzed organs, with the highest amounts in liver, lung, kidney, and spleen. Neither Ad11 genomes nor Ad11 vector-mediated transgene expression were, however, detected at 72 h postinfusion. A large number of Ad11 particles were also found to be associated with circulating blood cells. We also discovered differences in in vitro transduction efficiencies and in vivo biodistributions between Ad11 vectors and chimeric Ad5 vectors possessing Ad11 fibers, indicating that Ad11 capsid proteins other than fibers influence viral infectivity and tropism. Overall, our study provides a basis for the application of Ad11 vectors for in vitro and in vivo gene transfer and for gaining an understanding of the factors that determine Ad tropism.
Expression of the Full-Length Form of gp2 of Equine Herpesvirus 1 (EHV-1) Completely Restores Respiratory Virulence to the Attenuated EHV-1 Strain KyA in CBA Mice
Authors: Smith Patrick M. , Kahan Shannon M. , Rorex Colin B. , von Einem Jens , Osterrieder Nikolaus , O'Callaghan Dennis J.
Published: March 2005

Wild-type equine herpesvirus 1 (EHV-1) strains express a large (250-kDa) glycoprotein, gp2, that is encoded by
EUs4
(gene 71) located within the unique short region of the genome. DNA sequence analysis revealed that
EUs4
of the pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp that encodes a protein of 791 amino acids. The attenuated EHV-1 vaccine strain KyA harbors an in-frame deletion of 1,242 bp from bp 222 to 1461 and expresses a truncated gp2 of 383 amino acids. To determine the relative contribution of gp2 to EHV-1 pathogenesis, we compared the course of respiratory infection of CBA mice infected with either wild-type RacL11, attenuated KyA, or a recombinant KyA that expresses the full-length gp2 protein (KyARgp2F). Mice infected with KyA lost a negligible amount of body weight (0.18% total weight loss) on day 1 postinfection and regained weight thereafter, whereas mice infected with KyARgp2F or RacL11 steadily lost weight beginning on day 1 and experienced a 20 and 18% loss in body weight, respectively, by day 3. Immunohistochemical and flow cytometric analyses revealed higher numbers of T and B lymphocytes and an extensive consolidation consisting of large numbers of Mac-1-positive cells in the lungs of animals infected with KyARgp2F compared to animals infected with KyA. RNase protection analyses revealed increased expression of numerous cytokines and chemokines, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2, interferon γ-inducible protein, monocyte chemotactic protein 1, and T-cell activation gene 3 at 12 h postinfection with KyARgp2F. Three independent DNA array experiments confirmed these results and showed a 2- to 13-fold increase in the expression of 31 inflammatory genes at 8 and 12 h postinfection with KyARgp2F compared to infection with KyA. Taken together, the results indicate that expression of full-length gp2 is sufficient to restore full respiratory virulence to the attenuated KyA strain and raise caution concerning the inclusion of full-length gp2 in the development of EHV-1 vaccines.
Inhibition of Dengue Virus Serotypes 1 to 4 in Vero Cell Cultures with Morpholino Oligomers
Authors: Kinney Richard M. , Huang Claire Y.-H. , Rose Becky C. , Kroeker Andrew D. , Dreher Theo W. , Iversen Patrick L. , Stein David A.
Published: March 2005

Five dengue (DEN) virus-specific R
5
F
2
R
4
peptide-conjugated phosphorodiamidate morpholino oligomers (P4-PMOs) were evaluated for their ability to inhibit replication of DEN virus serotype 2 (DEN-2 virus) in mammalian cell culture. Initial growth curves of DEN-2 virus 16681 were obtained in Vero cells incubated with 20 μM P4-PMO compounds. At 6 days after infection, a P4-PMO targeting the 3′-terminal nucleotides of the DEN-2 virus genome and a random-sequence P4-PMO showed relatively little suppression of DEN-2 virus titer (0.1 and 0.9 log
10
, respectively). P4-PMOs targeting the AUG translation start site region of the single open reading frame and the 5′ cyclization sequence region had moderate activity, generating 1.6- and 1.8-log
10
reductions. Two P4-PMO compounds, 5′SL and 3′CS (targeting the 5′-terminal nucleotides and the 3′ cyclization sequence region, respectively), were highly efficacious, each reducing the viral titer by greater than 5.7 log
10
compared to controls at 6 days after infection with DEN-2 virus. Further experiments showed that 5′SL and 3′CS inhibited DEN-2 virus replication in a dose-dependent and sequence-specific manner. Treatment with 10 μM 3′CS reduced the titers of all four DEN virus serotypes, i.e., DEN-1 (strain 16007), DEN-2 (16681), DEN-3 (16562), and DEN-4 (1036) viruses by over 4 log
10
, in most cases to below detectable limits. The extent of 3′CS efficacy was affected by the timing of compound application in relation to viral infection of the cells. The 5′SL and 3′CS P4-PMOs did not suppress the replication of West Nile virus NY99 in Vero cells. These data indicate that further evaluation of the 5′SL and 3′CS compounds as potential DEN virus therapeutics is warranted.
Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication
Authors: Jia Qingmei , Chernishof Vasili , Bortz Eric , Mchardy Ian , Wu Ting-Ting , Liao Hsiang-I , Sun Ren
Published: March 2005

Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the
Gammaherpesvirinae
subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in
trans
or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the
Gammaherpesvirinae
subfamily are critical for the function of ORF45 in viral replication.
A Peptide Pertaining to the Loop Segment of Human Immunodeficiency Virus gp41 Binds and Interacts with Model Biomembranes: Implications for the Fusion Mechanism
Authors: Pascual Roberto , Moreno Miguel R. , Villalaín José
Published: March 2005

The human immunodeficiency virus gp41 envelope protein mediates the entry of the virus into the target cell by promoting membrane fusion. In order to gain new insights into the viral fusion mechanism, we studied a 35-residue peptide pertaining to the loop domain of gp41, both in solution and membrane bound, by using infrared and fluorescence spectroscopy. We show here that the peptide, which has a membrane-interacting surface, binds and interacts with phospholipid model membranes and tends to aggregate in the presence of a membranous medium and induce the leakage of vesicle contents. The results reported in this work, i.e., the destabilization and fusion of negatively charged model membranes, suggest an essential role of the loop domain in the membrane fusion process induced by gp41.
Plateau Levels of Viremia Correlate with the Degree of CD4+-T-Cell Loss in Simian Immunodeficiency Virus SIVagm-Infected Pigtailed Macaques: Variable Pathogenicity of Natural SIVagm Isolates
Authors: Goldstein Simoy , Ourmanov Ilnour , Brown Charles R. , Plishka Ronald , Buckler-White Alicia , Byrum Russell , Hirsch Vanessa M.
Published: March 2005

Simian immunodeficiency virus from African green monkeys (SIVagm) results in asymptomatic infection in its natural host species. The virus is not inherently apathogenic, since infection of pigtailed (PT) macaques (Macaca nemestrina) with one isolate of SIVagm results in an immunodeficiency syndrome characterized by progressive CD4+-T-cell depletion and opportunistic infections. This virus was passaged once in a PT macaque and, thus, may not be entirely reflective of the virulence of the parental strain. The goal of the present study was to assess the pathogenicity of the PT-passaged isolate (SIVagm9063) and two primary SIVagm isolates in PT macaques, including the parental strain of the PT-passaged variant. Infection of macaques with any of the three isolates resulted in high levels of primary plasma viremia by 1 week after inoculation. Viremia was quickly controlled following infection with SIVagm155; these animals have maintained CD4+-T-cell subsets and remain healthy. The plateau levels among SIVagm90- and SIVagm9063-inoculated macaques varied widely from 100 to 1 million copies/ml of plasma. Three of four animals from each of these groups progressed to AIDS. Setpoint viremia and the degree of CD4+-T-cell loss at 6 months postinfection were not significantly different between macaques inoculated with SIVagm90 and SIVagm9063. However these parameters were significantly different in SIVagm155-inoculated macaques (Pvalues of <0.01). Considering all the macaques, the degree of CD4+-T-cell loss by 6 months postinfection correlated with the plateau levels of viremia. Thus, similar to SIVsm/mac infection of macaques and human AIDS, viral load is an excellent prognostic indicator of disease course. The inherent pathogenicity of natural SIVagm isolates varies, but such natural isolates are capable of inducing AIDS in macaques without prior macaque passage.
A Proline-Rich Region in the Coxsackievirus 3A Protein Is Required for the Protein To Inhibit Endoplasmic Reticulum-to-Golgi Transport
Authors: Wessels Els , Duijsings Daniël , Notebaart Richard A. , Melchers Willem J. G. , van Kuppeveld Frank J. M.
Published: March 2005

The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro
19
completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro
14
, Pro
17
, or Pro
20
also impaired this ability, but to a lesser extent. The mutation of Pro
18
had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.