bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hepatitis C Virus NS2 Protein Is Phosphorylated by the Protein Kinase CK2 and Targeted for Degradation to the Proteasome
Authors: Franck Nathalie , Le Seyec Jacques , Guguen-Guillouzo Christiane , Erdtmann Lars 
Published: February 2005

ABSTRACT
Hepatitis C virus (HCV) nonstructural 2 (NS2) protein is a hydrophobic transmembrane protein, described to be involved in different functions, such as apoptosis inhibition and gene transcription modulation. We investigated here NS2 protein turnover and found that NS2 was rapidly degraded by the proteasome in different cell lines, as in primary human hepatocytes. Since posttranslational modifications can influence protein turnover, we looked for potential phosphoacceptor sites in NS2. Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation. In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE). This motif was highly conserved since it could be found in the NS2 primary consensus sequences from all HCV genotypes. To verify whether CK2 is involved in NS2 phosphorylation, we showed by an in vitro kinase assay that CK2 phosphorylated NS2, as far as this CK2 motif was conserved. Interestingly, NS2 became resistant to protein degradation when the CK2 motif was modified by a single point mutation. Furthermore, inhibition of CK2 activity by curcumin decreased NS2 phosphorylation in vitro and stabilized NS2 expression in HepG2 cells. Finally, we showed in Huh-7.5 replicon cells that NS2, expressed in the context of the HCV polyprotein, was also sensitive to both proteasome-mediated degradation and CK2 inhibitor treatment. We suggest that NS2 is a short-lived protein whose degradation by the proteasome is regulated in a phosphorylation-dependent manner through the protein kinase CK2.
Early Antiretroviral Therapy for Simian Immunodeficiency Virus Infection Leads to Mucosal CD4+T-Cell Restoration and Enhanced Gene Expression Regulating Mucosal Repair and Regeneration
Authors: George Michael D. , Reay Elizabeth , Sankaran Sumathi , Dandekar Satya 
Published: February 2005

ABSTRACTSimian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections lead to rapid depletion of CD4+T cells from gut-associated lymphoid tissue (GALT). Although the administration of antiretroviral therapy (ART) has been shown to increase CD4+T-cell levels in the peripheral blood in both SIV and HIV infections, its efficacy in restoring intestinal mucosal CD4+T cells has not been well investigated. To gain insights into the molecular mechanisms of virally induced disruptions in the mucosal immune system, we have evaluated longitudinal changes in viral burden, T-cell subsets, and mucosal gene expression profiles in SIV-infected rhesus macaques in the absence or presence of ART. Our results demonstrate a dramatic suppression of mucosal viral loads and rapid reconstitution of CD4+T cells in GALT in animals receiving ART that were not observed in untreated SIV-infected animals. DNA microarray-based gene expression profiling indicated that CD4+T-cell restoration in GALT was associated with up regulation of growth factors and genes involved in repair and regeneration of the mucosal epithelium. In contrast, untreated SIV-infected animals increased expression of lymphocyte activation and inflammatory response-associated genes and did not up regulate mucosal growth and repair associated transcription. In conclusion, these data indicate that initiating ART in primary SIV infection may lead to the restoration of the mucosal immune system through reduction of inflammation and promotion of epithelial repair in the intestinal mucosa.
Nonstructural Protein σ1s Is a Determinant of Reovirus Virulence and Influences the Kinetics and Severity of Apoptosis Induction in the Heart and Central Nervous System
Authors: Hoyt Cristen C. , Richardson-Burns Sarah M. , Goody Robin J. , Robinson Bridget A. , DeBiasi Roberta L. , Tyler Kenneth L. 
Published: February 2005

ABSTRACT

The mechanisms by which viruses kill susceptible cells in target organs and ultimately produce disease in the infected host remain poorly understood. Dependent upon the site of inoculation and strain of virus, experimental infection of neonatal mice with reoviruses can induce fatal encephalitis or myocarditis. Reovirus-induced apoptosis is a major mechanism of tissue injury, leading to disease development in both the brain and heart. In cultured cells, differences in the capacity of reovirus strains to induce apoptosis are determined by the S1 gene segment, which also plays a major role as a determinant of viral pathogenesis in both the heart and the central nervous system (CNS) in vivo. The S1 gene is bicistronic, encoding both the viral attachment protein sigma-1 and the nonstructural protein sigma-1-small (σ1s). Although σ1s is dispensable for viral replication in vitro, we wished to investigate the expression of σ1s in the infected heart and brain and its potential role in reovirus pathogenesis in vivo. Two-day-old mice were inoculated intramuscularly or intracerebrally with either σ1s
−
or σ1s
+
reovirus strains. While viral replication in target organs did not differ between σ1s
−
and σ1s
+
viral strains, virus-induced caspase-3 activation and resultant histological tissue injury in both the heart and brain were significantly reduced in σ1s
−
reovirus-infected animals. These results demonstrate that σ1s is a determinant of the magnitude and extent of reovirus-induced apoptosis in both the heart and CNS and thereby contributes to reovirus pathogenesis and virulence.
Human Cytomegalovirus Labeled with Green Fluorescent Protein for Live Analysis of Intracellular Particle Movements
Authors: Sampaio Kerstin Laib , Cavignac Yolaine , Stierhof York-Dieter , Sinzger Christian 
Published: February 2005

ABSTRACT
Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.
Human Cytomegalovirus Protein US11 Provokes an Unfolded Protein Response That May Facilitate the Degradation of Class I Major Histocompatibility Complex Products
Authors: Tirosh Boaz , Iwakoshi Neal N. , Lilley Brendan N. , Lee Ann-Hwee , Glimcher Laurie H. , Ploegh Hidde L. 
Published: February 2005

ABSTRACT
The human cytomegalovirus (HCMV) glycoprotein US11 diverts class I major histocompatibility complex (MHC) heavy chains (HC) from the endoplasmic reticulum (ER) to the cytosol, where HC are subjected to proteasome-mediated degradation. In mouse embryonic fibroblasts that are deficient for X-box binding protein 1 (XBP-1), a key transcription factor in the unfolded protein response (UPR) pathway, we show that degradation of endogenous mouse HC is impaired. Moreover, the rate of US11-mediated degradation of ectopically expressed HLA-A2 is reduced when XBP-1 is absent. In the human astrocytoma cell line U373, turning on expression of US11, but not US2, is sufficient to induce a UPR, as manifested by upregulation of the ER chaperone Bip and by splicing of XBP-1 mRNA. In the presence of dominant-negative versions of XBP-1 and activating transcription factor 6, the kinetics of class I MHC HC degradation were delayed when expression of US11 was turned on. The magnitude of these effects, while reproducible, was modest. Conversely, in cells that stably express high levels of US11, the degradation of HC is not affected by the presence of the dominant negative effectors of the UPR. An infection of human foreskin fibroblasts with human cytomegalovirus induced XBP-1 splicing in a manner that coincides with US11 expression. We conclude that the contribution of the UPR is more pronounced on HC degradation shortly after induction of US11 expression and that US11 is sufficient to induce such a response.
Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Authors: Kino Tomoshige , Gragerov Alexander , Valentin Antonio , Tsopanomihalou Maria , Ilyina-Gragerova Galina , Erwin-Cohen Rebecca , Chrousos George P. , Pavlakis George N. 
Published: February 2005

ABSTRACT

Vpr and selected mutants were used in a
Saccharomyces cerevisiae
two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G
2
/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3σ. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G
2
/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status.
Detection of Antibody-Dependent Complement-Mediated Inactivation of both Autologous and Heterologous Virus in Primary Human Immunodeficiency Virus Type 1 Infection
Authors: Aasa-Chapman Marlén M. I. , Holuigue Sophie , Aubin Keith , Wong MaiLee , Jones Nicola A. , Cornforth David , Pellegrino Pierre , Newton Philippa , Williams Ian , Borrow Persephone , McKnight Áine 
Published: February 2005

ABSTRACTSpecific CD8 T-cell responses to human immunodeficiency virus type 1 (HIV-1) are induced in primary infection and make an important contribution to the control of early viral replication. The importance of neutralizing antibodies in containing primary viremia is questioned because they usually arise much later. Nevertheless antienvelope antibodies develop simultaneously with, or even before, peak viremia. We determined whether such antibodies might control viremia by complement-mediated inactivation (CMI). In each of seven patients studied, antibodies capable of CMI appeared at or shortly after the peak in viremia, concomitantly with detection of virus-specific T-cell responses. The CMI was effective on both autologous and heterologous HIV-1 isolates. Activation of the classical pathway and direct viral lysis were at least partly responsible. Since immunoglobulin G (IgG)-antibodies triggered the CMI, specific memory B cells could also be induced by vaccination. Thus, consideration should be given to vaccination strategies that induce IgG antibodies capable of CMI.
Functional Interaction of the Adenovirus IVa2 Protein with Adenovirus Type 5 Packaging Sequences
Authors: Ostapchuk Philomena , Yang Jihong , Auffarth Ece , Hearing Patrick 
Published: February 2005

ABSTRACT

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the
cis
-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting
trans
-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.
Positive and Negative Regulation of Chicken Anemia Virus Transcription
Authors: Miller Myrna M. , Jarosinski Keith W. , Schat Karel A. 
Published: February 2005

ABSTRACT
Chicken anemia virus (CAV) is a small circular single-stranded DNA virus with a single promoter-enhancer region containing four consensus cyclic AMP response element sequences (AGCTCA), which are similar to the estrogen response element (ERE) consensus half-sites (A)GGTCA. These sequences are arranged as direct repeats, an arrangement that can be recognized by members of the nuclear receptor superfamily. Transient-transfection assays which use a short CAV promoter construct that ended at the transcription start site and drive expression of enhanced green fluorescent protein (EGFP) showed high basal activity in DF-1, LMH, LMH/2A, and primary theca and granulosa cells. The estrogen receptor-enhanced cell line, LMH/2A, had significantly greater expression than LMH cells, and this expression was significantly increased with estrogen treatment. A long promoter construct which included GGTCA-like sequences downstream of the first CAV protein translation start site was found to have significantly less EGFP expression in DF-1 cells than the short promoter, which was largely due to decreased RNA transcription. DNA-protein binding assays indicated that proteins recognizing a consensus ERE palindrome also bind GGTCA-like sequences in the CAV promoter. Estrogen receptor and other members of the nuclear receptor superfamily may provide a mechanism to regulate CAV activity in situations of low virus copy number.
Identification of Cytomegalovirus-Specific Cytotoxic T Lymphocytes In Vitro Is Greatly Enhanced by the Use of Recombinant Virus Lacking the US2 to US11 Region or Modified Vaccinia Virus Ankara Expressing Individual Viral Genes
Authors: Khan Naeem , Bruton Rachel , Taylor Graham S. , Cobbold Mark , Jones Thomas R. , Rickinson Alan B. , Moss Paul A. H. 
Published: February 2005

ABSTRACT
Cytomegalovirus (CMV) elicits a potent T-cell response in humans that appears to protect the host from virus-associated disease. Despite facing strong host defense mechanisms, CMV remains as a lifelong infection that may reactivate and cause life-threatening disease in immunocompromised individuals. This persistence is probably assisted by expression of immune subversion proteins of the virus encoded by genes belonging to the US gene family. These proteins modulate major histocompatibility complex expression in infected cells and bias in vitro experiments toward the detection of only certain specificities. We have combined the use of recombinant CMV, lacking the US2 to US11 region genes, and cytoplasmic gamma interferon staining to define a more accurate assessment of CMV-specific responses in vivo. Recombinant CMV stimulation reveals a CD8 response much larger than that of parental virus in all donors tested. In some cases, this represented up to 10-fold increases in the number of cells detected. Responses were directed mainly against pp65, IE-1, and pp50 in the majority of donors. In addition, previously unreported IE-2-specific T-cell responses could be detected in a minority of cases. Furthermore, we observed a less marked increase in the response to mutant CMV by CD4 T cells in some donors. This suggests that a much broader T-cell response to CMV exists in vivo than is revealed by restimulation with wild-type virus and adds to the evidence that the efficacy of immune evasion strategies may not be as absolute as previously believed.
Establishment and Maintenance of Long-Term Murine Gammaherpesvirus 68 Latency in B Cells in the Absence of CD40
Authors: Willer David O. , Speck Samuel H. 
Published: February 2005

ABSTRACTMurine gammaherpesvirus 68 (γHV68), like Epstein-Barr virus (EBV), establishes a chronic infection in its host by gaining access to the memory B-cell reservoir, where it persists undetected by the host's immune system. EBV encodes a membrane protein, LMP1, that appears to function as a constitutively active CD40 receptor, and is hypothesized to play a central role in EBV-driven differentiation of infected naive B cells to a memory B-cell phenotype. However, it has recently been shown that there is a critical role for CD40-CD40L interaction in B-cell immortalization by EBV (K.-I. Imadome, M. Shirakata, N. Shimizu, S. Nonoyama, and Y. Yamanashi, Proc. Natl. Acad. Sci. USA 100:7836-7840, 2003), indicating that LMP1 does not adequately recapitulate all of the necessary functions of CD40. The role of CD40 receptor expression on B cells for the establishment and maintenance of γHV68 latency is unclear. Data previously obtained with a competition model, demonstrated that in the face of CD40-sufficient B cells, γHV68 latency in CD40-deficient B cells waned over time in chimeric mice (I.-J. Kim, E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and M. A. Blackman, J. Immunol. 171:886-892, 2003). To further investigate the role of CD40 in γHV68 latency in vivo, we have characterized the infection of CD40 knockout (CD40−/−) mice. Here we report that, consistent with previous observations, γHV68 efficiently established a latent infection in B cells of CD40−/−mice. Notably, unlike the infection of normal C57BL/6 mice, significant ex vivo reactivation from splenocytes harvested from infected CD40−/−mice 42 days postinfection was observed. In addition, in contrast to γHV68 infection of C57BL/6 mice, the frequency of infected naive B cells remained fairly stable over a 3-month period postinfection. Furthermore, a slightly higher frequency of γHV68 infection was observed in immunoglobulin D (IgD)-negative B cells, which was stably maintained over a period of 3 months postinfection. The presence of virus in IgD-negative B cells indicates that γHV68 may either directly infect memory B cells present in CD40−/−mice or be capable of driving differentiation of naive CD40−/−B cells. A possible explanation for the apparent discrepancy between the failure of γHV68 latency to be maintained in CD40-deficient B cells in the presence of CD40-sufficient B cells and the stable maintenance of γHV68 B-cell latency in CD40−/−mice came from examining virus replication in the lungs of infected CD40−/−mice, where we observed significantly higher levels of virus replication at late times postinfection compared to those in infected C57BL/6 mice. Taken together, these findings are consistent with a model in which chronic virus infection of CD40−/−mice is maintained through virus reactivation in the lungs and reseeding of latency reservoirs.
Cellular and Humoral Immunity following Snow Mountain Virus Challenge
Authors: Lindesmith Lisa , Moe Christine , LePendu Jacques , Frelinger Jeffrey A. , Treanor John , Baric Ralph S. 
Published: February 2005

ABSTRACTLittle is known about the immune response to noroviruses. To elucidate the immunobiology of norovirus infection in humans, 15 volunteers were challenged with Snow Mountain virus (SMV), a genogroup 2 norovirus. We assessed the cellular and humoral immune response and infection by analyzing stool, serum, saliva, and peripheral blood mononuclear cell (PBMC) responses pre- and postchallenge. In contrast to Norwalk virus (NV), SMV infection was not dependent upon blood group secretor status. Nine of 15 volunteers were infected and showed a ≥4-fold increase over the prechallenge anti-SMV serum immunoglobulin G (IgG) titer, mostly subclass IgG1. Although serum IgG elicited by SMV infection was cross-reactive with Hawaii virus (HV), another genogroup 2 norovirus, salivary IgA was less cross-reactive. Neither SMV-elicited serum IgG nor salivary IgA cross-reacted with NV, a genogroup 1 norovirus. Significant increases in serum gamma interferon (IFN-γ) and IL-2, but not IL-6 or IL-10, were noted on day 2 postchallenge. For the majority of volunteers, both infected and uninfected, PBMCs stimulated with norovirus virus-like particles secreted IFN-γ and other Th1 cytokines, suggesting previous norovirus exposure in most volunteers. Like the IgG antibodies, the SMV-activated T cells were cross-reactive with HV but not NV. IFN-γ production was dependent upon CD4+cells, consistent with a predominant, but not exclusive, Th1 response. To our knowledge, this is the first report characterizing T-cell and cytokine responses following live norovirus challenge.