bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Interferon Gamma by a Recombinant Rabies Virus Strongly Attenuates the Pathogenicity of the Virus via Induction of Type I Interferon
Authors: Barkhouse Darryll A. , Garcia Samantha A. , Bongiorno Emily K. , Lebrun Aurore , Faber Milosz , Hooper D. Craig 
Published: October 2014

ABSTRACT

Previous animal model experiments have shown a correlation between interferon gamma (IFN-γ) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN-γ by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN-γ gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBNγ. Morbidity and mortality were monitored in mice infected intranasally with SPBNγ or SPBN(−) control virus to determine the degree of attenuation caused by the expression of IFN-γ. Incorporation of IFN-γ into the rabies virus genome highly attenuated the virus. SPBNγ has a 50% lethal dose (LD
50)
more than 100-fold greater than SPBN(−).
In vitro
and
in vivo
mouse experiments show that SPBNγ infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNAR
−/−
) cannot control the SPBNγ infection and rapidly die. These data suggest that IFN-γ production has antiviral effects in rabies, largely due to the induction of type I interferons.

IMPORTANCE
Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN-γ production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN-γ triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN-γ directly in the infected tissue reduces rabies virus replication and spread, limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN-γ to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus.
Nonstructural Protein 5A (NS5A) and Human Replication Protein A Increase the Processivity of Hepatitis C Virus NS5B Polymerase Activity
In Vitro
Authors: Mani Nagraj , Yuzhakov Alexander , Yuzhakov Olga , Coll Joyce T. , Black Jim , Saxena Kumkum , Fulghum John R. , Lippke Judith A. , Rao B. Govinda , Rijnbrand Rene , Kwong Ann D. 
Published: October 2014

ABSTRACT

The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel
in vitro
replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A
(S25-C447)
, but not NS5A
(S25-K215)
, enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner
in vitro
. This new
in vitro
HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication.

IMPORTANCE
The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an
in vitro
HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A
(S25-C447)
and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5BΔ21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.
Complex T-Cell Receptor Repertoire Dynamics Underlie the CD8
+
T-Cell Response to HIV-1
Authors: Costa Ana I. , Koning Dan , Ladell Kristin , McLaren James E. , Grady Bart P. X. , Schellens Ingrid M. M. , van Ham Petra , Nijhuis Monique , Borghans José A. M. , Keşmir Can , Price David A. , van Baarle Debbie 
Published: October 2014

ABSTRACT

Although CD8
+
T cells are important for the control of HIV-1
in vivo
, the precise correlates of immune efficacy remain unclear. In this study, we conducted a comprehensive analysis of viral sequence variation and T-cell receptor (TCR) repertoire composition across multiple epitope specificities in a group of antiretroviral treatment-naive individuals chronically infected with HIV-1. A negative correlation was detected between changes in antigen-specific TCR repertoire diversity and CD8
+
T-cell response magnitude, reflecting clonotypic expansions and contractions related to alterations in cognate viral epitope sequences. These patterns were independent of the individual, as evidenced by discordant clonotype-specific transitions directed against different epitopes in single subjects. Moreover, long-term asymptomatic HIV-1 infection was characterized by evolution of the TCR repertoire in parallel with viral replication. Collectively, these data suggest a continuous bidirectional process of adaptation between HIV-1 and virus-specific CD8
+
T-cell clonotypes orchestrated at the TCR-antigen interface.

IMPORTANCE
We describe a relation between viral epitope mutation, antigen-specific T-cell expansion, and the repertoire of responding clonotypes in chronic HIV-1 infection. This work provides insights into the process of coadaptation between the human immune system and a rapidly evolving lentivirus.
Insight into the Mechanism of Inhibition of Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex
Authors: Lentz Thomas B. , Samulski R. Jude 
Published: October 2014

ABSTRACT

Adeno-associated virus (AAV) is a dependent virus of the family
Parvoviridae
. The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable.

IMPORTANCE
Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on coinfecting helper virus, such as adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications for understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance and likely translate to other viruses that must navigate the DDR.
Novel Mode of ISG15-Mediated Protection against Influenza A Virus and Sendai Virus in Mice
Authors: Morales David J. , Monte Kristen , Sun Lulu , Struckhoff Jessica J. , Agapov Eugene , Holtzman Michael J. , Stappenbeck Thaddeus S. , Lenschow Deborah J. 
Published: October 2014

ABSTRACTISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15−/−mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replicationin vivo. A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting the virus burden. In order to better understand the function of ISG15in vivo, we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15−/−mice. We found that ISG15 protects mice from virus induced lethality by a conjugation-dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugation-dependent, but nonantiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15in vivo.IMPORTANCEIt has previously been demonstrated that ISG15−/−mice are more susceptible to a number of viral infections. Since ISG15 is one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. Although a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protectionin vivo. In these studies we have found that while ISG15−/−mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus, in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15in vivo.
Quantitative Proteomics Identifies Host Factors Modulated during Acute Hepatitis E Virus Infection in the Swine Model
Authors: Rogée Sophie , Le Gall Morgane , Chafey Philippe , Bouquet Jérôme , Cordonnier Nathalie , Frederici Christian , Pavio Nicole 
Published: October 2014

ABSTRACT

Hepatitis E virus (HEV) causes acute enterically transmitted hepatitis. In industrialized
c
ountries, it is a zoonotic disease, with swine being the major reservoir of human HEV contamination. The occurrence and severity of the disease are variable, with clinical symptoms ranging from asymptomatic to self-limiting acute hepatitis, chronic infection, or fulminant hepatitis. In the absence of a robust cell culture system or small-animal models, the HEV life cycle and pathological process remain unclear. To characterize HEV pathogenesis and virulence mechanisms, a quantitative proteomic analysis was carried out to identify cellular factors and pathways modulated during acute infection of swine. Three groups of pigs were inoculated with three different strains of swine HEV to evaluate the possible role of viral determinants in pathogenesis. Liver samples were analyzed by a differential proteomic approach, two-dimensional difference in gel electrophoresis, and 61 modulated proteins were identified by mass spectroscopy. The results obtained show that the three HEV strains replicate similarly in swine and that they modulate several cellular pathways, suggesting that HEV impairs several cellular processes, which can account for the various types of disease expression. Several proteins, such as heterogeneous nuclear ribonucleoprotein K, apolipoprotein E, and prohibitin, known to be involved in other viral life cycles, were upregulated in HEV-infected livers. Some differences were observed between the three strains, suggesting that HEV's genetic variability may induce variations in pathogenesis. This comparative analysis of the liver proteome modulated during infection with three different strains of HEV genotype 3 provides an important basis for further investigations on the factors involved in HEV replication and the mechanism of HEV pathogenesis.

IMPORTANCE
Hepatitis E virus (HEV) is responsible for acute hepatitis, with clinical symptoms ranging from asymptomatic to self-limiting acute hepatitis, chronic infection, or fulminant hepatitis. In industrialized countries, HEV is considered an emerging zoonotic disease, with swine being the principal reservoir for human contamination. The viral and cellular factors involved in the replication and/or pathogenesis of HEV are still not fully known. Here we report that several cellular pathways involved in cholesterol and lipid metabolism or cell survival were modulated during HEV infection in the swine model. Moreover, we observed a difference between the different swine strains, suggesting that HEV's genetic variability could play a role in pathogenesis. We also identified some proteins known to be involved in other viral cycles. Our study provides insight into the mechanisms modulated during HEV infection and constitutes a useful reference for future work on HEV pathogenesis and virulence.
Rhabdovirus-Based Vaccine Platforms against Henipaviruses
Authors: Kurup Drishya , Wirblich Christoph , Feldmann Heinz , Marzi Andrea , Schnell Matthias J. 
Published: October 2014

ABSTRACT

The emerging zoonotic pathogens Hendra virus (HeV) and Nipah virus (NiV) are in the genus
Henipavirus
in the family
Paramyxoviridae
. HeV and NiV infections can be highly fatal to humans and livestock. The goal of this study was to develop candidate vaccines against henipaviruses utilizing two well-established rhabdoviral vaccine vector platforms, recombinant rabies virus (RABV) and recombinant vesicular stomatitis virus (VSV), expressing either the codon-optimized or the wild-type (wt) HeV glycoprotein (G) gene. The RABV vector expressing the codon-optimized HeV G showed a 2- to 3-fold increase in incorporation compared to the RABV vector expressing wt HeV G. There was no significant difference in HeV G incorporation in the VSV vectors expressing either wt or codon-optimized HeV G. Mice inoculated intranasally with any of these live recombinant viruses showed no signs of disease, including weight loss, indicating that HeV G expression and incorporation did not increase the neurotropism of the vaccine vectors. To test the immunogenicity of the vaccine candidates, we immunized mice intramuscularly with either one dose of the live vaccines or 3 doses of 10 μg chemically inactivated viral particles. Increased codon-optimized HeV G incorporation into RABV virions resulted in higher antibody titers against HeV G compared to inactivated RABV virions expressing wt HeV G. The live VSV vectors induced more HeV G-specific antibodies as well as higher levels of HeV neutralizing antibodies than the RABV vectors. In the case of killed particles, HeV neutralizing serum titers were very similar between the two platforms. These results indicated that killed RABV with codon-optimized HeV G should be the vector of choice as a dual vaccine in areas where rabies is endemic.

IMPORTANCE
Scientists have been tracking two new viruses carried by the Pteropid fruit bats: Hendra virus (HeV) and Nipah virus (NiV). Both viruses can be fatal to humans and also pose a serious risk to domestic animals. A recent escalation in the frequency of outbreaks has increased the need for a vaccine that prevents HeV and NiV infections. In this study, we performed an extensive comparison of live and killed particles of two recombinant rhabdoviral vectors, rabies virus and vesicular stomatitis virus (VSV), expressing wild-type or codon-optimized HeV glycoprotein, with the goal of developing a candidate vaccine against HeV. Based on our data from the presented mouse immunogenicity studies, we conclude that a killed RABV vaccine would be highly effective against HeV infections and would make an excellent vaccine candidate in areas where both RABV and henipaviruses pose a threat to human health.
Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration
Authors: Knight Laura M. , Stakaityte Gabriele , Wood Jennifer, J. , Abdul-Sada Hussein , Griffiths David A. , Howell Gareth J. , Wheat Rachel , Blair G. Eric , Steven Neil M. , Macdonald Andrew , Blackbourn David J. , Whitehouse Adrian 
Published: October 2014

ABSTRACT
Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV small and large tumor antigens (ST and LT, respectively). Although a number of molecular mechanisms have been attributed to MCPyV tumor antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organization and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilization, leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilization and cell motility and implicate the cellular phosphatase catalytic subunit protein phosphatase 4C (PP4C) in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC.

IMPORTANCE
Merkel cell polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally induced cancer development have yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV small tumor antigen (ST) promotes the destabilization of the host cell microtubule network, which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule-associated protein stathmin by its known association with the cellular phosphatase catalytic subunit PP4C. These findings highlight stathmin as a possible biomarker of MCC and as a target for novel antitumoral therapies.
Topical Herpes Simplex Virus 2 (HSV-2) Vaccination with Human Papillomavirus Vectors Expressing gB/gD Ectodomains Induces Genital-Tissue-Resident Memory CD8+T Cells and Reduces Genital Disease and Viral Shedding after HSV-2 Challenge
Authors: Çuburu Nicolas , Wang Kening , Goodman Kyle N. , Pang Yuk Ying , Thompson Cynthia D. , Lowy Douglas R. , Cohen Jeffrey I. , Schiller John T. 
Published: October 2014

ABSTRACTNo herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8+T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8+T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection.IMPORTANCEGenital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a nonreplicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV-based vectors as a platform for the induction of genital-tissue-resident memory T cell responses and the control of local manifestations of primary HSV infection.