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Chi-Ping Chan, Chun-Kit Yuen, Pak-Hin Hinson Cheung, Sin-Yee Fung, Pak-Yin Lui, Honglin Chen, Kin-Hang Kok, Dong-Yan Jin
PACT is a double-stranded RNA-binding protein that has been implicated in host-influenza A virus (IAV) interaction. PACT facilitates the action of RIG-I in the activation of the type I IFN response, which is suppressed by the viral nonstructural protein NS1. PACT is also known to interact with the IAV RNA polymerase subunit PA. Exactly how PACT exerts its antiviral activity during IAV infection remains to be elucidated. In the current study, we demonstrated the interplay between PACT and IAV polymerase. Induction of IFN-β by the IAV RNP complex was most robust when both RIG-I and PACT were expressed...
March 7, 2018: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
Aneth S Canale, Sergey V Venev, Troy W Whitfield, Daniel R Caffrey, Wayne A Marasco, Celia A Schiffer, Timothy F Kowalik, Jeffrey D Jensen, Robert W Finberg, Konstantin B Zeldovich, Jennifer P Wang, Daniel N A Bolon
The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious in both bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects...
February 18, 2018: Journal of Molecular Biology
Joshua J Vasquez, Rajaa Hussien, Brandon Aguilar-Rodriguez, Henrik Junger, Dejan Dobi, Timothy J Henrich, Cassandra Thanh, Erica Gibson, Louise E Hogan, Joseph McCune, Peter W Hunt, Cheryl A Stoddart, Zoltan G Laszik
Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal...
February 1, 2018: Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society
Graham D Williams, Dana Townsend, Kristine M Wylie, Preston J Kim, Gaya K Amarasinghe, Sebla B Kutluay, Adrianus C M Boon
Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Here we applied photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP-vRNA interactions in infected human cells. NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging...
January 31, 2018: Nature Communications
Sunil More, Xiaoyun Yang, Zhengyu Zhu, Gayan Bamunuarachchi, Yujie Guo, Chaoqun Huang, Keith Bailey, Jordan P Metcalf, Lin Liu
Wnt/β-catenin signaling is an essential pathway in cell cycle control. Dysregulation of the Wnt/β-catenin signaling pathway during viral infection has been reported. In this study, we examined the effect of modulating Wnt/β-catenin signaling during influenza virus infection. The activation of the Wnt/β-catenin pathway by Wnt3a increased influenza virus mRNA and virus production in in vitro in mouse lung epithelial E10 cells and mRNA expresson of influenza virus genes in vivo in the lungs of mice infected with influenza virus A/Puerto Rico/8/34...
2018: PloS One
Autumn T LaPointe, Natasha N Gebhart, Megan E Meller, Richard W Hardy, Kevin J Sokoloski
Arthropod-borne viruses, such as the members of genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish, and complete, their viral lifecycles. Despite considerable efforts to define the host/pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA:protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized...
January 10, 2018: Journal of Virology
Takeshi Noda, Shin Murakami, Sumiho Nakatsu, Hirotaka Imai, Yukiko Muramoto, Keiko Shindo, Hiroshi Sagara, Yoshihiro Kawaoka
The influenza A virus genome is composed of eight single-stranded negative-sense RNAs. Eight distinct viral RNA segments (vRNAs) are selectively packaged into progeny virions, with eight vRNAs in ribonucleoprotein complexes (RNPs) arranged in a specific "1+7" pattern, that is, one central RNP surrounded by seven RNPs. Here we report the genome packaging of an artificially generated seven-segment virus that lacks the hemagglutinin (HA) vRNA. Electron microscopy shows that, even in the presence of only seven vRNAs, the virions efficiently package eight RNPs arranged in the same "1+7" pattern as wild-type virions...
January 4, 2018: Nature Communications
Milena A Wasik, Luca Eichwald, Yvonne Genzel, Udo Reichl
Defective interfering particles (DIPs) lack an essential portion of the virus genome, but retain signals for replication and packaging, and therefore, interfere with standard virus (STV) replication. Due to this property, DIPs can be potential antivirals. The influenza A virus DIP DI244, generated during propagation in chicken eggs, has been previously described as a potential candidate for influenza antiviral therapy. As a cell culture-based manufacturing process would be more suitable to fulfill large-scale production needs of an antiviral and enables full process control in closed systems, we investigated options to produce DI244 in the avian cell line AGE1...
February 2018: Applied Microbiology and Biotechnology
Cheng-Kai Chang, Chi-Jene Chen, Chih-Ching Wu, Shiau-Wen Chen, Shin-Ru Shih, Rei-Lin Kuo
The viral ribonucleoprotein (vRNP) of influenza A virus is formed by virion RNA (vRNA), viral polymerase complex, and nucleoprotein (NP). The NP plays an important role in facilitating the replication and stabilization of viral RNA. To explore host factors that may be involved in the regulation of viral replication through interactions with NP, we conducted an immunoprecipitation experiment followed by mass spectrometry to identify NP-associated cellular proteins. Here, we demonstrate that NP can interact and colocalize with heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in mammalian cells and that the interaction may occur via direct binding to the glycine-rich domain (GRD) of hnRNP A2/B1...
2017: PloS One
Mykola Pinkevych, Christine M Fennessey, Deborah Cromer, Martin Tolstrup, Ole S Søgaard, Thomas A Rasmussen, Brandon F Keele, Miles P Davenport
HIV viremia rebounds rapidly after treatment interruption, and a variety of strategies are being explored to reduce or control viral reactivation post-treatment. This viral rebound arises from reactivation of individual latently infected cells, which spread during ongoing rounds of productive infection. The level of virus produced by the initial individual reactivating cells is not known, although it may have major implications for the ability of different immune interventions to control viral rebound. Here we use data from both HIV and SIV treatment interruption studies to estimate the initial viral load post-interruption and thereby the initial individual reactivation event...
November 8, 2017: Journal of Virology
Judith Oymans, Aartjan J W Te Velthuis
The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2 and PA. To copy the viral RNA (vRNA) genome segments and the complementary RNA (cRNA) segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3' terminus, while on the cRNA promoter the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full...
November 8, 2017: Journal of Virology
Y D Liu, L Wang
Recurrence rate after cessation of nucleos(t)ide analogues remains high in clinical practice. According to current evidences, age, HBsAg level, HB VRNA level, time of consolidation therapy and HBV DNA load were considered to be associated with off-treatment responses after cessation of nucleos(t)ide analogues. Combinative factors of several predictors might perform better in future accompanying with further studies of HBV related markers.
July 20, 2017: Zhonghua Gan Zang Bing za Zhi, Zhonghua Ganzangbing Zazhi, Chinese Journal of Hepatology
Nicolás Cifuentes-Muñoz, Jean Branttie, Kerri Beth Slaughter, Rebecca Ellis Dutch
Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm...
December 15, 2017: Journal of Virology
Markus M Knodel, Sebastian Reiter, Paul Targett-Adams, Alfio Grillo, Eva Herrmann, Gabriel Wittum
Mathematical models of virus dynamics have not previously acknowledged spatial resolution at the intracellular level despite substantial arguments that favor the consideration of intracellular spatial dependence. The replication of the hepatitis C virus (HCV) viral RNA (vRNA) occurs within special replication complexes formed from membranes derived from endoplasmatic reticulum (ER). These regions, termed membranous webs, are generated primarily through specific interactions between nonstructural virus-encoded proteins (NSPs) and host cellular factors...
September 30, 2017: Viruses
Syed Benazir Alam, Ron Reade, Jane Theilmann, D'Ann Rochon
Cucumber necrosis virus (CNV) is a T = 3 icosahedral virus with a (+)ssRNA genome. The N-terminal CNV coat protein arm contains a conserved, highly basic sequence ("KGRKPR"), which we postulate is involved in RNA encapsidation during virion assembly. Seven mutants were constructed by altering the CNV "KGRKPR" sequence; the four basic residues were mutated to alanine individually, in pairs, or in total. Virion accumulation and vRNA encapsidation were significantly reduced in mutants containing two or four substitutions and virion morphology was also affected, where both T = 1 and intermediate-sized particles were produced...
December 2017: Virology
Tania S Bonny, John P Driver, Taylor Paisie, Marco Salemi, John Glenn Morris, Lisa A Shender, Lisa Smith, Carolyn Enloe, Kevin Oxenrider, Jeffery A Gore, Julia C Loeb, Chang-Yu Wu, John A Lednicky
Bats are natural reservoirs of coronaviruses and other viruses with zoonotic potential. Florida has indigenous non-migratory populations of Brazilian free-tailed bats (Tadarida brasiliensis) that mostly roost in colonies in artificial structures. Unlike their counterparts in Brazil and Mexico, the viruses harbored by the Florida bats have been underexplored. We report the detection of an alphacoronavirus RNA-dependent RNA polymerase (RdRp) gene sequence in the feces of two of 19 different T. brasiliensis that were capture/release bats that had been evaluated for overall health...
February 27, 2017: Diseases (Basel)
Subbiah Jeeva, Sean Pador, Brittany Voss, Safder Saieed Ganaie, Mohammad Ayoub Mir
Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA...
2017: PloS One
Nara Lee, Valerie Le Sage, Adalena V Nanni, Dan J Snyder, Vaughn S Cooper, Seema S Lakdawala
Influenza A virus (IAV) genomes are composed of eight single-stranded RNA segments that are coated by viral nucleoprotein (NP) molecules. Classically, the interaction between NP and viral RNA (vRNA) is depicted as a uniform pattern of 'beads on a string'. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), we identified the vRNA binding profiles of NP for two H1N1 IAV strains in virions. Contrary to the prevailing model for vRNA packaging, NP does not bind vRNA uniformly in the A/WSN/1933 and A/California/07/2009 strains, but instead each vRNA segment exhibits a unique binding profile, containing sites that are enriched or poor in NP association...
September 6, 2017: Nucleic Acids Research
David G Courtney, Edward M Kennedy, Rebekah E Dumm, Hal P Bogerd, Kevin Tsai, Nicholas S Heaton, Bryan R Cullen
Many viral RNAs are modified by methylation of the N(6) position of adenosine (m(6)A). m(6)A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m(6)A-modified RNAs, but the effects of m(6)A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m(6)A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m(6)A "reader" protein YTHDF2 increases IAV gene expression and replication...
September 13, 2017: Cell Host & Microbe
Wanyin Tao, Tianyu Gan, Mingzhe Guo, Yongfen Xu, Jin Zhong
Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available, which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL-4) laboratories. EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL-2 conditions. Here, we developed a novel EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe EBOV minigenome...
September 6, 2017: Journal of Virology
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