The purpose of this project is to understand the molecular mechanisms involved in the replication of picornaviruses in susceptible target cells. This virus family includes numerous human pathogens (poliovirus, coxsackievirus, echovirus, rhinoviruses, hepatitis A virus). Infection of cells with these viruses leads to drastic changes in the host cell's structure and metabolic activity: Cellular protein and RNA synthesis are inhibited;the intracellular membrane network becomes rearranged into a network of vesicles that surround and provide a scaffold for viral RNA replication complexes;cellular proteins are subverted into facilitating viral protein and RNA synthesis. The unique combination of viral and cellular proteins together accomplishes a highly efficient production of viral RNA, proteins, and particles. This year in collaboration with Dr. vanKuppeveld's laboratory we showed how coxsackievirus, an enterovirus, can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIbeta (PI4KIIIbeta) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid microenvironment is essential for enterovirus RNA replication;PI4KIIIbeta inhibition interferes with this process;and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication. The replication of coxsackieviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. This year we showed that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. This study identified a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study. The cellular protein GBF1 is critical for poliovirus replication. This year we found that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP-ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. This year we generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of Discosoma sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved;fluorescent 2A proteinase retained protease activity in trans and supported translation and replication of viral RNA, both in vitro and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indistinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin. This year we performed transposon-mediated insertion mutagenesis of the entire nonstructural protein-coding region of the poliovirus genome, followed by selection of genomes capable of generating infectious, viable viruses in order to identify regions in the poliovirus polyprotein that could potentially accommodate insertion of additional RNA. This procedure allowed us to identify at least one site in each viral nonstructural protein, except protein 2C, in which a minimum of five amino acids could be inserted. The distribution of these sites was analyzed from the perspective of their protein structural context and from the perspective of virus evolution.
| Teterina, Natalya L; Lauber, Chris; Jensen, Kenneth S et al. (2011) Identification of tolerated insertion sites in poliovirus non-structural proteins. Virology 409:1-11 |
| Teterina, Natalya L; Pinto, Yuval; Weaver, Joseph D et al. (2011) Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells. J Virol 85:4284-96 |
| Hsu, Nai-Yun; Ilnytska, Olha; Belov, Georgiy et al. (2010) Viral reorganization of the secretory pathway generates distinct organelles for RNA replication. Cell 141:799-811 |
| Teterina, Natalya L; Levenson, Eric A; Ehrenfeld, Ellie (2010) Viable polioviruses that encode 2A proteins with fluorescent protein tags. J Virol 84:1477-88 |
| Belov, George A; Kovtunovych, Gennadiy; Jackson, Catherine L et al. (2010) Poliovirus replication requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1. Cell Microbiol : |
| Lanke, Kjerstin H W; van der Schaar, Hilde M; Belov, George A et al. (2009) GBF1, a guanine nucleotide exchange factor for Arf, is crucial for coxsackievirus B3 RNA replication. J Virol 83:11940-9 |
| Ehrenfeld, Ellie; Modlin, John; Chumakov, Konstantin (2009) Future of polio vaccines. Expert Rev Vaccines 8:899-905 |
| Adams, Peter; Kandiah, Eaazhisai; Effantin, Grégory et al. (2009) Poliovirus 2C protein forms homo-oligomeric structures required for ATPase activity. J Biol Chem 284:22012-21 |
| Ehrenfeld, Ellie; Chumakov, Konstantin (2008) Monovalent oral poliovirus vaccines--a good tool but not a total solution. N Engl J Med 359:1726-7 |
| Belov, George A; Feng, Qian; Nikovics, Krisztina et al. (2008) A critical role of a cellular membrane traffic protein in poliovirus RNA replication. PLoS Pathog 4:e1000216 |
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