Sindbis Virus Usurps the Cellular HuR Protein to Stabilize Its Transcripts and Promote Productive Infections in Mammalian and Mosquito Cells

Sokoloski, Kevin J.; Dickson, Alexa; Chaskey, Emily L; Garneau, Nicole L.; Wilusz, Carol J.; Wilusz, Jeffrey

doi:10.1016/j.chom.2010.07.003

Cited by 95 publications

(116 citation statements)

References 48 publications

(60 reference statements)

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“…This study also highlights how the same protein can be exploited to exert different functions in the context of different viruses. In this case, it is HuR which can regulate the viral life cycle either by stabilizing the viral RNA (Sindbis virus) (55) or by regulating viral translation (HIV-1) (56) or by altering protein binding at the HCV 3= UTR, as shown in the present study using HCV as a model system.…”

Section: Discussionmentioning

confidence: 94%

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

Shwetha

Kumar

Mullick

et al. 2015

J Virol

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HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3= untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3= UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3= UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3= UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA.Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3= untranslated region (UTR) of HCV RNA. At the 3= UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions. Hepatitis C virus (HCV) was discovered in 1989 as a major cause of chronic non-A non-B hepatitis (1). HCV is an enveloped positive-strand RNA virus classified in the Hepacivirus genus within the Flaviviridae family. The single-stranded uncapped 9.6-kb RNA codes for a long polyprotein of ϳ3,000 amino acids which is then processed to structural and nonstructural proteins. The RNA genome is flanked by the 5= and 3= untranslated regions (UTRs), which are essential for translation and replication of the viral RNA. The viral proteins are synthesized by internal ribosome entry site (IRES)-mediated translation. These proteins initiate extensive remodeling of the intracellular membranes to create a detergent-resistant scaffold for viral replication, termed as the "membranous web" (2). The progeny viral genomes a...

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Section: Discussionmentioning

confidence: 94%

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

Shwetha

Kumar

Mullick

et al. 2015

J Virol

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“…This may assist in the stabilization of viral transcripts as well as cause a disruption of the regulation of host cell gene expression. The data described here, along with other recently reported examples of viral RNA interactions with the cellular RNA decay machinery (Sokoloski et al 2010;Dougherty et al 2011;Palusa et al 2012), demonstrate interesting aspects of viral RNA-host interactions that have potentially broad biological implications. These findings also present a potential target for the development of broadspectrum antivirals as well as discovery tools for novel insights into mechanisms and regulation of cellular mRNA stability.…”

Section: Discussionmentioning

confidence: 92%

“…Some RNA viruses that encode polyadenylated transcripts appear to usurp cellular RNA-binding proteins to stabilize their transcripts (Sokoloski et al 2010;Palusa et al 2012). Poliovirus, interestingly, appears to cause the accelerated turnover of several proteins involved in the 59-39 RNA decay pathway, including XRN1 and the auxiliary decapping factor DCP1a (Dougherty et al 2011).…”

Section: Introductionmentioning

confidence: 99%

A noncoding RNA produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease XRN1 and alters host mRNA stability

Moon

Anderson

Kumagai

et al. 2012

RNA

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180

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All arthropod-borne flaviviruses generate a short noncoding RNA (sfRNA) from the viral 39 untranslated region during infection due to stalling of the cellular 59-to-39 exonuclease XRN1. We show here that formation of sfRNA also inhibits XRN1 activity. Cells infected with Dengue or Kunjin viruses accumulate uncapped mRNAs, decay intermediates normally targeted by XRN1. XRN1 repression also resulted in the increased overall stability of cellular mRNAs in flavivirus-infected cells. Importantly, a mutant Kunjin virus that cannot form sfRNA but replicates to normal levels failed to affect host mRNA stability or XRN1 activity. Expression of sfRNA in the absence of viral infection demonstrated that sfRNA formation was directly responsible for the stabilization of cellular mRNAs. Finally, numerous cellular mRNAs were differentially expressed in an sfRNA-dependent fashion in a Kunjin virus infection. We conclude that flaviviruses incapacitate XRN1 during infection and dysregulate host mRNA stability as a result of sfRNA formation.

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“…The unbound RNAs were then phenol extracted and ethanol precipitated prior to use as a template for cDNA synthesis via RT with Random Hexamer. The resulting cDNAs were assayed via qRT-PCR to determine the relative abundances of the incoming SINV genomic RNAs normalized to the cellular 18S rRNA as previously described (27,28). RNA half-lives were calculated via nonlinear regression.…”

Section: Methodsmentioning

confidence: 99%

“…After incubation at 4°C for 1 h, the beads were collected via centrifugation and washed six times prior to being phenol extracted and ethanol precipitated. The resulting immunoprecipitate was used as the template for cDNA synthesis and qRT-PCR detection as previously described (24,27).…”

Section: = End Characterizationmentioning

confidence: 99%

Noncapped Alphavirus Genomic RNAs and Their Role during Infection

Sokoloski

Haist

Morrison

et al. 2015

J Virol

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Alphaviruses are enveloped positive-sense RNA viruses that exhibit a wide host range consisting of vertebrate and invertebrate species. Previously we have reported that the infectivity of Sindbis virus (SINV), the model alphavirus, was largely a function of the cell line producing the viral particles. Mammalian-cell-derived SINV particles, on average, exhibit a higher particle-to-PFU ratio than mosquito cell-derived SINV particles. Nevertheless, the outcome of nonproductive infection, the molecular traits that determine particle infectivity and the biological importance of noninfectious particles were, prior to this study, unknown. Here, we report that the incoming genomic RNAs of noninfectious SINV particles undergo rapid degradation following infection. Moreover, these studies have led to the identification of the absence of the 5= cap structure as a primary molecular determinant of particle infectivity. We show that the genomic RNAs of alphaviruses are not universally 5= capped, with a significant number of noncapped genomic RNA produced early in infection. The production of noncapped viral genomic RNAs is important to the establishment and maintenance of alphaviral infection. IMPORTANCEThis report is of importance to the field of virology for three reasons. First, these studies demonstrate that noncapped Sindbis virus particles are produced as a result of viral RNA synthesis. Second, this report is, to our knowledge, the first instance of the direct measurement of the half-life of an incoming genomic RNA from a positive-sense RNA virus. Third, these studies indicate that alphaviral infection is likely a concerted effort of infectious and noninfectious viral particles.A lphaviruses are enveloped, positive-sense RNA viruses that are cyclically transmitted between sylvatic vertebrate reservoir hosts and a mosquito vector during the enzootic cycle. The maintenance of this cyclical transmission is vital to viral fitness, as prolonged serial passage within a single host results in attenuation in the alternate host (1-4). Moreover, the ultimate outcome of alphaviral infection differs between vertebrate and invertebrate hosts, as infection of a vertebrate host results in acute cytolytic infection whereas infection of invertebrate hosts often results in persistent infection with minimal cell death (5-11). The widespread geographic distribution of competent vector mosquito species leading to contact with immunologically naive human populations has resulted in several significant outbreaks of alphaviral disease (12)(13)(14). Perhaps most notable is the ongoing reemergence of chikungunya virus, which caused significant morbidity during the height of the 2006 epidemic, with as many as 40,000 new cases per week (13).Despite the range of diseases and morbidity associated within the genus, the underlying molecular life cycles are highly similar in the two hosts. Since alphaviruses are positive-sense RNA viruses, they function similarly to cellular mRNAs, relying on the translation of the incoming viral genome to ini...

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Sindbis Virus Usurps the Cellular HuR Protein to Stabilize Its Transcripts and Promote Productive Infections in Mammalian and Mosquito Cells

Cited by 95 publications

References 48 publications

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

A noncoding RNA produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease XRN1 and alters host mRNA stability

Noncapped Alphavirus Genomic RNAs and Their Role during Infection

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