Nuclear Localization of Cytoplasmic Poly(A)-Binding Protein upon Rotavirus Infection Involves the Interaction of NSP3 with eIF4G and RoXaN

The mRNAs of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, possess a 5 cap structure but lack a 3 poly(A) tail, a common feature of eukaryotic mRNAs that greatly enhances translation efficiency. Viral mRNAs also contain untranslated regions (UTRs) that flank the coding sequence. Using model virus-like mRNAs that harbor the Renilla luciferase reporter gene, we found that the 3 UTR of the BUNV small-segment mRNA mediated efficient translation in the absence of a poly(A) tail. Viral UTRs did not increase RNA stability, and polyadenylation did not significantly enhance reporter activity. Translation of virus-like mRNAs in transfected cells was unaffected by knockdown of poly(A)-binding protein (PABP) but was markedly reduced by depletion of eukaryotic initiation factor 4G, suggesting a PABP-independent process for translation initiation. In BUNV-infected cells, translation of polyadenylated but not virus-like mRNAs was inhibited. Furthermore, we demonstrate that the viral nucleocapsid protein binds to, and colocalizes with, PABP in the cytoplasm early in infection, followed by nuclear retention of PABP. Our results suggest that BUNV corrupts PABP function in order to inhibit translation of polyadenylated cellular mRNAs while its own mRNAs are translated in a PABP-independent process.

Section: Discussionmentioning

confidence: 99%

Bunyamwera Orthobunyavirus S-Segment Untranslated Regions Mediate Poly(A) Tail-Independent Translation

Blakqori

Knippenberg

Elliott

2009

“…In fact, during rotavirus infection, PABP is evicted from eIF4G by NSP3 (9) and relocalizes to the cell nucleus (16). These observations led to the idea that the shutdown of cellular protein synthesis is due to the eviction of PABP and, hence, to the displacement of cellular polyadenylated mRNAs from eIF4G.…”

mentioning

confidence: 94%

Rotavirus NSP3 Is a Translational Surrogate of the Poly(A) Binding Protein-Poly(A) Complex

Gratia

Sarot

Vende

et al. 2015

Self Cite

Through its interaction with the 5= translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5=-capped and 3=-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3= GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3= end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)-and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCETo control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery. W hen delivered into or synthesized by the host cell, viral mRNAs compete with cellular mRNAs already present in the cytoplasm for access to the protein synthesis machinery. Recruitment of the 40S ribosomal subunit onto mRNA (translation initiation) is the rate-limiting and most controlled step of eukaryotic protein biosynthesis; hence, it is highly competitive for both cellular and viral mRNAs. The 5= cap and 3= poly(A) tail of most cellular mRNAs are joined by a protein complex containing the cap binding pr...

“…Additionally, PABP1 partially relocalizes to the nucleus during HSV-1 infection (40,41). Rotavirus protein NSP3A functionally replaces and evicts PABP1 from the eIF4F complex and leads to nuclear relocalization of PABP1 (42,43). Kaposi's sarcoma-associated herpesvirus SOX protein expression leads to PABP1 relocalization to the nucleus (44).…”

mentioning

confidence: 99%

Nuclear Relocalization of Polyadenylate Binding Protein during Rift Valley Fever Virus Infection Involves Expression of the NSs Gene

Copeland

Altamura

Deusen

et al. 2013

Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production.