Importin α-Mediated Nuclear Import of Cytoplasmic Poly(A) Binding Protein Occurs as a Direct Consequence of Cytoplasmic mRNA Depletion

HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.IMPORTANCE HIV-1 infects more than 34 million people worldwide causing Ͼ1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.KEYWORDS CRM1, Gag, RNA trafficking, Rev, exportin-1, human immunodeficiency virus, nuclear export signal, nuclear pore complex, nucleolus, retroviruses A core challenge to eukaryotic gene expression is ensuring strong but transient interactions between newly transcribed messenger RNAs (mRNAs) in the nucleus and export receptors at nuclear pore complexes (NPCs) (1-3). For spliced mRNAs, posttranscriptional regulatory factors program export receptor recruitment, the formation of export complexes, and subsequent transit through the hydrophobic core of the NPC (4, 5). mRNA dissociation from the NPC is also crucial and is regulated by RNA

Section: Discussionsupporting

confidence: 72%

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Behrens

Aligeti

Pocock

et al. 2017

“…They propose a model in which poly(A) RNA binds PABP1, masking an NLS and thus preventing translocation into the nucleus. Conversely, when PABP1 is not bound by poly(A) RNA, it is translocation competent (63). It follows that, under normal conditions, when cytoplasmic levels of poly(A) RNA are relatively high, PABP1 remains predominantly cytoplasmic.…”

Section: Discussionmentioning

confidence: 99%

“…It has recently been hypothesized that a nontraditional nuclear localization sequence (NLS) resides within the RRM. Kumar et al demonstrated that the presence of poly(A) RNA antagonizes PABP1's ability to interact with nuclear import factors and a depletion of poly(A) RNA from the cytoplasm results in PABP1 relocalization to the nucleus (63). They propose a model in which poly(A) RNA binds PABP1, masking an NLS and thus preventing translocation into the nucleus.…”

Section: Discussionmentioning

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.

“…Recent data implicate that the nuclear accumulation of PABPC is resulting from virus-induced mRNA depletion from the cytoplasm (32). Interestingly, cellular expression of PABPC is altered upon infection by members of all herpesvirus subfamilies.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, interaction of PABPC with mRNAs retains PABPC in the cytoplasm (32). Upon expression of KSHV SOX and subsequent mRNA depletion from the cytoplasm, PABPC is relocalized to the nucleus (32,35), where it induces hyperadenylation and nuclear retention of Macacine herpesvirus 4 gi|24943119…”

mentioning

confidence: 99%

The “Bridge” in the Epstein-Barr Virus Alkaline Exonuclease Protein BGLF5 Contributes to Shutoff Activity during Productive Infection

Horst

Burmeister

Boer

et al. 2012

Replication of the human herpesvirus Epstein-Barr virus drastically impairs cellular protein synthesis. This shutoff phenotype results from mRNA degradation upon expression of the early lytic-phase protein BGLF5. Interestingly, BGLF5 is the viral DNase, or alkaline exonuclease, homologues of which are present throughout the herpesvirus family. During productive infection, this DNase is essential for processing and packaging of the viral genome. In contrast to this widely conserved DNase activity, shutoff is only mediated by the alkaline exonucleases of the subfamily of gammaherpesviruses. Here, we show that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation. Furthermore, shutoff by BGLF5 induces nuclear relocalization of the cytosolic poly(A) binding protein. Guided by the recently resolved BGLF5 structure, mutants were generated and analyzed for functional consequences on DNase and shutoff activities. On the one hand, a point mutation destroying DNase activity also blocks RNase function, implying that both activities share a catalytic site. On the other hand, other mutations are more selective, having a more pronounced effect on either DNA degradation or shutoff. The latter results are indicative of an oligonucleotide-binding site that is partially shared by DNA and RNA. For this, the flexible “bridge” that crosses the active-site canyon of BGLF5 appears to contribute to the interaction with RNA substrates. These findings extend our understanding of the molecular basis for the shutoff function of BGLF5 that is conserved in gammaherpesviruses but not in alpha- and betaherpesviruses.