Abstract

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

Highlights

  • Human immunodeficiency virus type 1 (HIV-1) replication requires a tightly orchestrated series of post-transcriptional regulatory events encompassing the nuclear export, translation and packaging of viral full-length positive sense genomic RNAs [1,2,3]

  • We use fluorescence microscopy to visualize HIV-1 messenger RNAs (mRNAs) nuclear export, translation, trafficking, and virus particle assembly in single living cells, focusing on the fulllength viral genomic RNAs that are packaged into virus particles

  • We demonstrate that HIV-1 genomic RNAs (gRNAs) exhibit dramatic “burst”-like transitions from the nucleus to fill the cytoplasm prior to the onset of virus particle assembly

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Summary

Introduction

Human immunodeficiency virus type 1 (HIV-1) replication requires a tightly orchestrated series of post-transcriptional regulatory events encompassing the nuclear export, translation and packaging of viral full-length positive sense genomic RNAs (gRNAs) [1,2,3]. Viral gRNAs are transcribed in the nucleus as pre-mRNAs by cellular RNA polymerase II, 5’ 7mG capped and 3’ polyadenylated The bulk of these transcripts undergo complex alternative splicing to generate the viral messenger RNAs (mRNAs). The virus must regulate a switch in the cytoplasm to terminate Gag/ Gag-Pol translation and initiate gRNA packaging [6,20]. How these activities involving Rev, gRNAs, Gag, and cellular factors are coordinated spatially and temporally is poorly understood

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