HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA.

Lara Ajamian,Karen Abel,Shringar Rao,Ricardo Soto-Rifo,Francisco García-De-Gracia,Kishanda Vyboh,Andreas Kulozik,Niels Gehring,Andrew Mouland

doi:10.3390/biom5042808

Abstract

Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking.

Highlights

The nucleocytoplasmic export of macromolecules (RNA and protein) and RNA-protein (RNP)complexes is one of the critical steps necessary to ensure normal cellular function
Our earlier work showed that the expression of either the wild-type upframeshift protein 1 (UPF1) (UPF1WT) or the trans-dominant negative (TDN) nonsense-mediated mRNA decay (NMD)-null RNA helicase mutant, UPF1R844C, increased HIV-1 Gag and vRNA levels, indicating that UPF1’s function in vRNA metabolism is independent of its role in NMD [5]
While NMD does require an interaction between UPF1 and UPF2 in most circumstances [53], this association is not essential for the resulting effects on HIV-1 gene expression as UPF2 is excluded from a cytoplasmic Gag ribonucleoprotein complexes (RNP) [5] and UPF1 mutants that do not interact with UPF2 maintain Gag and vRNA

Summary

Introduction

The nucleocytoplasmic export of macromolecules (RNA and protein) and RNA-protein (RNP)complexes is one of the critical steps necessary to ensure normal cellular function. The recruitment of viral and host factors to the unspliced viral genomic RNAs (vRNA is used ) helps this molecule to overcome nuclear retention and promote nucleocytoplasmic export [3,4]. Bypassing host RNA surveillance machineries that target and clear unspliced and intron-containing, aberrantly spliced RNA substrates leads to the preservation of viral genomic RNAs and guarantees structural viral protein synthesis once they arrive in the cytoplasm [5,6,7]. While the nuclear RNA export factor 1 (NXF1) is required for the constitutive export of the 2-kb, completely-spliced viral RNA species [8], the 9-kb, unspliced, vRNA and the 4-kb, singly-spliced. RNAs harbor the cis-acting Rev-responsive element (RRE) that are bound by the viral protein Rev to program nucleocytoplasmic RNA trafficking [9]. Many studies have highlighted the requirement for various host proteins in these transport processes. Eukaryotic translation initiation factor 5A (eIF5A) acts as an adaptor for Rev and the mammalian nuclear export factor Chromosomal Maintenance 1

Methods

Results

Conclusion