Summary The cellular and viral determinants required for HIV-1 infection of nondividing cells have been a subject of intense scrutiny. Here we identify the 68 kDa subunit of cleavage factor Im, CPSF6, as an inhibitor of HIV-1 infection. When enriched in the cytoplasm by high level expression or mutation, CPSF6 prevents nuclear entry of the virus. Similar to TRIM5 and Fv1 type restrictions, CPSF6 targets the viral capsid (CA). N74D mutation of the HIV-1 CA leads to a loss of interaction with CPSF6 and evasion of the nuclear import restriction. Interestingly, N74D mutation of CA changes HIV-1 nucleoporin (NUP) requirements. Whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics the NUP requirements of feline immunodeficiency virus (FIV) and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs.

Inherently unstable mRNAs contain AU-rich elements (AREs) in their 3 untranslated regions that act as mRNA stability determinants by interacting with ARE-binding proteins (ARE-BPs). We have destabilized two mRNAs by fusing sequence-specific RNA-binding proteins to KSRP, a decay-promoting ARE-BP, in a tethering assay. These results support a model that KSRP recruits mRNA decay machinery/factors to elicit decay. The ability of tethered KSRP to elicit mRNA decay depends on functions of known mRNA decay enzymes. By targeting the Rev response element of human immunodeficiency virus type 1 by using Rev-KSRP fusion protein, we degraded viral mRNA, resulting in a dramatic reduction of viral replication. These results provide a foundation for the development of novel therapeutic strategies to inhibit specific gene expression in patients with acquired or hereditary diseases.mRNA stability varies considerably from one mRNA species to another and plays an important role in determining levels of gene expression (19,46,47). Differential mRNA decay rates are determined by specific cis-acting elements within the mRNA molecule. The most common cis element responsible for rapid mRNA decay in mammalian cells is the AU-rich element (ARE) present within the 3Ј untranslated regions (UTRs) of a variety of short-lived mRNAs (2, 10). Recent computational analysis of the 3Ј UTRs revealed that as many as 8% of human mRNAs contain AREs (2). This finding suggests that AREs may account for degradation of most unstable mRNAs in human cells.

Exogenous retroviruses are obligate cellular parasites that co-opt a number of host proteins and functions to enable their replication and spread. Several host factors that restrict HIV and other retroviral infections have also recently been described. Here we demonstrate that Mov10, a protein associated with P-bodies that has a putative RNA-helicase domain, when overexpressed in cells can inhibit the production of infectious retroviruses. Interestingly, reducing the endogenous Mov10 levels in virus-producing cells through siRNA treatment also modestly suppresses HIV infectivity. The actions of Mov10 are not limited to HIV, however, as ectopic expression of Mov10 restricts the production of other lentiviruses as well as the gammaretrovirus, murine leukemia virus. We found that HIV produced in the presence of high levels of Mov10 is restricted at the pre-reverse transcription stage in target cells. Finally, we show that either helicase mutation or truncation of the C-terminal half of Mov10, where a putative RNA-helicase domain is located, maintained most of its HIV inhibition; whereas removing the N-terminal half of Mov10 completely abolished its activity on HIV. Together these results suggest that Mov10 could be required during the lentiviral lifecycle and that its perturbation disrupts generation of infectious viral particles. Because Mov10 is implicated as part of the P-body complex, these findings point to the potential role of cytoplasmic RNA processing machinery in infectious retroviral production.

No abstract