HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA

Abstract

HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-B activation through binding to A20, and the loss of Naf1 controlled NF-B activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.HIV-1 depends on host factors to complete its life cycle (1-8). Hundreds of human proteins involved in the interaction with HIV-1 for modulating viral replication have been identified by genome-wide RNA interference screen or affinitytagged protein purification using mass spectrometry (1,(3)(4)9). A comprehensive understanding of how host machineries are manipulated during HIV-1 infection can facilitate the identification of host targets for antiviral drugs or gene therapy.The CRM1 (chromosome region maintenance 1, 2 also known as exportin 1) nuclear export pathway is typically used to transport host protein cargoes and small RNAs, which has been found to engage HIV-1 regulatory protein Rev for directing the nuclear export of unspliced and partially spliced HIV-1 RNAs (10 -15). These incompletely spliced HIV-1 RNAs contain a Rev response element (RRE), and the coordinate assembly of multiple Rev molecules on RRE recruits human protein complex containing CRM1 and Ran-GTP (GTP-binding nuclear protein Ran). Rev contains a leucine-rich nuclear export signal (NES) domain located near the carboxyl terminus, through which Rev interacts with CRM1, and the Rev-RRE-CRM1/Ran-GTP complex is then transported to the cytoplasm (6, 11-16). Similarly, other retroviruses such as mouse mammary tumor virus and human T cell lymphotropic virus encode Rev-like regulatory proteins Rem or Rex, respectively, for hijacking the host CRM1 pathway to accomplish nuclear export of unspliced viral transcripts (17)(18)(19)(20)(21)(22).Several other cellular facto...

Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4 + T Cells

“…Naf1 has been reported to be a shuttling protein that moves between the cytoplasm and the nucleus (17,20). The cytoplasmic and nuclear localization of Naf1 was confirmed by confocal microscopy (Fig.…”

“…These data may suggest that Naf1 possesses multifaceted and likely cell-type-dependent roles in regulating HIV-1 infection. Naf1 has been shown to be a nucleocytoplasmic shuttling protein (17,20). Recently, we found that nuclear Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production for viral assembly and release (20).…”

“…Naf1 has been shown to be a nucleocytoplasmic shuttling protein (17,20). We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production (in that study, a Gag-expressing plasmid was used) (20).…”

“…We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production (in that study, a Gag-expressing plasmid was used) (20). To further determine the overall effect of endogenous Naf1 on HIV-1 replication in this study, the replication-competent HIV-1 NL4-3 strain was used to infect primary CD4 ϩ T cells (Fig.…”

“…Naf1 is expressed ubiquitously in human tissues, with high levels of expression in peripheral blood lymphocytes (16,17). Naf1 can increase cell surface CD4 expression (16), inhibit NF-B activation (15,18,19), and modulate HIV-1 infection and viral production (17,20). Naf1 also interacts with the HIV-1 matrix protein, and the overexpression of Naf1 inhibits HIV-1 infection (17).…”