Mining the Human Complexome Database Identifies RBM14 as an XPO1-Associated Protein Involved in HIV-1 Rev Function

Abstract

By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 depletion also reduced p24 expression during viral infection, indicating that RBM14 is limiting for Rev function. RBM14 has previously been shown to localize to nuclear paraspeckles, a structure implicated in retaining unspliced HIV-1 transcripts for either Rev-mediated nuclear export or degradation. We found that depletion of NEAT1 RNA, a long noncoding RNA required for paraspeckle integrity, abolished the ability of overexpressed RBM14 to enhance Rev function, indicating the dependence of RBM14 function on paraspeckle integrity. Our study extends the known host cell interactome of Rev and XPO1 and further substantiates a critical role for paraspeckles in the mechanism of action of Rev. Our study also validates the nuclear complexome as a database from which viral cofactors can be mined. IMPORTANCEThis study mined a database of nuclear protein complexes to identify a cellular protein named RBM14 that is associated with XPO1 (CRM1), a nuclear protein that binds to the HIV-1 Rev protein and mediates nuclear export of incompletely spliced viral RNAs. Functional assays demonstrated that RBM14, a protein found in paraspeckle structures in the nucleus, is involved in HIV-1 Rev function. This study validates the nuclear complexome database as a reference that can be mined to identify viral cofactors. Since the HIV-1 genome encodes only 15 proteins, the virus must exploit the function of host cofactors at every step in its replication cycle (1). A meta-analysis of genome-wide small interfering RNA (siRNA) screens suggests that more than 2,410 proteins, or 9.5% of human genes, may be involved in HIV-1 replication (2). While data from siRNA screens provide insight into virus-host interactions and provide an opportunity to identify "druggable" targets to inhibit virus replication, these screens have limitations. Comparison of different genome-wide siRNA screens reveal minimal overlap in the host genes involved in HIV-1 replication, and these screens may frequently yield false-positive results due to siRNA off-target effects (3).In the present study, we utilized a novel strategy to identify host factors involved in HIV-1 replication. The human nuclear "complexome" was recently described in a high-throughput immunoprecipitation/mass spectrometry (IP/MS) study (4). This complexome describes endogenous protein complexes defined by identification of proteins that coimmunoprecipitated in more than 3,000 immunoprecipitations of HeLa cell nuclear extracts. We mined the nuclear complexome to identify cellular proteins found in complexe...

The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

“…Examples include ACIN1 which is identified as a HIV-1 Tat interacting protein52, CHMP4B which is critical for HIV-1 membrane budding via ESCRT pathway53, NMT1 which is involved in targeting and assembly of HIV-1 Gag proteins to plasma membrane via N-myristoylation54, MAPK1 which interacts with 10 HIV-1 proteins and may play multiple roles in HIV-1 replication55. NFAT5 which interacts with an enhancer binding site in LTR of HIV-1 to enable replication in human primary macrophages56, MED1, a subunit of mediator complex which plays a critical role in HIV-1 transcription and infectivity5758 and RBM14 which encodes a nucleic acid binding protein that associates with XPO1 to export incompletely spliced HIV-1 transcripts59. Although combined effects of RNA editing of hundreds of genes on HIV-1 replication is unknown, A3G may alter the host environment by means of RNA editing to antagonize HIV-1 infection.…”

Paraspeckles: Where Long Noncoding RNA Meets Phase Separation

“…In most cases the evidence suggests that NEAT1_2/paraspeckles increase as a cell defence mechanism. More abundant paraspeckles lead to increased sequestration of the paraspeckle proteins, thereby preventing the hijacking of these proteins by the virus [48,49], or triggering gene regulatory changes that help the cell [38,47].…”

Roles of microRNAs and long‐noncoding RNAs in human immunodeficiency virus replication

“…A paraspeckle protein termed RBM14 has recently been shown to coimmunoprecipitate with CRM1 and Rev and be involved in export of viral transcripts. 58 These recent findings of paraspeckle proteins and Rev suggest the rather speculative model shown in Figure 3 for nuclear export of incompletely spliced HIV transcripts. In this model, unspliced viral transcripts are retained in paraspeckles through the RNA-binding proteins p54nrb, PSF, and RBM14.…”

“…The Rev export pathway utilizes the export factor CRM1 (XPO1) and its cofactor RAN‐GTP. A paraspeckle protein termed RBM14 has recently been shown to coimmunoprecipitate with CRM1 and Rev and be involved in export of viral transcripts . These recent findings of paraspeckle proteins and Rev suggest the rather speculative model shown in Figure for nuclear export of incompletely spliced HIV transcripts.…”