The RNA helicase DDX1 is involved in restricted HIV-1 Rev function in human astrocytes

Abstract

Productive infection by human immunodeficiency virus type I (HIV-1) in the central nervous system (CNS) involves mainly macrophages and microglial cells. A frequency of less than 10% of human astrocytes is estimated to be infectable with HIV-1. Nonetheless, this relatively low percentage of infected astrocytes, but associated with a large total number of astrocytic cells in the CNS, makes human astrocytes a critical part in the analyses of potential HIV-1 reservoirs in vivo. Investigations in astrocytic cell lines and primary human fetal astrocytes revealed that limited HIV-1 replication in these cells resulted from low-level viral entry, transcription, viral protein processing, and virion maturation. Of note, a low ratio of unspliced versus spliced HIV-1-specific RNA was also investigated, as Rev appeared to act aberrantly in astrocytes, via loss of nuclear and/or nucleolar localization and diminished Rev-mediated function. Host cellular machinery enabling Rev function has become critical for elucidation of diminished Rev activity, especially for those factors leading to RNA metabolism. We have recently identified a DEAD-box protein, DDX1, as a Rev cellular co-factor and now have explored its potential importance in astrocytes. Cells were infected with HIV-1 pseudotyped with envelope glycoproteins of amphotropic murine leukemia viruses (MLV). Semi-quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) for unspliced, singly-spliced, and multiply-spliced RNA clearly showed a lower ratio of unspliced/singly-spliced over multiply-spliced HIV-1-specific RNA in human astrocytes as compared to Rev-permissive, non-glial control cells. As well, the cellular localization of Rev in astrocytes was cytoplasmically dominant as compared to that of Rev-permissive, non-glial controls. This endogenous level of DDX1 expression in astrocytes was demonstrated directly to lead to a shift of Rev sub-cellular distribution dominance from nuclear and/or nucleolar to cytoplasmic, as input of exogenous DDX1 significantly altered both Rev sub-cellular localization from cytoplasmic to nuclear predominance and concomitantly increased HIV-1 viral production in these human astrocytes. We conclude that altered DDX1 expression in human astrocytes is, at least in part, responsible for the unfavorable cellular microenvironment for Rev function in these CNS-based cells. Thus, these data suggest a molecular mechanism(s) for restricted replication in astrocytes as a potential low-level site of residual HIV-1 in vivo.

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1

“…To investigate whether alteration of Rev subcellular localization induced by Nullbasic also affects the subcellular distributions of DDX1, we visualized its subcellular localization in HeLa cells using fluorescence microscopy. In cells transfected with parental pcDNA3.1 plasmid, endogenous DDX1 was primarily found in the nucleus ( Figure 4A, row 1), as observed by others [28]. As expected, the localization of MYC-Rev expressed alone was predominantly nucleolar of Nullbasic-mCherry and MYC-Rev had no apparent effect on DDX3 and DDX17 subcellular distribution [see Additional file 3].…”

“…The expression level of DDX1 in cells has been shown to highly regulate HIV-1 Rev's subcellular distribution. In the HeLa, Cos-1 and 293 T cell lines, HIV-1 Rev predominantly localizes in the nucleolus but also to the nucleoplasm to a lesser extent [26,28,33,35,37]. Whereas when DDX1 is expressed at lower levels, for instance in human astrocytes and DDX1-downregulated cells, Rev subcellular distribution is altered the nucleus to cytoplasm [26,28].…”

“…Overexpression of DDX1 in HIV-1 infected cells results in increased virus production, while downregulation of DDX1 by RNAi not only inhibits viral replication but also unexpectedly alters the subcellular localization of Rev from predominantly nucleolus to nucleus and cytoplasm, resulting in inhibition of nuclear export of incompletely spliced viral mRNAs [26,27]. Of note, low DDX1 levels in primary human astrocytes have effects on Rev subcellular localization similar to DDX1 RNAi [28]. Similar to DDX1, DDX3, known as a nucleocytoplasmic shuttle protein, directly binds to CRM1 and mediates Rev-dependent viral mRNA transport [29].…”

RNA helicases in infection and disease

“…4) functions in transcription, 53,54 premRNA processing 55,56 and mRNA translation. 57 It interacts with the oligomerization domain of HIV-1 Rev, 58 and promotes Revoligomerization on the RRE. 59 The binding of DDX1 to Rev may target DDX1 to incompletely spliced transcripts.…”

Identification of DDX1 as a JC Virus Transcriptional Control Region‐Binding Protein

“…Although DDX1 has been implicated in transcription, pre-mRNA processing, ribosome biogenesis, RNA export, translation initiation and RNA degradation (1), the biological roles played by this protein remain poorly understood. Furthermore, association between DDX1 and viral genomes has been reported, and it is involved in the regulation of growth of viruses such as HIV (7). Binding between DDX1 and JCV-TCR may provide a novel clue toward understanding the physiological function of DDX1 in JCV infection.…”