Abstract
Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5’ untranslated region (UTR) with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5’ UTR, especially with the transactivation response (TAR) hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5’ UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.
Highlights
Human DDX3 belongs to the DEAD-box protein family of RNA helicases, which are characterized by nine conserved motifs that mediate ATP hydrolysis and RNA helicase activities
Immunoblotting showed that the level of DDX3 protein was significantly reduced by three different DDX3-targeting shRNAs (Figure 1, lanes 2-4) of which sh-DDX3#2 was the most effective inhibiting the expression of endogenous DDX3 by ~90% (Figure 1, lane 3)
Previous reports have indicated that DDX3 is required for the replication of Human immunodeficiency virus type 1 (HIV-1) [16,18] and hepatitis C virus (HCV) [40], but the underlying molecular mechanisms remain poorly understood
Summary
Human DDX3 belongs to the DEAD-box protein family of RNA helicases, which are characterized by nine conserved motifs that mediate ATP hydrolysis and RNA helicase activities. The role of DDX3 in translation initiation seems to be evolutionarily conserved from yeast to humans [10]. Cig mRNA has an unusually long 5’ untranslated region (UTR), while the 5’ UTR of Cdc mRNA is predicted to contain a complex secondary structure. Consistent with this notion, we recently demonstrated that DDX3 facilitates the translation of mRNAs with a long or structured 5’ UTR [7,8]. DDX3 and its homologs may facilitate translation initiation by resolving secondary structures in the 5’ UTR of selected mRNAs
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