HIV-1 Replication and APOBEC3 Antiviral Activity Are Not Regulated by P Bodies

Abstract

The APOBEC3 cytidine deaminases play a critical role in host-mediated defense against exogenous viruses, most notably, human immunodeficiency virus type-1 (HIV-1) and endogenous transposable elements. APOBEC3G and APOBEC3F interact with numerous proteins that regulate cellular RNA metabolism, including components of the RNA-induced silencing complex (RISC), and colocalize with a subset of these proteins to mRNA processing bodies (P bodies), which are sites of mRNA translational repression and decay. We sought to determine the role of P bodies and associated proteins in HIV-1 replication and APOBEC3 antiviral activity. While we established a positive correlation between APOBEC3 protein incorporation into virions and localization to P bodies, depletion of the P-body components DDX6 or Lsm1 did not affect HIV-1 replication, APOBEC3 packaging into virions or APOBEC3 protein mediated inhibition of HIV-1 infectivity. In addition, neither HIV-1 genomic RNA nor Gag colocalized with P-body proteins. However, simultaneous depletion of multiple Argonaute family members, the effector proteins of RISC, could modestly increase viral infectivity. Because some APOBEC3 proteins interact with several Argonaute proteins, we also tested whether they could modulate microRNA (miRNA) activity. We found no evidence for the specific regulation of miRNA function by the APOBEC3 proteins, though more general effects on transfected gene expression were observed. In sum, our results indicate that P bodies and certain associated proteins do not regulate HIV-1 replication or APOBEC3 protein antiviral activity. Localization to P bodies may therefore provide a means of sequestering APOBEC3 enzymatic activity away from cellular DNA or may be linked to as yet unidentified cellular functions.

Mov10 and APOBEC3G Localization to Processing Bodies Is Not Required for Virion Incorporation and Antiviral Activity

“…The results indicated that inhibiting RISC function The copy numbers of HIV-1 gag RNA in the presence or absence of FLAG-Mov10 were adjusted for RNA input by determination of cellular ␤-actin RNA copy had no effect on Mov10 virion incorporation. As expected from previous studies (30,59), AGO2 knockdown alone increased virus infectivity approximately 2.5-fold; however, AGO2 knockdown did not significantly influence the ability of Mov10 or A3G to inhibit virus infectivity (Fig. 8F).…”

“…We further investigated the role of P bodies in Mov10 virion incorporation and antiviral activity by siRNA knockdown of DDX6, which has been shown to deplete microscopically visible P bodies (57)(58)(59). Transfection of HeLa cells with the DDX6 siRNA, but not control siRNA, resulted in substantial reduction in the number of P bodies, as determined by the detection of cytoplasmic granules containing eYFP-Mov10 or eYFP-V866A (Fig.…”

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism

“…First, RNA editing is a bona fide function for at least two family members, APOBEC1 (Teng et al, 1993) and A3A (Sharma et al, 2015). Second, RNA has been implicated in governing the cytoplasmic localization of A3G and A3F, including accumulation in stress granules and RNA processing bodies (Gallois-Montbrun et al, 2007; Izumi et al, 2013; Kozak et al, 2006; Phalora et al, 2012; Stenglein et al, 2008; Wichroski et al, 2006). Third, an RNA-dependent interaction is required for A3G and A3F to interact with HIV-1 Gag, gain access to assembling viral particles, and exert antiviral activity (Apolonia et al, 2015; Bogerd and Cullen, 2008; Cen et al, 2004; Huthoff and Malim, 2007; Khan et al, 2005; Schafer et al, 2004; Svarovskaia et al, 2004; Wang et al, 2007; Wang et al, 2008; York et al, 2016; Zennou et al, 2004).…”

Manipulation of Cellular Processing Bodies and Their Constituents by Viruses

“…APOBEC3F and 3G localize to PBs as well as SGs (Wichroski et al, 2006;Gallois-Montbrun et al, 2007) where they interact with different proteins, including the RISC protein (Ago2), PB proteins, (Dcp1 and Dcp2) and PB-SG protein (DDX6) (Kozak et al, 2006). Although discrepancies exist with regard to interactions among various PB constituents and HIV-1 RNA, mRNA, and Gag proteins (Nathans et al, 2009;Yu et al, 2011;Bouttier et al, 2012;Phalora et al, 2012;Reed et al, 2012), the majority of the studies show that many PB components suppress HIV-1 replication by multiple mechanisms. Similar to APOBEC3, Mov10, an RNA helicase, is incorporated into HIV-1 particles.…”