Interaction with 7SL RNA but Not with HIV-1 Genomic RNA or P Bodies Is Required for APOBEC3F Virion Packaging

Abstract

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F), like APOBEC3G, has broad antiviral activity against diverse retroelements, including Vif-deficient human immunodeficiency virus (HIV)-1. Its antiviral functions are known to rely on its virion encapsidation and be suppressed by HIV-1 Vif, which recruits Cullin5-based E3 ubiquitin ligases. However, the factors that mediate A3F virion packaging have not yet been identified. In this study, we demonstrate that A3F specifically interacts with cellular signal recognition particle (SRP) RNA (7SL RNA), which is selectively packaged into HIV-1 virions. Efficient packaging of 7SL RNA as well as A3F was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. Reducing 7SL RNA packaging by overexpression of SRP19 protein inhibited A3F virion packaging. Although A3F has been shown to interact with P bodies and viral genomic RNA, our data indicated that P bodies and HIV-1 genomic RNA were not required for A3F packaging. Thus, in addition to its well-known function in SRPs, 7SL RNA, which is encapsidated into diverse retroviruses, also participates in the innate antiviral function of host cytidine deaminases.

Introduction

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G, hereinafter A3G) and other APOBEC3 proteins¹ are related to a family of proteins that also includes APOBEC1 (apolipoprotein B-editing catalytic subunit 1), APOBEC2, and activation-induced cytidine deaminase.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 These proteins have cytidine deaminase activities that modify RNA or DNA. To date, seven members of the APOBEC3 family (A3A–A3H) have been described in human with varying degrees of inhibitory activity against retroviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus,13, 14, 15, 16, 17, 18, 19, 20, 21 retrotransposons LINE1,22, 23, 24, 25, 26, 27 Alu,22, 23, 28 Ty elements of yeast,29, 30 endogenous retroviruses,³¹ hepatitis B virus,32, 33, 34, 35, 36, 37 and adeno-associated virus.²⁴

The most prominent member of the APOBEC3 family, human A3G, was the first protein in this family of cytidine deaminases to be identified as a potent inhibitor of HIV-1 in the absence of Vif.¹³ When A3G is successfully incorporated into newly formed virions, it mediates antiviral activity by inducing hypermutations in newly synthesized viral minus-strand DNA.18, 20, 21, 38, 39, 40, 41 Virion-packaged A3G and A3F can also reduce the accumulation of viral DNA by inhibiting reverse transcription42, 43, 44, 45, 46 or inducing viral DNA degradation.47, 48 However, this reduction in viral DNA cannot fully account for the antiviral activities of A3G and A3F,20, 21, 44, 45, 46 and a potent inhibitory effect of A3G on the formation of proviral DNA has been described.44, 45 A3F also inhibits HIV-1 reverse transcription45, 49, 50 and the formation of proviral DNA.⁴⁵

HIV-1 Vif suppresses APOBEC3 antiviral function by hijacking the cellular Cullin5 (Cul5)–ElonginB–ElonginC E3 ubiquitin ligase⁵¹ to target A3G for proteasomal degradation.51, 51, 53, 54, 55, 56, 57, 58 Vif molecules of HIV-1 and simian immunodeficiency virus are newly identified substrate receptor proteins that assemble with Cul5, ElonginB, ElonginC, and Rbx1 to form an E3 ubiquitin ligase.51, 52, 58, 59, 60, 61 The most conserved motif among all lentiviral Vif proteins, SLQxLA, is a virus-specific BC-box motif that mediates the interaction with ElonginC, which, in turn, interacts with ElonginB and Cul5. Primate lentiviral Vif molecules use another highly conserved Hx₅Cx_17–18Cx_3–5H motif to selectively bind Cul5.⁶⁰ This motif binds zinc and stabilizes a highly conserved hydrophobic interface in Vif that mediates Cul5 selection.60, 62, 63 Interaction of HIV-1 Vif with substrate A3G or A3F maps to its N-terminal region.54, 64, 65, 66, 67

In the absence of the Vif protein, APOBEC3 proteins are packaged into diverse retroviruses and mediate antiviral functions in newly infected target cells. The virion-packaging mechanisms of the APOBEC3 proteins are not yet fully understood. Encapsidation of A3G into HIV-1 particles is mediated by the Gag protein.68, 69, 70, 71, 72, 73, 74 Most studies have found that the RNA-binding nucleocapsid (NC) domain of Gag molecules is required for efficient A3G packaging.68, 69, 70, 71, 72, 73, 74 Several groups have reported that the interaction between HIV-1 Gag and A3G requires RNA,45, 69, 72, 75, 76 suggesting a role for RNA in mediating A3G packaging. An RNase-resistant interaction between HIV-1 Gag and A3G has also been observed.70, 73 While two studies have reported that viral genomic RNA is required for efficient packaging of A3G into virions,72, 77 many studies have found that viral genomic RNA is largely dispensable for A3G packaging,68, 69, 70, 71, 72, 73, 74, 75, 77 suggesting a role for cellular RNA in the virion packaging of A3G.

An interaction of A3G and A3F with mRNA processing (P) bodies has been observed and has been proposed to play a role in mediating the packaging of these cytidine deaminases into HIV-1 virions.⁷⁸ A3G and A3F have also been shown to bind mRNAs in polyribosomes,⁷⁹ stress granules,79, 80 and Staufen granules.²⁸ However, whether P bodies, stress granules, or Staufen granules are important for A3F packaging into HIV-1 virions is not yet clear.

In this study, we demonstrate that A3F selectively interacts with 7SL RNA in virus-producing cells. We further show that packaging of both A3F and 7SL RNA requires the NC domain of HIV-1 Gag, and inhibiting 7SL RNA packaging into HIV-1 virions impairs A3F packaging. On the other hand, our data indicate that viral genomic RNA and P bodies are not required for A3F packaging into HIV-1 virions.