Journal of Molecular Biology
Interaction with 7SL RNA but Not with HIV-1 Genomic RNA or P Bodies Is Required for APOBEC3F Virion Packaging
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 Hx5Cx17–18Cx3–5H 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.
Section snippets
Viral genomic RNA is not required for A3F virion packaging
It has been reported that A3F interacts with mRNAs, including HIV-1 RNA. To address the question of whether viral genomic RNA plays a role in A3F packaging into HIV-1 virions, we have compared the level of virion-associated A3F-HA in wild-type pNL4-3 and the viral genomic RNA packaging mutant pRB653-47,81 which has mutations of the cysteine residues in the zinc fingers of the HIV-1 NC domain (Fig. 1a). A3F-HA was relatively resistant to HIV-1 Vif-induced degradation (Fig. 1a), consistent with
Discussion
In the present study, careful evaluation of various A3F-associated factors, including 7SL RNA, viral genomic RNA, and P bodies, indicated that an interaction with 7SL RNA plays an important role in the virion packaging of A3F. Although the role of viral genomic RNA in mediating A3G packaging into HIV-1 virions has been controversial, several lines of evidence suggest that viral genomic RNA is not required for A3F virion packaging. First, HIV-1 NC zinc finger mutant viruses packaged only a
Plasmid construction
Infectious molecular clones of the parental wild-type HIV-1pNL4-3 and Vif mutant (pNL4-3ΔVif) constructs were obtained from the AIDS Research Reagents Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. pΔPolΔEnv and pGagINS have been described previously.68 pGagINS expresses Gag in the absence of Rev.101 Gag-NC+ and Gag-NC− have been described previously as pNCS and P2SLZ.68 The HIV-1 NC mutant construct pRB653-4781 was a gift from
Acknowledgements
We thank Michael H. Malim for kindly providing the A3F-HA expression vector, Robert Gorelick for kindly providing the pRB653-47 vector, Elana Ehrlich and Anna Maria Niewiadomska for technical assistance and thoughtful discussions, and Deborah McClellan for editorial assistance. This work was supported by a grant from the National Institutes of Health (AI062644), a grant from the Johns Hopkins Center for AIDS Research, and funding from the National Science Foundation of China (NSFC-30425012) and
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