Functional Analysis and Structural Modeling of Human APOBEC3G Reveal the Role of Evolutionarily Conserved Elements in the Inhibition of Human Immunodeficiency Virus Type 1 Infection and
            <i>Alu</i>
            Transposition

Many murine leukemia viruses (MLVs) are partially resistant to restriction by mouse APOBEC3 (mA3) and essentially fully resistant to induction of G-to-A mutations by mA3. In contrast, Vif-deficient HIV-1 (⌬Vif HIV-1) is profoundly restricted by mA3, and the restriction includes high levels of G-to-A mutation. Human APOBEC3G (hA3G), unlike mA3, is fully active against MLVs. We produced a glutathione S-transferase-mA3 fusion protein in insect cells and demonstrated that it possesses cytidine deaminase activity, as expected. This activity is localized within the N-terminal domain of this 2-domain protein; the C-terminal domain is enzymatically inactive but required for mA3 encapsidation into retrovirus particles. We found that a specific arginine residue and several aromatic residues, as well as the zinc-coordinating cysteines in the C-terminal domain, are necessary for mA3 packaging; a structural model of this domain suggests that these residues line a potential nucleic acid-binding interface. Mutation of a few potential phosphorylation sites in mA3 drastically reduces its antiviral activity by impairing either deaminase activity or its encapsidation. mA3 deaminates short single-stranded DNA oligonucleotides preferentially toward their 3= ends, whereas hA3G exhibits the opposite polarity. However, when packaged into infectious ⌬Vif HIV-1 virions, both mA3 and hA3G preferentially induce deaminations toward the 5= end of minus-strand viral DNA, presumably because of the sequence of events during reverse transcription in vivo. Despite the fact that mA3 in MLV particles does not induce detectable deaminations upon infection, its deaminase activity is easily detected in virus lysates. We still do not understand how MLV resists mA3-induced G-to-A mutation. IMPORTANCEOne way that mammalian cells defend themselves against infection by retroviruses is with APOBEC3 proteins. These proteins convert cytidine bases to uridine bases in retroviral DNA. However, mouse APOBEC3 protein blocks infection by murine leukemia viruses without catalyzing this base change, and the mechanism of inhibition is not understood in this case. We have produced recombinant mouse APOBEC3 protein for the first time and characterized it here in a number of ways. Our mutational studies shed light on the mechanism by which mouse APOBEC3 protein is incorporated into retrovirus particles. While mouse APOBEC3 does not catalyze base changes in murine leukemia virus DNA, it can be recovered from these virus particles in enzymatically active form; it is still not clear why it fails to induce base changes when these viruses infect new cells. M ammals possess several innate mechanisms for defense against retroviruses. One of these restriction factors is the APOBEC3 system. APOBEC3 proteins are cytidine deaminases (1). In many cases, they are encapsidated into assembling virions; when these virions infect a new cell and copy their RNA into DNA, the APOBEC3 protein from the virion binds to the singlestranded DNA (ssDNA; the initial DNA product) and deamin...

Section: Resultsmentioning

confidence: 99%

Biochemical and Biological Studies of Mouse APOBEC3

Nair

Sánchez-Martínez

et al. 2014

“…The resistance of human A3G to SIVagm Vif could serve as an effective barrier and may explain why SIVagm has not colonized humans. Several studies have shown that A3G residue 128 directly affects the binding of HIV-1 and SIVagm Vif to their respective A3G proteins, suggesting that this residue is part of the Vif binding site (19,20,22,24,25). However, adjacent amino acids, such as those at positions 129 and 130, appear to also be required for HIV-1 Vif/A3G binding (25)(26)(27), and A3G position 130 is also involved in African green monkey subspecies-specific adaptions to Vif degradation (15).…”

mentioning

confidence: 99%

Vif Proteins from Diverse Primate Lentiviral Lineages Use the Same Binding Site in APOBEC3G

Letko

Silvestri

Hahn

et al. 2013

f APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) and other lentiviruses. Most of these viruses encode a Vif protein that directly binds A3G and leads to its proteasomal degradation. Both Vif proteins of HIV-1 and African green monkey simian immunodeficiency virus (SIVagm) bind residue 128 of A3G. However, this position does not control the A3G degradation by Vif variants derived from HIV-2 and SIVmac, which both originated from SIV of sooty mangabey monkeys (SIVsmm), suggesting that the A3G binding site for Vif proteins of the SIVsmm/HIV-2 lineage differs from that of HIV-1. To map the SIVsmm Vif binding site of A3G, we performed immunoprecipitations of individual A3G domains, Vif/A3G degradation assays and a detailed mutational analysis of human A3G. We show that A3G residue 129, but not the adjacent position 128, confers susceptibility to degradation by SIVsmm Vif. An artificial A3G mutant, the P129D mutant, was resistant to degradation by diverse Vifs from HIV-1, HIV-2, SIVagm, and chimpanzee SIV (SIVcpz), suggesting a conserved lentiviral Vif binding site. Gorilla A3G naturally contains a glutamine (Q) at position 129, which makes its A3G resistant to Vifs from diverse lineages. We speculate that gorilla A3G serves as a barrier against SIVcpz strains. In summary, we show that Vif proteins from distinct lineages bind to the same A3G loop, which includes positions 128 and 129. The multiple adaptations within this loop among diverse primates underscore the importance of counteracting A3G in lentiviral evolution.

“…The observation that Bet prevented A3G dimerization allowed us to identify relevant residues in A3G regulating the Bet interaction. Amino acids in the putative A3G dimer interface were recently described (36,46), so we tested the Y124A, Y125A, F126L, and W127A A3G mutants (36) that partake in stabilizing the A3G dimer for interaction with Bet. Of these mutants, the W127A mutant is unable to homodimerize and to inhibit HIV-1 ⌬vif due to a lack of encapsidation (36,46).…”

Section: Resultsmentioning

confidence: 99%

“…Amino acids in the putative A3G dimer interface were recently described (36,46), so we tested the Y124A, Y125A, F126L, and W127A A3G mutants (36) that partake in stabilizing the A3G dimer for interaction with Bet. Of these mutants, the W127A mutant is unable to homodimerize and to inhibit HIV-1 ⌬vif due to a lack of encapsidation (36,46). The Y124A and F126L A3G mutants coimmunoprecipitated with Bet, like wild-type A3G, whereas the F126A and W127A A3G mutants showed partial and complete loss of coimmunoprecipitation, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For structure-guided mutation analysis, the previously published homology model of the A3G N terminus-N terminus dimer interface (36) was used. This model was generated using the MODELLER program (47); the crystal structure of the human A3G Cterminal domain (48) served as the template (for detailed methods, see reference 36).…”

Section: Coimmunoprecipitation Assays (I) Interaction Of Purified A3mentioning

confidence: 99%

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Prototype Foamy Virus Bet Impairs the Dimerization and Cytosolic Solubility of Human APOBEC3G

Vasudevan

Perković

Bulliard

et al. 2013

Self Cite

c Cellular cytidine deaminases from the APOBEC3 family are potent restriction factors that are able to block the replication of retroviruses. Consequently, retroviruses have evolved a variety of different mechanisms to counteract inhibition by APOBEC3 proteins. Lentiviruses such as human immunodeficiency virus (HIV) express Vif, which interferes with APOBEC3 proteins by targeting these restriction factors for proteasomal degradation, hence blocking their ability to access the reverse transcriptase complex in the virions. Other retroviruses use less-well-characterized mechanisms to escape the APOBEC3-mediated cellular defense. Here we show that the prototype foamy virus Bet protein can protect foamy viruses and an unrelated simian immunodeficiency virus against human APOBEC3G (A3G). In our system, Bet binds to A3G and prevents its encapsidation without inducing its degradation. Bet failed to coimmunoprecipitate with A3G mutants unable to form homodimers and dramatically reduced the recovery of A3G proteins from soluble cytoplasmic cell fractions. The Bet-A3G interaction is probably a direct binding interaction and seems to be independent of RNA. Together, these data suggest a novel model whereby Bet uses two possibly complementary mechanisms to counteract A3G: (i) Bet prevents encapsidation of A3G by blocking A3G dimerization, and (ii) Bet sequesters A3G in immobile complexes, impairing its ability to interact with nascent virions.