Quantification of Deaminase Activity-Dependent and -Independent Restriction of HIV-1 Replication Mediated by APOBEC3F and APOBEC3G through Experimental-Mathematical Investigation

Abstract

f APOBEC3F and APOBEC3G cytidine deaminases potently inhibit human immunodeficiency virus type 1 (HIV-1) replication by enzymatically inserting G-to-A mutations in viral DNA and/or impairing viral reverse transcription independently of their deaminase activity. Through experimental and mathematical investigation, here we quantitatively demonstrate that 99.3% of the antiviral effect of APOBEC3G is dependent on its deaminase activity, whereas 30.2% of the antiviral effect of APOBEC3F is attributed to deaminase-independent ability. This is the first report quantitatively elucidating how APOBEC3F and APOBEC3G differ in their anti-HIV-1 modes. Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, hijacks lines of cellular proteins for its replication (reviewed in reference 1). On the other hand, accumulating evidence has revealed that human cells intrinsically possess anti-HIV-1 proteins (reviewed in reference 2). Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cellular cytidine deaminases, and certain APOBEC3 proteins, particularly APOBEC3F (3, 4) and APOBEC3G (5), are the intrinsic restriction factors against HIV-1. APOBEC3F and APOBEC3G are incorporated into assembling HIV-1 particles and brought into the newly infected cells. During reverse transcription (RT) in the infected cells, these APOBEC3 proteins enzymatically convert C in the viral minus-strand DNA to U, resulting in G-to-A mutation in the nascent plus-strand DNA. These mutations can become nonsynonymous or even lethal, which severely debilitates subsequent viral replication.To antagonize these APOBEC3F-and APOBEC3G-mediated antiviral effect, viral infectivity factor (Vif), an accessory protein of HIV-1, degrades APOBEC3F and APOBEC3G through a ubiquitin/proteasome-dependent pathway (reviewed in references 6 and 7). Previous studies have directly demonstrated that Vif is a prerequisite for HIV-1 replication in vitro (e.g., human primary CD4 ϩ T lymphocytes and macrophages) (8, 9) and in vivo (e.g., humanized mouse models) (10, 11). Moreover, G-to-A mutations have been clearly observed in the proviral DNA of HIV-1-infected patients, although the frequencies of G-to-A mutations seem to vary among individuals (12-23).It has been reported that the incorporated APOBEC3F and APOBEC3G potently impair the HIV-1 RT process even prior to the insertion of G-to-A mutations (24-26). Moreover, this inhibition is independent of these APOBEC3s' deaminase activity (26). These findings indicate that APOBEC3F and APOBEC3G potently inhibit HIV-1 replication through at least two distinct modes: (i) deaminase activity-dependent G-to-A mutations of viral DNA and (ii) impairment of viral RT independent of deaminase activity (Fig. 1). However, how much of the inhibition of HIV-1 replication by APOBEC3 proteins is attributed to each of these two modes has not been quantitatively revealed.In order to quantitatively determine the relative extent of deaminase activity-dependent and -independent antiviral activity of ...

Long-term passage of Vif-null HIV-1 in CD4 + T cells expressing sub-lethal levels of APOBEC proteins fails to develop APOBEC resistance

“…Importantly, increasing A3G expression using a deaminase-defective variant of A3G did not alter the semi-permissive phenotype of the A3.01 cells. These results suggest that viral restriction is associated with increased A3G deaminase activity consistent with previous reports showing that efficient inhibition of HIV-1 requires enzymatically active A3G (Browne et al, 2009; Kobayashi et al, 2014; Miyagi et al, 2007; Schumacher et al, 2008). On the other hand, a previous study proposed that A3G-mediated viral restriction requires a cellular cofactor that may be lacking in certain cell lines such as CEM-T4 (Han et al, 2008).…”

APOBECs and virus restriction

“…This deaminase activity-independent effect appears to be greater for A3F than for A3G (Albin et al, 2014; Browne et al, 2009; Holmes et al, 2007a; Kobayashi et al, 2014; Schumacher et al, 2008). Primary cell studies also suggest a deaminase-independent component (Gillick et al, 2013).…”

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…A3F was also shown to inhibit the production of full-length products of RT more efficiently than A3G, and can inhibit the accumulation of late reverse transcriptase products [74]. Interestingly, the deaminationindependent mode represents less than 1% of the restriction activity for A3G, whereas for A3F it represents approximately 30% [162]. Therefore, these studies on the deamination-independent mechanisms have highlighted the contribution of these mechanisms to the restriction of HIV, but this mode of restriction is notably less potent than the restriction caused by cytosine deamination.…”