Incorporation of Mouse APOBEC3 into Murine Leukemia Virus Virions Decreases the Activity and Fidelity of Reverse Transcriptase

Abstract

bAPOBEC3 proteins are restriction factors that induce G¡A hypermutation in retroviruses during replication as a result of cytidine deamination of minus-strand DNA transcripts. However, the mechanism of APOBEC inhibition of murine leukemia viruses (MuLVs) does not appear to be G¡A hypermutation and is unclear. In this report, the incorporation of mA3 in virions resulted in a loss in virion reverse transcriptase (RT) activity and RT fidelity that correlated with the loss of virion-specific infectivity.A POBEC3G (hA3G) in humans and APOBEC3 (mA3) in mice are cytidine deaminases that act on single-stranded DNA during reverse transcription, resulting in G¡A hypermutation of newly synthesized proviral DNA (1, 2). Although exogenous murine leukemia viruses (MuLVs) are relatively insensitive to the actions of mA3 (2-7), several studies have reported partial inhibition of exogenous MuLVs after incorporation of mA3 (2,3,6,(8)(9)(10)(11). Furthermore, the finding that Rfv3, a resistance gene for Friend erythroleukemia, encodes mA3 and is responsible for a decreased infectious titer of the Friend MuLV (Fr-MuLV) (11-13) strongly suggests that mA3 inhibits the replication of exogenous MuLVs in vivo. Exogenous ecotropic MuLVs, such as the FrMuLV and Moloney MuLV (Mo-MuLV), are inhibited through mechanisms that do not appear to involve cytidine deamination (2, 9, 10). In this study, the effects of mA3 on the infectivity, reverse transcriptase (RT) activity, and frequency of mutations of the ecotropic MuLV CasFr KP were examined. Virion-associated mA3 suppresses CasFr KP MuLV infectivity. In order to examine the effects of mA3 incorporated into MuLV virions, we derived clonal cell lines infected with CasFr KP (14). The 3T3mA3 cells were derived by transfection of a plasmid encoding the full-length mA3 derived from the BALB/c mouse strain and was tagged at the C termini with hemagglutinin (HA) (5). Infected clonal cell lines were obtained from 3T3 cells as well as from 3T3 cells expressing mA3 (3T3mA3) and were designated 3T3/CasFr KP and 3T3mA3/CasFr KP , respectively. In agreement with earlier reports (2, 7, 9, 13, 15), the clonal cell line expressing mA3 (3T3mA3/CasFr KP ) released virions that had incorporated an easily detectable level of mA3 (Fig. 1).The infectivity of CasFr KP containing mA3 was compared to that of CasFr KP devoid of mA3 by a focal immunofluorescence assay (FIA) (16) and normalized for virion number using the level of p30 CA protein (Fig. 2A). The specific infectivity of virions released from cells expressing mA3 exhibited over a 90% reduction in infectivity (Fig. 2B), corroborating earlier studies of inhibition by mA3 (2,3,6,(8)(9)(10)(11).Decrease in RT activity in virions containing mA3. A recent study examined the efficiency of virion reverse transcription by monitoring the appearance of strong-stop DNA during the course of the RT reaction using virions isolated from C57BL/6 and BALB/c mice as well as those from mA3 knockout (KO) mice (17). Both mouse strains exhibited a similar decrease in RT activity...

APOBECs and virus restriction

“…Recent data indicate that glyco-Gag affords protection from the anti-viral effects of murine A3 (Boi et al, 2014; Kolokithas et al, 2010; Nitta et al, 2012; Stavrou et al, 2013). Almost all MuLVs encode a longer glycosylated form of the capsid precursor protein Gag (glyco-Gag), which originates from translation initiation at a CUG start codon upstream of the normal cytoplasmic Gag start codon (Berlioz and Darlix, 1995).…”

“…Several studies have shown that glyco-Gag defective particles are less infectious than wild-type MuLV particles (Boi et al, 2014; Kolokithas et al, 2010; Nitta et al, 2012; Stavrou et al, 2013). This restriction phenotype is largely alleviated in A3-deficient cells and animals (Boi et al, 2014; Kolokithas et al, 2010; Stavrou et al, 2013).…”

“…This restriction phenotype is largely alleviated in A3-deficient cells and animals (Boi et al, 2014; Kolokithas et al, 2010; Stavrou et al, 2013). Moreover, glyco-Gag-defective viruses reverted to wild-type function during infections of A3-expressing animals, but not A3-null animals, demonstrating the importance of glyco-Gag in antagonizing A3-dependent restriction (Stavrou et al, 2013).…”

“…Interestingly, glyco-Gag-mutant virions are less stable than wild-type particles during ultracentrifugation with detergent (Stavrou et al, 2013). Further, A3 incorporation during cell culture and in vivo replication caused defects in reverse transcription when glyco-Gag was absent (Boi et al, 2014; Stavrou et al, 2013). These studies combined to suggest a mechanism in which glyco-Gag stabilizes the viral core and shields viral reverse transcription complexes from the restrictive activities of A3, as well as affording protection from other innate immune effector proteins such as the DNA nuclease Trex1 (Stavrou et al, 2013) (Figure 3).…”

APOBEC3 enzymes restrict marginal zone B cells

“…Apobec3 (A3) enzymes restrict exogenous and endogenous retroelements by a variety of mechanisms , including hypermutating the first‐strand cDNA of endogenous mouse leukemia virus (MLV) , aggregating retroelement mRNA , and decreasing the activity and fidelity of reverse transcriptase . As a result, these enzymes prevent the spread of infection by exogenous retroviruses, and help maintain the integrity of the cellular genome and transcriptome by suppressing insertional mutagenesis of endogenous retroelements.…”