Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase

Abstract

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses.

Figure 1. Deamination is not required for antiviral activity of A3A.

(A) Schematic of A3A and active site mutants. Domains marked are the cytidine deaminase domain (CDD), the linker (LINK), the pseudoactive site (PAS) and the hemagglutinin epitope tag (H). Active site residues conserved among APOBEC3 proteins (H-X-E-X₂₈-PC-X₄-C) are indicated in bold. Asterisks indicate specific mutations generated for this study; F75 is indicated with a red asterisk. (B) Immunofluorescence to detect HA-tagged APOBEC3 proteins (red) expressed by transfection in U2OS cells. (C) In vitro assay for cytidine deaminase activity. Proteins were generated by IVT, immuno-precipitated by the HA epitope, and incubated with a radiolabeled substrate (T₂₈TCAT₂₉). The deaminated molecules were cleaved by treatment with UDG followed by high pH, and the products were resolved by PAGE (upper panel). Arrows indicate the substrate and deaminated product. The panel below shows an immunoblot to detect in vitro translated proteins. (D) Inhibition of AAV. Production of rAAVLuc was assessed by transfection of 293T cells with AAV plasmids in the presence of APOBEC3 expression constructs (1 µg). Production of rAAV was assessed by transduction of target cells and quantitation of luciferase activity. Presented is the average of three independent experiments normalized to vector only control (mock). The panels below show immunoblots to detect HA-tagged wild type and mutant A3A proteins in transfected 293T cell lysates. Ku86 served as a loading control.

Figure 2. A3A mutants inhibit AAV DNA replication.

(A) Titration of wild-type and mutant A3A expression vectors in rAAVLuc production assays. Production of rAAVLuc was assessed by transduction of target cells and quantitation of luciferase activity. Presented is the average of four independent experiments normalized to vector alone control (mock). The panels below show immunoblots to detect HA-tagged wild-type and mutant A3A proteins in transfected 293T cell lysates. (B) Southern blot detection of low molecular weight DNA extracted from 293T cells transfected for rAAVLuc production in the presence of mock (1 µg) A3G (1 µg), A3A (1, 0.1, 0.01 and 0.001 µg) and mutant A3A expression vectors (1 µg). The DNA was digested with Dpn-I, separated by gel electrophoresis, and hybridized with a radiolabeled luciferase probe.

Figure 3. A3A inhibits AAV2 and MVM DNA replication.

(A) Inhibition of wild-type AAV replication. U2OS cells were transfected with plasmids for APOBEC3 proteins and then infected with AAV and adenovirus. HA-tagged APOBEC3 (red) and AAV Rep proteins (green) were detected by immunofluorescence using specific antibodies. (B) Inhibition of wild-type MVM replication. Southern blot detection of low molecular weight DNA extracted from A9 cells cotransfected with an infectious MVM clone together with APOBEC3A expression plasmids. The DNA was digested with Dpn-I, separated by gel electrophoresis and hybridized with a radiolabeled MVM probe. Left line is a marker (M). Replicative intermediates of ssDNA (SS), monomer (M), and dimer (D) are indicated to the right. Panel below shows immunoblots for APOBEC3 and the NS1 protein of MVM.

Figure 4. Alignment of APOBEC3 amino acid sequences for A3A with the C-terminus of A3B and A3G.

Residues exchanged in A3A are boxed and the mutant designation is indicated above. The PmlI site and stretches of variable sequence VS1 and VS2 switched in the chimeras are also indicated. The asterisks mark individual amino acid mutants, and the diamond indicates the start of A3G-CT (residues 197–384). Residue numbers are indicated on the right side. Predicted secondary structure of A3A is indicated below the alignment with α-helices in black and stranded β-sheets in grey. A3A secondary structure modeling was generated by Swiss-Model using the crystal structure of the C-terminal fragment of A3G (Protein Data Bank accession number 3E1A) as template .

Figure 5. Activity of A3A/A3G PmlI based chimeras.

(A) Schematic of A3A (dark grey) and A3G (light grey). Domains marked are the cytidine deaminase domains (CDD), the linker (LINK), the pseudoactive site (PAS), and the hemagglutinin epitope tag (H). Below are the chimeras generated at the PmlI site. (B) In vitro assays for cytidine deaminase activity. Proteins were immunoprecipitated from transfected cells by the HA epitope and incubated with the indicated radiolabeled substrate in UDG-dependent assays. The upper panel uses a substrate oligonucleotide with target sequence CCCG, and the middle panel uses an oligonucleotide with the specific A3A target sequence TCA. The substrate and deaminated products are indicated. The bottom panel shows an immunoblot to detect proteins in immunoprecipitates. A band corresponding to the light-chain IgG used for immunoprecipitation is indicated (*). (C) Production of rAAV in the presence of APOBEC3 proteins. 293T cells were transfected with APOBEC3 constructs (1 µg, except for A3A 0.1 µg), together with plasmids required for rAAVLuc production. Virus production was assessed by transduction of target cells and quantitation by luciferase assay. Panels below show immunoblots for APOBEC3 proteins (HA) in transfected cells and Ku86 as a loading control. (D) Dose-response for A3A and the A3ApmlA3G chimera in the rAAV production assay. Panels below show immunoblots for APOBEC3 (HA) and Ku86 proteins in transfected cells.

Figure 6. Mutants of A3A with residues replaced with the corresponding sequences of A3G.

(A) Schematic of variable segments VS1 and VS2, and the chimeric mutants generated for A3A in these regions. The VS1 segment corresponds to A3A residues 60 to 67, and VS2 corresponds to A3A residues 132 to 137. (B) In vitro deamination assay. Proteins were immunoprecipitated from transfected cells and incubated with radiolabeled oligonucleotide (T₂₈CCCGT₂₈) for 16 h in UDG-dependent assays. Arrows indicate the substrate and deaminated product. The panel below shows an immunoblot to detect proteins in immunoprecipitates. (C) Production of rAAV in the presence of wild-type (0.1 µg) and mutant A3A proteins (1 µg). Panels below show immunoblots for APOBEC3 proteins (HA) in transfected cells and Ku86 as a loading control.

Figure 7. Identification of a gain-of-function mutant for A3G.

(A) Schematic of full-length A3G and the C-terminal fragment A3G-CT. Chimeras of A3G-CT were generated with variable segments VS1 and VS2 replaced with sequences of A3A. (B) Immunofluorescence to detect localization of HA-tagged APOBEC3 and chimeric proteins (red) expressed by transfection in U2OS cells. Cell nuclei were detected by staining with DAPI (blue). (C) Production of rAAV in the presence of APOBEC3 proteins. Virus production was assessed by transduction of target cells and quantitation by luciferase assay. Immunoblots show similar expression levels for APOBEC proteins in transfected cells. Ku86 served as a loading control. The A3G-CT/KNLLCGFY mutant demonstrated activity against AAV. The asterisks indicate that the inhibition with A3A and A3G-CT/KNLLCGFY was statistically significant (p<0.001) when compared to mock by Student t test.