Host restriction of murine gammaherpesvirus 68 replication by human APOBEC3 cytidine deaminases but not murine APOBEC3

Abstract

Humans encode seven APOBEC3 (A3A-A3H) cytidine deaminase proteins that differ in their expression profiles, preferred nucleotide recognition sequence and capacity for restriction of RNA and DNA viruses. We identified APOBEC3 hotspots in numerous herpesvirus genomes. To determine the impact of host APOBEC3 on herpesvirus biology in vivo, we examined whether murine APOBEC3 (mA3) restricts murine gammaherpesvirus 68 (MHV68). Viral replication was impaired by several human APOBEC3 proteins, but not mA3, upon transfection of the viral genome. The restriction was abrogated upon mutation of the A3A and A3B active sites. Interestingly, virus restriction by A3A, A3B, A3C, and A3DE was lost if the infectious DNA was delivered by the virion. MHV68 pathogenesis, including lung replication and splenic latency, was not altered in mice lacking mA3. We infer that mA3 does not restrict wild type MHV68 and restriction by human A3s may be limited in the herpesvirus replication process.

Keywords: APOBEC3; Cytidine deaminase; Herpesvirus; Host restriction; Latency; Murine gammaherpesvirus; Pathogenesis; Replication.

Figure 1. APOBEC3 hotspots in herpesvirus genomes

A. Percentage of 1 kb windows neutral, overrepresented or underrepresented for human and mouse APOBEC3 hotspots in the indicated viral genome. B. Heatmap of A3 hotspots in herpesvirus genomes. Scale indicates the percentage of 1 kb windows of the viral genome with overrepresentation or underrepresentation of hotspots as described in A. C. Distribution of 1 kb regions of the MHV68 genome overrepresented (red bars) or underrepresented (blue bars) for the indicated A3 hotspots. The boundary of each bar corresponds to the midpoint of the first and last 1 kb window represented within the bar. The gray trace represents the distribution of the hotspot occurrence in a region compared to the occurrence in 1,000 randomly shuffled 1 kb genomic regions. The scale of the trace is from −0.5 to 0.5, the numbers on the y-axis indicate the distance from the mean of each distribution (y=0). Genomic regions are significantly overrepresented (red bars) or underrepresented (blue bars) if they are above 0.45 or below −0.45 respectively.

Figure 2. Restriction of MHV68 spread by human APOBEC3A, but not murine APOBEC3

HEK 293T cells were cotransfected with MHV68-H2bYFP BAC and the indicated human (hA3) and murine (mA3) APOBEC3 constructs and virus spread was visualized by fluorescence microscopy (upper panels) and bright field microscopy (lower panels) 48 hrs later. HA3A E72A is an active site mutant of human A3A. MA3 E73A and mA3 E253A are active site mutants of mA3. Images shown are cropped fields of pictures taken at 20X magnification. The images of MHV68-H2bYFP+ foci were false-colored green.

Figure 3. Restriction of MHV68 replication by human APOBEC3

A. Variable restriction by human APOBEC3 constructs as measured by plaque assay 48 hrs after HEK 293T cells were cotransfected with infectious MHV68-H2bYFP BAC DNA and the indicated APOBEC3 constructs. B. Murine A3 did not significantly impair MHV68 replication in HEK293Ts. Below each bar graph, immunoblots using antibodies against the indicated epitopes were used to validate APOBEC3 expression. Lysates from the empty vectors pcDNA3.1 (control vector for hA3B-hA3H) and pcDNA6 (control vector for hA3A and hA3C) were included in each blot and revealed no specific signal. Bars represent the mean +/− standard deviation of three replicates (A) and five replicates (B); *p≤0.05 and **p≤0.005.

Figure 4. APOBEC3A and APOBEC3B active sites are required for restriction of viral replication and viral DNA synthesis

A. Loss of restriction by mutant human APOBEC3A (hA3) and APOBEC3B (hA3B) constructs as measured by plaque assay and quantitative PCR (hA3B) 48 hrs after HEK 293T cells were cotransfected with infectious MHV68-H2bYFP BAC DNA and the indicated APOBEC3 constructs. Below each bar graph, immunoblots using antibodies against the indicated epitopes were used to validate APOBEC3 expression. Lysates from the empty vectors pcDNA6 (control vector for WT and mutant hA3A) and pcDNA3.1 (control vector for WT and mutant hA3B) were included in each blot and revealed no specific signal. Bars represent the mean +/− standard deviation of three replicates; *p≤0.05 and **p≤0.005. B. Quantitation of MHV68 genomes by qPCR analysis of DpnI-digested DNA. DNA levels were normalized to levels upon cotransfection with the empty vector, pCDNA6; *p≤0.05 and **p≤0.005.

Figure 5. Loss of APOBEC3 restriction in the context of de novo infection with virus particles

HEK 293T cells were transfected with the indicated APOBEC3 constructs 24 hrs prior to de novo infection with MHV68-H2bYFP virus. MHV68 replication was measured by plaque assay 48 hpi. Below each bar graph, immunoblots using antibodies against the indicated epitopes were used to validate APOBEC3 expression. Lysates from the empty vectors pcDNA3.1 (control vector for hA3B, hA3DE-hA3H) and pcDNA6 (control vector for hA3A and hA3C) were included in each blot and revealed no specific signal.

Figure 6. Absence of murine APOBEC3 does not alter MHV68 pathogenesis

A. Schematic depicting the disruption of murine APOBEC3 (mA3) by the insertion of a β-geo cassette to generate mA3 knockout mice (mA3^−/−). B. Genotype PCR analysis of DNA harvested from the splenocytes of infected WT C57BL/6 or mA3^−/− mice. The locations of primers for either the WT or mutant mA3 gene are shown in panel A. C. Immunoblot demonstrating expression of mA3 in WT but not mA3^−/− mice. D. Acute replication in the lungs of WT or mA3^−/− lungs 4 and 7 days post intranasal infection with 1000 PFU of WT MHV68. E. Spleen weights from naïve WT, infected mA3^−/− mice or infected WT mice 16 dpi. F. Latency is determined as the frequency of viral genome positive splenocytes determined by limiting dilution PCR. G. Frequency of reactivation from intact splenocytes determined by a limiting dilution explant reactivation assay 16 dpi. Open symbols represent preformed infectious virus from mechanically disrupted cells plated in parallel. The data shown represent three independent experiments with splenocytes pooled from three to five mice per experimental group.