Positive selection and increased antiviral activity associated with the PARP-containing isoform of human zinc-finger antiviral protein

Abstract

Intrinsic immunity relies on specific recognition of viral epitopes to mount a cell-autonomous defense against viral infections. Viral recognition determinants in intrinsic immunity genes are expected to evolve rapidly as host genes adapt to changing viruses, resulting in a signature of adaptive evolution. Zinc-finger antiviral protein (ZAP) from rats was discovered to be an intrinsic immunity gene that can restrict murine leukemia virus, and certain alphaviruses and filoviruses. Here, we used an approach combining molecular evolution and cellular infectivity assays to address whether ZAP also acts as a restriction factor in primates, and to pinpoint which protein domains may directly interact with the virus. We find that ZAP has evolved under positive selection throughout primate evolution. Recurrent positive selection is only found in the poly(ADP-ribose) polymerase (PARP)-like domain present in a longer human ZAP isoform. This PARP-like domain was not present in the previously identified and tested rat ZAP gene. Using infectivity assays, we found that the longer isoform of ZAP that contains the PARP-like domain is a stronger suppressor of murine leukemia virus expression and Semliki forest virus infection. Our study thus finds that human ZAP encodes a potent antiviral activity against alphaviruses. The striking congruence between our evolutionary predictions and cellular infectivity assays strongly validates such a combined approach to study intrinsic immunity genes.

Figure 1. ZAP(S) and ZAP(L) Isoforms in Mammals

(A) Schematic of the genomic structure of human ZAP(L) and the protein structure with previously defined domains (CCCH fingers, TPH, WWE, and PARP-like). The asterisk indicates the stop codon for an alternative transcript that has been reportedly isolated from multiple cDNA libraries. An additional transcript with an alternatively spliced exon (366 bp) inserted after exon 4 was also identified in several primates (not shown) but not in human, so our evolutionary analysis was limited to the ZAP(L) isoform shown. The rat NZAP (254 aa) protein structure is shown for comparison. A longer ZAP(L) isoform was also detected by RT-PCR from rat liver RNA using a forward primer in the WWE domain (exon 7) and a reverse primer in the PARP domain (exon 13) (see arrows). The resulting product was directly sequenced and corresponded to exons 7–13 of rat ZAP (gel inset). (B) Human ZAP isoforms are expressed in a wide range of tissues, including germline tissues and peripheral blood lymphocytes (PBLs). Results from PCR amplification from a human multiple tissue cDNA panel with primers specific to the human ZAP(S) and (L) isoforms (top and bottom panels, respectively). Lanes are labeled according to the template tissue (M, DNA standard marker; blank, no template).

Figure 2. Positive Selection of ZAP(L) in Primates

(A) Ancient signature of positive selection in primate ZAP. A sequence alignment of ZAP(L) from four New World monkeys, three Old World monkeys, and six hominoids was analyzed using the free-ratio model from PAML, which allows dN/dS to vary along each branch. The corresponding dN/dS values are shown for each branch, and bold numbers indicate those branches with dN/dS > 1. In the case of no observed synonymous changes (inf), a dN/dS ratio could not be calculated. (B) An alignment of a segment of the PARP domains from these primate species is shown. Identical and similar residues are indicated with asterisks (*) and colons (:), respectively. The 3 residues identified with high confidence as evolving under positive selection are indicated with exclamation marks and lie in close proximity to the NAD+ binding contact residues (indicated by yellow boxes).

Figure 3. ZAP Viral Inhibition Is Specific to MLV but Not HIV, and the PARP Domain Enhances the Viral Restriction

Co-transfections of ZAP isoforms with the LlucSN (MLV) or pwtLTR-luc1 (HIV) promoter constructs were used to assess the ability of the ZAP isoforms to restrict HIV- and MLV-driven luciferase. (A) ZAP inhibits MLV in cells transfected with equal amounts of the ZAP expression constructs. Equal amounts of each ZAP expression construct were co-transfected with LlucSN (MLV). All transfections were equalized for DNA amount with the addition of pcDNA. Lysates from transfected cells were collected after 24 h, and luciferase activity was measured. (B) Effect of increasing amounts of transfected human ZAP(L) or ZAP(S) DNA on MLV-LTR driven luciferase expression. Lysates from transfected cells were harvested at 48 h, and luciferase activity was measured. Results are shown as percent luciferase expression of the control transfection (no ZAP). The results from one representative experiment are shown, with error bars reflecting the variation between triplicate infections within one experiment. (C) Equivalent amounts of lysates from 293T cells transiently co-transfected with different amounts of ZAP expression constructs and the MLV LTR-luciferase construct were analyzed by Western blot analysis to determine the relative amount of protein. Samples were harvested 48 hours after transfection. Sizes of HA-tagged ZAP(L), ZAP(S), and rat NZAP are ∼115 kDa, ∼80 kDa, and ∼30 kDa, respectively. Actin was the loading control. To determine the relative amounts of ZAP(L) and ZAP(S), Western blot analysis was performed on a dilution series of lysates from 293T cells transfected with the ZAP constructs. (D) For ZAP and HIV co-transfections, pwtLTR-luc1 (HIV) was co-transfected with either no ZAP, ZAP(L), ZAP(S), or rat NZAP (a CMV-Tat construct was also transfected to improve expression levels of HIV). Transfections and luciferase assays were performed as in the MLV experiment.

Figure 4. Human ZAP Restricts SFV Infection in Human Cells through the PARP Domain

(A) Western analysis with anti-HA antibody shows expression of HA-tagged human and rat ZAP isoforms in stably expressing HeLa cell lines. Equal amounts of protein were loaded in each lane. (B) SFV infection was strongly inhibited (7-fold) in HeLa cells stably expressing ZAP(L), but only weakly restricted (2-fold) in HeLa cells stably expressing ZAP(S) or rat NZAP. Cells were challenged with four 3-fold serial dilutions of virus in quadruplicate and infectivity was determined by measuring SFV-driven β-galactosidase activity as relative light units using a luminescent substrate. The data are plotted as the means ± standard deviation on a log scale. (C) HIV infection was not attenuated in HeLA cells stably expressing the ZAP isoforms. HeLa cells stably expressing the ZAP isoforms were challenged by five 3-fold serial dilutions of virus, and infectivity was determined by measuring HIV-driven luciferase activity (relative light units). The data are plotted as the means ± standard deviations on a log scale.