In humans, homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing protein 5 (HERC5) is an interferon-induced protein that inhibits replication of evolutionarily diverse viruses, including human immunodeficiency virus type 1 (HIV-1). To better understand the origin, evolution, and function of HERC5, we performed phylogenetic, structural, and functional analyses of the entire human small-HERC family, which includes HERC3, HERC4, HERC5, and HERC6. We demonstrated that the HERC family emerged >595 million years ago and has undergone gene duplication and gene loss events throughout its evolution. The structural topology of the RCC1-like domain and HECT domains from all HERC paralogs is highly conserved among evolutionarily diverse vertebrates despite low sequence homology. Functional analyses showed that the human small HERCs exhibit different degrees of antiviral activity toward HIV-1 and that HERC5 provides the strongest inhibition. Notably, coelacanth HERC5 inhibited simian immunodeficiency virus (SIV), but not HIV-1, particle production, suggesting that the antiviral activity of HERC5 emerged over 413 million years ago and exhibits species- and virus-specific restriction. In addition, we showed that both HERC5 and HERC6 are evolving under strong positive selection, particularly blade 1 of the RCC1-like domain, which we showed is a key determinant of antiviral activity. These studies provide insight into the origin, evolution, and biological importance of the human restriction factor HERC5 and the other HERC family members.IMPORTANCE Intrinsic immunity plays an important role as the first line of defense against viruses. Studying the origins, evolution, and functions of proteins responsible for effecting this defense will provide key information about virus-host relationships that can be exploited for future drug development. We showed that HERC5 is one such antiviral protein that belongs to an evolutionarily conserved family of HERCs with an ancient marine origin. Not all vertebrates possess all HERC members, suggesting that different HERCs emerged at different times during evolution to provide the host with a survival advantage. Consistent with this, two of the more recently emerged HERC members, HERC5 and HERC6, displayed strong signatures of having been involved in an ancient evolutionary battle with viruses. Our findings provide new insights into the evolutionary origin and function of the HERC family in vertebrate evolution, identifying HERC5 and possibly HERC6 as important effectors of intrinsic immunity in vertebrates.

TYRO3, AXL, and MERTK (TAM) receptors are a family of receptor tyrosine kinases that maintain homeostasis through the clearance of apoptotic cells, and when defective, contribute to chronic inflammatory and autoimmune diseases such as atherosclerosis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and Crohn's disease. In addition, certain enveloped viruses utilize TAM receptors for immune evasion and entry into host cells, with several viruses preferentially hijacking MERTK for these purposes. Despite the biological importance of TAM receptors, little is understood of their recent evolution and its impact on their function. Using evolutionary analysis of primate TAM receptor sequences, we identified strong, recent positive selection in MERTK's signal peptide and transmembrane domain that was absent from TYRO3 and AXL. Reconstruction of hominid and primate ancestral MERTK sequences revealed three nonsynonymous single nucleotide polymorphisms in the human MERTK signal peptide, with a G14C mutation resulting in a predicted non-B DNA cruciform motif, producing a significant decrease in MERTK expression with no significant effect on MERTK trafficking or half-life. Reconstruction of MERTK's transmembrane domain identified three amino acid substitutions and four amino acid insertions in humans, which led to significantly higher levels of self-clustering through the creation of a new interaction motif. This clustering counteracted the effect of the signal peptide mutations through enhancing MERTK avidity, whereas the lower MERTK expression led to reduced binding of Ebola virus-like particles. The decreased MERTK expression counterbalanced by increased avidity is consistent with antagonistic coevolution to evade viral hijacking of MERTK.

Survival following Ebola virus (EBOV) infection correlates with the ability to mount an early and robust interferon (IFN) response. The host IFN-induced proteins that contribute to controlling EBOV replication are not fully known. Among the top genes with the strongest early increases in expression after infection in vivo is IFN-induced HERC5. Using a transcription- and replication-competent VLP system, we showed that HERC5 inhibits EBOV virus-like particle (VLP) replication by depleting EBOV mRNAs. The HERC5 RCC1-like domain was necessary and sufficient for this inhibition and did not require zinc finger antiviral protein (ZAP). Moreover, we showed that EBOV (Zaire) glycoprotein (GP) but not Marburg virus GP antagonized HERC5 early during infection. Our data identify a novel ‘protagonist–antagonistic’ relationship between HERC5 and GP in the early stages of EBOV infection that could be exploited for the development of novel antiviral therapeutics.

Survival following Ebola virus (EBOV) infection correlates with the ability to mount an early and robust interferon (IFN) response. The host IFN-induced proteins that contribute to controlling EBOV replication are not fully known. Among the top genes with the strongest early increases in expression after infection in vivo is IFN-induced HERC5. Using a transcription- and replication-competent VLP system, we showed that HERC5 inhibits EBOV virus-like particle (VLP) replication by depleting EBOV mRNAs. The HERC5 RCC1-like domain was necessary and sufficient for this inhibition and did not require zinc finger antiviral protein (ZAP). Moreover, we showed that EBOV (Zaire) glycoprotein (GP) but not Marburg virus GP antagonized HERC5 early during infection. Our data identifies a novel ‘protagonist-antagonistic’ relationship between HERC5 and GP in the early stages of EBOV infection that could be exploited for the development of novel antiviral therapeutics.