A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits

Abstract

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes. IMPORTANCE The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.

Footprint of the host restriction factors APOBEC3 on the genome of human viruses

“…The A3 footprint on EBV is spatially limited to the lagging strands around the lytic replication origins. Interestingly, both EBV and KSHV were recently demonstrated to encode viral proteins capable of inhibiting A3B activity [ 28 , 65 ]. Those viral proteins (EBV BORF2 and KSHV ORF61) are both the large subunit of the ribonucleotide reductase, expressed during lytic replication and providing the precursors necessary for viral DNA synthesis.…”

Retroviruses drive the rapid evolution of mammalianAPOBEC3genes

“…Indeed, remnants of vif genelike ORFs have been identified in endogenous lentiviruses (43)(44)(45). In addition, it has recently been reported that herpesviruses encode ribonucleotide reductase large subunits that degrade human A3 proteins (5,46,47) and that the A3 antagonists of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus specifically recognize the loop 7 structure of A3B (5). Therefore, A3 antagonists encoded by viruses other than retroviruses may also have exerted selective pressure on the loop 7 structures of A3 genes.…”

HPV Meets APOBEC: New Players in Head and Neck Cancer

“…In this regard, our group has recently uncovered a novel mechanism of HCMV evasion from A3 restriction activity based on the progressive loss of APOBEC hot spots from its genome during evolution [ 10 ]. A3 inactivation as a means to evade host immune surveillance does not seem to be solely restricted to HCMV as other viruses, such as HSV-1 and Kaposi’s sarcoma-associated herpesvirus (KSHV), can delocalize A3 proteins and, in doing so, neutralize their antiviral activity through a conserved ribonucleotide reductase (RNR)-dependent mechanism [ 64 ]. More recently, APOBECs have been involved in SARS-CoV-2 RNA editing [ 65 ].…”