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

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Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (⌬UNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68⌬UNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs. IMPORTANCEHerpesviruses establish chronic lifelong infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the initial site of acute replication was associated with a substantial delay of latency establishment in the spleen. The levels of host UNG were substantially lower in the lung compared to the spleen, suggesting that herpesviruses encode a viral UNG to compensate for reduced host enzyme levels in some cell types and tissues. These data suggest that intervention at the site of initial replicative expansion can delay the establishment of latency, a hallmark of chronic herpesvirus infection.A ll herpesvirus genomes encode a homolog of the host uracil DNA glycosylase. If not repaired, uracil misincorporation during DNA replication (1, 2) or conversion upon cytosine deamination (2, 3) will lead to a C:G-to-T:A transition mutation that may have severe consequences for genomic integrity (1). In the host, the recognition and removal of uracils is mediated by members of the uracil DNA glycosylase (UDG) superfamily. UNG2 is the predominant nuclear enzyme responsible for initiating repair (2-6). The role of a seemingly redundant herpesvirus UNG in viral replication is ...

Section: Discussioncontrasting

confidence: 57%

Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

Minkah

Macaluso

Oldenburg

et al. 2015

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“…9C and D), it is possible that inhibition of papillomaviruses is an APOBEC3 function that developed late during vertebrate evolution. In fact, mA3 was also found to have no impact on murine gammaherpesvirus 68, another DNA virus (66). We speculate that other type I interferon-responsive proteins may further augment the antiviral effect in early MmuPV1 infection in mice (53,67,68).…”

Section: Discussionmentioning

confidence: 83%

APOBEC3A Functions as a Restriction Factor of Human Papillomavirus

Warren

Guo

et al. 2015

171

203

Human papillomaviruses (HPVs) are small DNA viruses causally associated with benign warts and multiple cancers, including cervical and head-and-neck cancers. While the vast majority of people are exposed to HPV, most instances of infection are cleared naturally. However, the intrinsic host defense mechanisms that block the early establishment of HPV infections remain mysterious. Several antiviral cytidine deaminases of the human APOBEC3 (hA3) family have been identified as potent viral DNA mutators. While editing of HPV genomes in benign and premalignant cervical lesions has been demonstrated, it remains unclear whether hA3 proteins can directly inhibit HPV infection. Interestingly, recent studies revealed that HPV-positive cervical and head-and-neck cancers exhibited higher rates of hA3 mutation signatures than most HPV-negative cancers. Here, we report that hA3A and hA3B expression levels are highly upregulated in HPV-positive keratinocytes and cervical tissues in early stages of cancer progression, potentially through a mechanism involving the HPV E7 oncoprotein. HPV16 virions assembled in the presence of hA3A, but not in the presence of hA3B or hA3C, have significantly decreased infectivity compared to HPV virions assembled without hA3A or with a catalytically inactive mutant, hA3A/E72Q. Importantly, hA3A knockdown in human keratinocytes results in a significant increase in HPV infectivity. Collectively, our findings suggest that hA3A acts as a restriction factor against HPV infection, but the induction of this restriction mechanism by HPV may come at a cost to the host by promoting cancer mutagenesis. IMPORTANCEHuman papillomaviruses (HPVs) are highly prevalent and potent human pathogens that cause >5% of all human cancers, including cervical and head-and-neck cancers. While the majority of people become infected with HPV, only 10 to 20% of infections are established as persistent infections. This suggests the existence of intrinsic host defense mechanisms that inhibit viral persistence. Using a robust method to produce infectious HPV virions, we demonstrate that hA3A, but not hA3B or hA3C, can significantly inhibit HPV infectivity. Moreover, hA3A and hA3B were coordinately induced in HPV-positive clinical specimens during cancer progression, likely through an HPV E7 oncoprotein-dependent mechanism. Interestingly, HPV-positive cervical and head-and-neck cancer specimens were recently shown to harbor significant amounts of hA3 mutation signatures. Our findings raise the intriguing possibility that the induction of this host restriction mechanism by HPV may also trigger hA3A-and hA3B-induced cancer mutagenesis. Human papillomaviruses (HPVs) are small, nonenveloped DNA viruses known as one of the most prevalent sexually transmitted pathogens. Among almost 200 different genotypes, ϳ24 high-risk HPV genotypes are causally associated with multiple human cancers, including nearly all cervical cancers and a portion of head-and-neck squamous cell carcinomas (1). From 1988 to 2004, the incidence of HPV-associ...

“…MHV68 (10 3 PFU; WUMS strain) was intranasally inoculated into mice. The mice were sacrificed at 7 and 16 dpi for acute-and latent-infection analysis, respectively (34). Plaque assays were conducted to measure the virus titers during acute infection in the lungs.…”

Section: Mice A3ko A3amentioning

confidence: 99%

“…Evidence of APOBEC3A-, 3C-, and 3G-mediated editing of herpes simplex virus 1 (HSV-1) was detected when these factors were overexpressed in cultured cells, and HSV-1 and Epstein-Barr virus (EBV) showed signs of cytidine deamination in buccal swabs or immortalized cell lines, respectively (30). Moreover, a recent study showed that several human APOBEC3s, including APOBEC3A, inhibited replication of murine gammaherpesvirus 68 (MHV68) when the viral genome was transfected into cultured cells but not when it was introduced by infection (34). However, APOBEC3 knockout (A3KO) and wild-type (WT) mice were equally resistant to infection by this virus (34).…”

mentioning

confidence: 99%

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In Vivo Examination of Mouse APOBEC3- and Human APOBEC3A- and APOBEC3G-Mediated Restriction of Parvovirus and Herpesvirus Infection in Mouse Models

Nakaya

Stavrou

Blouch

et al. 2016

APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo. IMPORTANCEIt is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo. Given the frequent encounters of vertebrates with viruses, it is not surprising that there has been selection for host defense systems. In 2002, human apolipoprotein B editing complex 3G (APOBEC3G) was discovered and shown to confer intrinsic immunity to HIV-1 (1). The APOBEC3G gene belongs to a family of genes encoding DNA-and RNA-editing enzymes characterized by the presence of at least one cytidine deaminase (CDA) domain (2). The number of APOBEC3 genes varies from species to species, from 1 gene in mice to 7 genes (the APOBEC3A, -3B, -3C, -3DE, -3F, -3G, and -3H genes) in primates (2). Moreover, there are multiple allelic differences in APOBEC3 gene coding regions; notably, there are at least 2 alleles in mice that differ in both their protein-coding regions and levels of expression (3, 4). These allelic variants confer weak or strong resistance to murine retrovirus infection in vivo (3, 5-7).Several human APOBEC3 proteins inhibit HIV-1 lacking the vif gene, which encodes a protein expressed at high levels late in infection (8-12). In vif-deficient-HIV producer cells, APOBEC3 proteins are packaged into progeny virions via interaction with the nucleocapsid (NC) protein and viral RNA. Vif prevents packaging by binding APOBEC3 proteins, targeting them for ubiquitination and degradation in the proteasome (13-17). APOBEC3 proteins inhibit infection by deaminating deoxycytidine residues on minus-strand DNA, inducing hypermutation in newly reversetranscribed HIV-1 DNA; this leads both to degradation of viral DNA prior to integration and to G-to-A coding-strand mutations in viral genes in the integrated provirus (18). APOBEC3 proteins also inhibit replication by a number of CDA-independent mechanisms (19). APOBEC3 proteins restrict animal retroviruses (20-2...