Mutational pressure by host APOBEC3s more strongly affects genes expressed early in the lytic phase of herpes simplex virus-1 (HSV-1) and human polyomavirus (HPyV) infection.

Herpes-Simplex Virus 1 (HSV-1) infects most humans when they are young, sometimes with fatal consequences. Gene expression occurs in a temporal order upon lytic HSV-1 infection: immediate early (IE) genes are expressed, then early (E) genes, followed by late (L) genes. During this infection cycle, the HSV-1 genome has the potential for exposure to APOBEC3 (A3) proteins, a family of cytidine deaminases that cause C>U mutations on single-stranded DNA (ssDNA), often resulting in a C>T transition. We developed a computational model for the mutational pressure of A3 on the lytic cycle of HSV-1 to determine which viral kinetic gene class is most vulnerable to A3 mutations. Using in silico stochastic methods, we simulated the infectious cycle under varying intensities of A3 mutational pressure. We found that the IE and E genes are more vulnerable to A3 than L genes. We validated this model by analyzing the A3 evolutionary footprints in 25 HSV-1 isolates. We find that IE and E genes have evolved to underrepresent A3 hotspot motifs more so than L genes, consistent with greater selection pressure on IE and E genes. We extend this model to two-step infections, such as those of polyomavirus, and find that the same pattern holds for over 25 human Polyomavirus (HPyVs) genomes. Genes expressed earlier during infection are more vulnerable to mutations than those expressed later.

A-to-I editing of Malacoherpesviridae RNAs supports the antiviral role of ADAR1 in mollusks.

BACKGROUND: Adenosine deaminase enzymes of the ADAR family are conserved in metazoans. They convert adenine into inosine in dsRNAs and thus alter both structural properties and the coding potential of their substrates. Acting on exogenous dsRNAs, ADAR1 exerts a pro- or anti-viral role in vertebrates and Drosophila. RESULTS: We traced 4 ADAR homologs in 14 lophotrochozoan genomes and we classified them into ADAD, ADAR1 or ADAR2, based on phylogenetic and structural analyses of the enzymatic domain. Using RNA-seq and quantitative real time PCR we demonstrated the upregulation of one ADAR1 homolog in the bivalve Crassostrea gigas and in the gastropod Haliotis diversicolor supertexta during Ostreid herpesvirus-1 or Haliotid herpesvirus-1 infection. Accordingly, we demonstrated an extensive ADAR-mediated editing of viral RNAs. Single nucleotide variation (SNV) profiles obtained by pairing RNA- and DNA-seq data from the viral infected individuals resulted to be mostly compatible with ADAR-mediated A-to-I editing (up to 97%). SNVs occurred at low frequency in genomic hotspots, denoted by the overlapping of viral genes encoded on opposite DNA strands. The SNV sites and their upstream neighbor nucleotide indicated the targeting of selected adenosines. The analysis of viral sequences suggested that, under the pressure of the ADAR editing, the two Malacoherpesviridae genomes have evolved to reduce the number of deamination targets. CONCLUSIONS: We report, for the first time, evidence of an extensive editing of Malacoherpesviridae RNAs attributable to host ADAR1 enzymes. The analysis of base neighbor preferences, structural features and expression profiles of molluscan ADAR1 supports the conservation of the enzyme function among metazoans and further suggested that ADAR1 exerts an antiviral role in mollusks.

A Needle in A Haystack: Tracing Bivalve-Associated Viruses in High-Throughput Transcriptomic Data.

Bivalve mollusks thrive in environments rich in microorganisms, such as estuarine and coastal waters, and they tend to accumulate various particles, including viruses. However, the current knowledge on mollusk viruses is mainly centered on few pathogenic viruses, whereas a general view of bivalve-associated viromes is lacking. This study was designed to explore the viral abundance and diversity in bivalve mollusks using transcriptomic datasets. From analyzing RNA-seq data of 58 bivalve species, we have reconstructed 26 nearly complete and over 413 partial RNA virus genomes. Although 96.4% of the predicted viral proteins refer to new viruses, some sequences belong to viruses associated with bivalve species or other marine invertebrates. We considered short non-coding RNAs (sncRNA) and post-transcriptional modifications occurring specifically on viral RNAs as tools for virus host-assignment. We could not identify virus-derived small RNAs in sncRNA reads obtained from the oyster sample richest in viral reads. Single Nucleotide Polymorphism (SNP) analysis revealed 938 A-to-G substitutions occurring on the 26 identified RNA viruses, preferentially impacting the AA di-nucleotide motif. Under-representation analysis revealed that the AA motif is under-represented in these bivalve-associated viruses. These findings improve our understanding of bivalve viromes, and set the stage for targeted investigations on the specificity and dynamics of identified viruses.

Evolutionary effects of the AID/APOBEC family of mutagenic enzymes on human gamma-herpesviruses.

The human gamma-herpesviruses, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, establish lifelong latency in B cells and are associated with multiple malignancies. Virus-host coevolution often drive changes in both host immunity and in the viral genome. We consider one host immune mechanism, the activation-induced deaminase (AID)/APOBEC family of cytidine deaminases, that induces mutations in viral DNA. AID, the ancestral gene in the family has a conserved role in somatic hypermutation, a key step in antibody affinity maturation. The APOBEC3 subfamily, of which there are seven genes in human, have evolved antiviral functions and have diversified in terms of their expression pattern, subcellular localization, and DNA mutation motifs (hotspots). In this study, we investigated how the human gamma-herpesviruses have evolved to avoid the action of the AID/APOBEC enzymes and determine if these enzymes are contributing to the ongoing evolution of the viruses. We used computational methods to evaluate observed versus expected frequency of AID/APOBEC hotspots in viral genomes and found that the viruses have evolved to limit the representation of AID and certain APOBEC3 motifs. At the same time, the remaining hotspots were highly likely to cause amino acid changes, suggesting prolonged evolutionary pressure of the enzymes on the viruses. To study current hypermutation, as opposed to historical mutation processes, we also analyzed putative mutations derived from alignments of published viral genomes and found again that AID and APOBEC3 appear to target the genome most frequently. New protein variants resulting from AID/APOBEC activity may have important consequences in health, including vaccine development (epitope evolution) and host immune evasion.

Combinatorial Loss of the Enzymatic Activities of Viral Uracil-DNA Glycosylase and Viral dUTPase Impairs Murine Gammaherpesvirus Pathogenesis and Leads to Increased Recombination-Based Deletion in the Viral Genome.

Misincorporation of uracil or spontaneous cytidine deamination is a common mutagenic insult to DNA. Herpesviruses encode a viral uracil-DNA glycosylase (vUNG) and a viral dUTPase (vDUT), each with enzymatic and nonenzymatic functions. However, the coordinated roles of these enzymatic activities in gammaherpesvirus pathogenesis and viral genomic stability have not been defined. In addition, potential compensation by the host UNG has not been examined in vivo The genetic tractability of the murine gammaherpesvirus 68 (MHV68) system enabled us to delineate the contribution of host and viral factors that prevent uracilated DNA. Recombinant MHV68 lacking vUNG (ORF46.stop) was not further impaired for acute replication in the lungs of UNG-/- mice compared to wild-type (WT) mice, indicating host UNG does not compensate for the absence of vUNG. Next, we investigated the separate and combinatorial consequences of mutating the catalytic residues of the vUNG (ORF46.CM) and vDUT (ORF54.CM). ORF46.CM was not impaired for replication, while ORF54.CM had a slight transient defect in replication in the lungs. However, disabling both vUNG and vDUT led to a significant defect in acute expansion in the lungs, followed by impaired establishment of latency in the splenic reservoir. Upon serial passage of the ORF46.CM/ORF54.CM mutant in either fibroblasts or the lungs of mice, we noted rapid loss of the nonessential yellow fluorescent protein (YFP) reporter gene from the viral genome, due to recombination at repetitive elements. Taken together, our data indicate that the vUNG and vDUT coordinate to promote viral genomic stability and enable viral expansion prior to colonization of latent reservoirs.IMPORTANCE Unrepaired uracils in DNA can lead to mutations and compromise genomic stability. Herpesviruses have hijacked host processes of DNA repair and nucleotide metabolism by encoding a viral UNG that excises uracils and a viral dUTPase that initiates conversion of dUTP to dTTP. To better understand the impact of these processes on gammaherpesvirus pathogenesis, we examined the separate and collaborative roles of vUNG and vDUT upon MHV68 infection of mice. Simultaneous disruption of the enzymatic activities of both vUNG and vDUT led to a severe defect in acute replication and establishment of latency, while also revealing a novel, combinatorial function in promoting viral genomic stability. We propose that herpesviruses require these enzymatic processes to protect the viral genome from damage, possibly triggered by misincorporated uracil. This reveals a novel point of therapeutic intervention to potentially block viral replication and reduce the fitness of multiple herpesviruses.

Correction to: The cytidine deaminase under-representation reporter (CDUR) as a tool to study evolution of sequences under deaminase mutational pressure.

Following publication of the original article [1], the authors reported that Figs. 1 and 3 were interchanged. The original article has been corrected.

The cytidine deaminase under-representation reporter (CDUR) as a tool to study evolution of sequences under deaminase mutational pressure.

BACKGROUND: Activation induced deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) are deaminases that mutate C to U on single-stranded DNA (ssDNA). AID is expressed primarily in germinal center B-cells, where it facilitates affinity maturation and class-switch recombination. APOBEC3 are a family of anti-viral proteins that act as part of the intrinsic immune response. In both cases, there are particular sequence motifs, also known as "mutation motifs", to which these deaminases prefer to bind and mutate. RESULTS: We present a program, the cytidine deaminase under-representation reporter (CDUR) designed to statistically determine whether a given sequence has an under/over-representation of these mutation motifs. CDUR shows consitency with other studies of mutation motifs, as we show by analyzing sequences from the adeno-associated virus 2 (AAV2) and human papillomavirus (HPV). CONCLUSION: Using various shuffling mechanisms to generate different null model distributions, we can tailor CDUR to correct for metrics such as GC-content, dinucleotide frequency, and codon bias.