Genetic Aspects Of Viral Oncogenesis In Inbred Strains and Wild Mouse Species

National Institute Of Allergy And Infectious Diseases

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Abstract

Our work has focused on mouse retroviruses, specifically mouse leukemia viruses (MLVs) and mouse mammary tumor viruses (MMTVs), some of which are pathogenic, along with the various host factors that restrict the replication of these viruses and the adaptive co-evolution of interactive host/pathogen pairings. We have used laboratory mouse strains as well as wild mice to exploit the genetic diversity of the viruses they harbor and the interacting host genes found in Mus. We have also expanded several studies to examine similar phenomena using human retroviruses. In FY25, we expanded our analysis of mouse mammary tumor viruses (MMTVs). Analysis of the sequenced genomes of 17 mouse strains, Mus musculus subspecies and Mus spretus identified 29 endogenous retroviruses (ERVs) of mouse mammary tumor viruses (MMTVs), termed Mtvs. The 15 laboratory mouse Mtvs are each present in multiple strains reflecting their common breeding history; most predate the development of inbred strains and were likely acquired by Mus musculus domesticus progenitors but have no orthologs in wild mice, while four, including the intact Mtv1, were likely endogenized more recently. One of the 14 Mtvs found in wild mice was distributed over a broad geographic range in southeast Asia. Most Mtvs are full-length, with multiple open reading frames, but Mtvs cloned from wild mice have an unusual envelope deletion that removes an intron of the viral rem gene suggesting its derivation from spliced MMTV cDNAs. These deleted envs have open reading frames, are found in globally distributed mice and show subspecies specific sequence variation consistent with their recurrent generation. The highly variable MMTV sag gene, responsible for resistance to exogenous infection, exhibits evidence of recombination as well as positive selection consistent with its role in antiviral defense. In contrast, the spread of Mtvs in Mus musculus populations is not marked by an active arms race pitting the MMTV envelope against its cellular receptor. Thus, acquisition of potentially disease-inducing Mtvs is a recent and ongoing process in Mus accompanied by recombination, positive selection and a recurrent envelope deletion. In FY25, we observed unusual mutagenesis patterns in the Mtvs we mined from sequenced mouse genomes, patterns attributable to the host restriction factor Apobec3. Apobec3 (A3) belongs to a family of cytidine deaminases with broadly antiretroviral activity. A3 is packaged in virions and converts cytidines to uracil (C>U) in viral minus strand DNA during reverse transcription resulting in guanosine to adenosine (G>A) mutations in the viral plus strand. Mouse Apobec3 (mA3) mutagenizes numerous infectious retroviruses and some families of endogenous retroviruses (ERVs). Here we show that endogenous mouse mammary tumor viruses, termed Mtvs, show a range of mA3 hypermutation from trace levels to an extreme example (Mtv21) that has sustained an extraordinary, genome-wide reduction of G content to 10.8%, a level partly attributable to the ongoing generation of new, immediately usable target sites by mutagenesis of consecutive cytidines. The otherwise identical long terminal repeats (LTRs) of four Mtvs have multiple G>A mutations unique to the 3’ LTR, likely acquired during reverse transcription. Individual hypermutated Mtvs display two different mA3 target site preferences corresponding to the sequence specificity of the two known inbred strain mA3 alleles. Mouse strains and Mtv-positive Mus musculus subspecies screened for these mA3 alleles unexpectedly revealed 20 distinct mA3 haplotypes with replacement mutations at the 15 sites that distinguish the two inbred strain alleles, eleven of which show signatures of positive selection. These data show that mA3 deamination can have a major impact on the genomic integrity of Mtvs, and that mA3 is actively evolving within this single mouse species suggestive of high impact genetic conflicts over a short evolutionary time frame. In FY25, we analyzed acquired deletions in mouse and human ERVs. Most ERVs are eventually inactivated by point mutations and deletions that can remove small or kilobase lengths of viral sequence. We examined deletions in mouse and human ERVs (HERVs) to describe their distribution in proviral genomes, the frequency and likely provenance of identical deletions, and their association with flanking DNA motifs. Mouse leukemia viruses (MLVs) and some HERV groups contain 9-4160 base pair (bp) internal deletions. These deletions are not randomly distributed or generated. First, 2-12 bp direct repeats (DRs) flank 85% of such deletions in MLVs and 87.5% in HERV-W, with fewer in HERV-K and HERV-H. The single DR copy retained at the deletion site is a hallmark of nonallelic homologous recombination. Second, the viral gene most impacted by deletion is envelope (env), expression of which is especially deleterious for the host, and loss of which can increase the likelihood of ERV retention and amplification. The bulk of env is removed either through different single env-spanning deletions or multiple adjacent deletions. An expanded analysis of the MLV ERVs in Mus musculus subspecies that contain functional variants of the MLV receptor show different patterns of env deletions. Deletions that are shared by two to 40+ ERVs can be variously attributed to recurrent deletion, proviral duplication or segmental retroviral recombination. The generation of env deletions may be facilitated by other factors because deletion-prone env DRs found elsewhere in these proviruses are not deletion-associated, and because multiple nearly identical MLV env deletions lack DRs. These data indicate that there is an evolutionary advantage to genome editing by targeted deletion genome editing by targeted deletions and provides insight into the processes that favor the adaptive co-existence of host and ERV. In FY25 we continued work on three additional projects: 1) a novel antiviral restriction agent that blocks replication of HIV and other retroviruses at late stages of their replication. Current efforts focus on determining the mechanism of this block, the responsible domains of the restriction factor and the target sites within retroviral genomes. 2) unusually well-preserved ERV genes in Mus including a gammaretroviral envelope preserved as an open reading frame within the mouse major histocompatibility complex and a capsid gene that resembles the Fv1 resistance gene, but which has been preserved over a longer evolutionary time frame. 3) analysis of conserved sequence motifs within the understudied C-terminal region of the surface domain of the retroviral envelope gene.