A simple phylogenetic approach to analyze hypermutated HIV proviruses reveals insights into their dynamics and persistence during antiretroviral therapy.

Hypermutated proviruses, which arise in a single HIV replication cycle when host antiviral APOBEC3 proteins introduce extensive G-to-A mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). But, the within-host evolutionary origins of hypermutated sequences are incompletely understood because phylogenetic inference algorithms, which assume that mutations gradually accumulate over generations, incorrectly reconstruct their ancestor-descendant relationships. Using >1400 longitudinal single-genome-amplified HIV env-gp120 sequences isolated from six women over a median 18 years of follow-up - including plasma HIV RNA sequences collected over a median 9 years between seroconversion and ART initiation, and >500 proviruses isolated over a median 9 years on ART - we evaluated three approaches for removing hypermutation from nucleotide alignments. Our goals were to 1) reconstruct accurate phylogenies that can be used for molecular dating and 2) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the tested approaches (stripping all positions containing putative APOBEC3 mutations from the alignment, or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought env -intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for env -intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, indicating that the corrected trees can be used for molecular dating. Use of these trees to infer the integration dates of hypermutated proviruses persisting during ART revealed that these spanned a wide age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection, and can persist for decades. In two of the six participants, hypermutated proviruses differed from env -intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus' evolutionary histories, allow insights into their in vivo origins and longevity, towards a more comprehensive understanding of HIV persistence during ART.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and this diversity persists within infected cell reservoirs during antiretroviral therapy (ART). Achieving a better understanding of on-ART proviral evolutionary dynamics, and a better appreciation of how the overall persisting pool of (largely genetically defective) proviruses differs from the much smaller replication-competent HIV reservoir, is critical to HIV cure efforts. We reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study who experienced HIV seroconversion, and used these data to characterize the diversity, lineage origins, and ages of proviral env-gp120 sequences sampled longitudinally up to 12 years on ART. We also studied HIV sequences emerging from the reservoir in two participants. We observed that proviral clonality generally increased over time on ART, with clones frequently persisting long term. While on-ART proviral integration dates generally spanned the duration of untreated infection, HIV emerging in plasma was exclusively younger (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained stable during ART in all but one participant, in whom there was evidence that younger proviruses had been preferentially eliminated after 12 years on ART. Analysis of the gag region in three participants corroborated our env-gp120-based observations, indicating that our observations are not influenced by the HIV region studied. Our results underscore the remarkable genetic stability of the distinct proviral sequences that persist in blood during ART. Our results also suggest that the replication-competent HIV reservoir is a genetically restricted, younger subset of this overall proviral pool.IMPORTANCECharacterizing the genetically diverse HIV sequences that persist in the reservoir despite antiretroviral therapy (ART) is critical to cure efforts. Our observations confirm that proviruses persisting in blood on ART, which are largely genetically defective, broadly reflect the extent of within-host HIV evolution pre-ART. Moreover, on-ART clonal expansion is not appreciably accompanied by the loss of distinct proviral lineages. In fact, on-ART proviral genetic composition remained stable in all but one participant, in whom, after 12 years on ART, proviruses dating to around near ART initiation had been preferentially eliminated. We also identified recombinant proviruses between parental sequence fragments of different ages. Though rare, such sequences suggest that reservoir cells can be superinfected with HIV from another infection era. Overall, our finding that the replication-competent reservoir in blood is a genetically restricted, younger subset of all persisting proviruses suggests that HIV cure strategies will need to eliminate a reservoir that differs in key respects from the overall proviral pool.

Genomic structures and regulation patterns at HPV integration sites in cervical cancer.

Human papillomavirus (HPV) integration has been implicated in transforming HPV infection into cancer, but its genomic consequences have been difficult to study using short-read technologies. To resolve the dysregulation associated with HPV integration, we performed long-read sequencing on 63 cervical cancer genomes. We identified six categories of integration events based on HPV-human genomic structures. Of all HPV integrants, defined as two HPV-human breakpoints bridged by an HPV sequence, 24% contained variable copies of HPV between the breakpoints, a phenomenon we termed heterologous integration. Analysis of DNA methylation within and in proximity to the HPV genome at individual integration events revealed relationships between methylation status of the integrant and its orientation and structure. Dysregulation of the human epigenome and neighboring gene expression in cis with the HPV-integrated allele was observed over megabase-ranges of the genome. By elucidating the structural, epigenetic, and allele-specific impacts of HPV integration, we provide insight into the role of integrated HPV in cervical cancer.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and members of these diverse forms persist within infected cell reservoirs, even during antiretroviral therapy (ART). Characterizing the diverse viral sequences that persist during ART is critical to HIV cure efforts, but our knowledge of on-ART proviral evolutionary dynamics remains incomplete, as does our understanding of the differences between the overall pool of persisting proviral DNA (which is largely genetically defective) and the subset of intact HIV sequences capable of reactivating. Here, we reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study (WIHS) who experienced HIV seroconversion. We measured diversity, lineage origins and ages of proviral sequences (env-gp120) sampled up to four times, up to 12 years on ART. We used the same techniques to study HIV sequences emerging from the reservoir in two participants. Proviral clonality generally increased over time on ART, with clones frequently persisting across multiple time points. The integration dates of proviruses persisting on ART generally spanned the duration of untreated infection (though were often skewed towards years immediately pre-ART), while in contrast, reservoir-origin viremia emerging in plasma was exclusively "younger" (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained highly stable during ART in all but one participant in whom, after 12 years, there was evidence that "younger" proviruses had been preferentially eliminated. Analysis of within-host recombinant proviral sequences also suggested that HIV reservoirs can be superinfected with virus reactivated from an older era, yielding infectious viral progeny with mosaic genomes of sequences with different ages. Overall, results underscore the remarkable genetic stability of distinct proviral sequences that persist on ART, yet suggest that replication-competent HIV reservoir represents a genetically-restricted and overall "younger" subset of the overall persisting proviral pool in blood.

Oncogenic Viruses and the Epigenome: How Viruses Hijack Epigenetic Mechanisms to Drive Cancer.

Globally, viral infections substantially contribute to cancer development. Oncogenic viruses are taxonomically heterogeneous and drive cancers using diverse strategies, including epigenomic dysregulation. Here, we discuss how oncogenic viruses disrupt epigenetic homeostasis to drive cancer and focus on how virally mediated dysregulation of host and viral epigenomes impacts the hallmarks of cancer. To illustrate the relationship between epigenetics and viral life cycles, we describe how epigenetic changes facilitate the human papillomavirus (HPV) life cycle and how changes to this process can spur malignancy. We also highlight the clinical impact of virally mediated epigenetic changes on cancer diagnosis, prognosis, and treatment.