HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer.

The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE: Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.

N-Substituted Bicyclic Carbamoyl Pyridones: Integrase Strand Transfer Inhibitors that Potently Inhibit Drug-Resistant HIV-1 Integrase Mutants.

HIV-1 integrase (IN) is an important molecular target for the development of anti-AIDS drugs. A recently FDA-approved second-generation integrase strand transfer inhibitor (INSTI) cabotegravir (CAB, 2021) is being marketed for use in long-duration antiviral formulations. However, missed doses during extended therapy can potentially result in persistent low levels of CAB that could select for resistant mutant forms of IN, leading to virological failure. We report a series of N-substituted bicyclic carbamoyl pyridones (BiCAPs) that are simplified analogs of CAB. Several of these potently inhibit wild-type HIV-1 in single-round infection assays in cultured cells and retain high inhibitory potencies against a panel of viral constructs carrying resistant mutant forms of IN. Our lead compound, 7c, proved to be more potent than CAB against the therapeutically important resistant double mutants E138K/Q148K (>12-fold relative to CAB) and G140S/Q148R (>36-fold relative to CAB). A significant number of the BiCAPs also potently inhibit the drug-resistant IN mutant R263K, which has proven to be problematic for the FDA-approved second-generation INSTIs.

The largest HIV-1-infected T cell clones in children on long-term combination antiretroviral therapy contain solo LTRs.

Combination antiretroviral therapy (cART) suppresses viral replication but does not cure HIV infection because a reservoir of infectious (intact) HIV proviruses persists in long-lived CD4+T cells. However, a large majority (>95%) of HIV-infected cells that persist on effective cART carry defective (non-infectious) proviruses. Defective proviruses consisting of only a single LTR (solo long terminal repeat) are commonly found as endogenous retroviruses in many animal species, but the frequency of solo-LTR HIV proviruses has not been well defined. Here we show that, in five pediatric donors whose viremia was suppressed on cART for at least 5 years, the proviruses in the nine largest clones of HIV-infected cells were solo LTRs. The sizes of five of these clones were assayed longitudinally by integration site-specific quantitative PCR. Minor waxing and waning of the clones was observed, suggesting that these clones are generally stable over time. Our findings show that solo LTRs comprise a large fraction of the proviruses in infected cell clones that persist in children on long-term cART. IMPORTANCE This work highlights that severely deleted HIV-1 proviruses comprise a significant proportion of the proviral landscape and are often overlooked.

Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants.

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

LINE1-mediated reverse transcription and genomic integration of SARS-CoV-2 mRNA detected in virus-infected but not in viral mRNA-transfected cells.

SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1,000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches for the host-virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10 - 20-fold more sensitive per tested cell, TagMap can interrogate 1,000 - 2,000-fold more cells and therefore can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected in contrast to transfected cells may be facilitated because virus infection in contrast to viral RNA transfection results in significantly higher viral RNA levels and stimulates LINE1-expression which causes cellular stress.

Current HIV/SIV Reservoir Assays for Preclinical and Clinical Applications: Recommendations from the Experts 2022 NIAID Workshop Summary.

Since the first HIV-cured person was reported in 2009, a strong interest in developing highly sensitive HIV and SIV reservoir assays has emerged. In particular, the question arose about the comparative value of state-of-the-art assays to measure and characterize the HIV reservoir, and how these assays can be applied to accurately detect changes in the reservoir during efforts to develop a cure for HIV infection. Second, it is important to consider the impact on the outcome of clinical trials if these relatively new HIV reservoir assays are incorporated into clinical trial endpoints and/or used for clinical decision-making. To understand the advantages and limitations and the regulatory implications of HIV reservoir assays, the National Institute of Allergy and Infectious Diseases (NIAID) sponsored and convened a meeting on September 16, 2022, to discuss the state of knowledge concerning these questions and best practices for selecting HIV reservoir assays for a particular research question or clinical trial protocol.

LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells.

SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole-genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences, but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches the host-virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10-20-fold more sensitive per tested cell, TagMap can interrogate 1000-2000-fold more cells and, therefore, can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected cells, in contrast to transfected cells, may be facilitated because virus infection, in contrast to viral RNA transfection, results in significantly higher viral RNA levels and stimulates LINE1 expression by causing cellular stress.

A Practical Approach to Bicyclic Carbamoyl Pyridones with Application to the Synthesis of HIV-1 Integrase Strand Transfer Inhibitors.

An efficient one-pot synthetic method has been developed for the preparation of bicyclic carbamoyl pyridones from the known common intermediate methyl 5-((2,4-difluorobenzyl)carbamoyl)-1-(2,2-dimethoxyethyl)-3-methoxy-4-oxo-1,4-dihydropyridine-2-carboxylate (8). The scalable protocol is facile and employs readily available reagents, needing only a single purification as the final step. The utility of the approach was demonstrated by preparing a library of HIV-1 integrase strand transfer inhibitors (INSTIs) that differ by the presence or absence of a double bond in the B-ring of the bicyclic carbamoyl pyridines 6 and 7. Several of the analogs show good antiviral potencies in single-round HIV-1 replication antiviral assays and show no cytotoxicity in cell culture assays. In general, the compounds with a B-ring double bond have higher antiviral potencies than their saturated congeners. Our methodology should be applicable to the synthesis of a range of new metal-chelating analogs.

HIV infected CD4+ T cell clones are more stable than uninfected clones during long-term antiretroviral therapy.

Although combination antiretroviral therapy (ART) blocks HIV replication, it is not curative because infected CD4+ T cells that carry intact, infectious proviruses persist. Understanding the behavior of clones of infected T cells is important for understanding the stability of the reservoir; however, the stabilities of clones of infected T cells in persons on long-term ART are not well defined. We determined the relative stabilities of clones of infected and uninfected CD4+ T cells over time intervals of one to four years in three individuals who had been on ART for 9-19 years. The largest clones of uninfected T cells were larger than the largest clones of infected T cells. Clones of infected CD4+ T cells were more stable than clones of uninfected CD4+ T cells of a similar size. Individual clones of CD4+ T cells carrying intact, infectious proviruses can expand, contract, or remain stable over time.

A Combination of Amino Acid Mutations Leads to Resistance to Multiple Nucleoside Analogs in Reverse Transcriptases from HIV-1 Subtypes B and C.

Resistance to anti-HIV drugs has been a problem from the beginning of antiviral drug treatments. The recent expansion of combination antiretroviral therapy worldwide has led to an increase in resistance to antiretrovirals; understanding the mechanisms of resistance is increasingly important. In this study, we analyzed reverse transcriptase (RT) variants based on sequences derived from an individual who had low-level rebound viremia while undergoing therapy with abacavir, azidothymidine (AZT) (zidovudine), and (-)-l-2',3'-dideoxy-3'-thiacytidine (3TC) (lamivudine). The RT had mutations at positions 64, 67, 70, 184, and 219 and a threonine insertion after amino acid 69 in RT. The virus remained partially susceptible to the nucleoside RT inhibitor (NRTI) regimen. We show how these mutations affect the ability of NRTIs to inhibit DNA synthesis by RT. The presence of the inserted threonine reduced the susceptibility of the RT mutant to inhibition by tenofovir.

INSTIs and NNRTIs Potently Inhibit HIV-1 Polypurine Tract Mutants in a Single Round Infection Assay.

Integrase strand transfer inhibitors (INSTIs) are a class of antiretroviral compounds that prevent the insertion of a DNA copy of the viral genome into the host genome by targeting the viral enzyme integrase (IN). Dolutegravir (DTG) is a leading INSTI that is given, usually in combination with nucleoside reverse transcriptase inhibitors (NRTIs), to treat HIV-1 infections. The emergence of resistance to DTG and other leading INSTIs is rare. However, there are recent reports suggesting that drug resistance mutations can occur at positions outside the integrase gene either in the HIV-1 polypurine tract (PPT) or in the envelope gene (env). Here, we used single round infectivity assays to measure the antiviral potencies of several FDA-approved INSTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) against a panel of HIV-1 PPT mutants. We also tested several of our promising INSTIs and NNRTIs in these assays. No measurable loss in potency was observed for either INSTIs or NNRTIs against the HIV-1 PPT mutants. This suggests that HIV-1 PPT mutants are not able, by themselves, to confer resistance to INSTIs or NNRTIs.

Insertional activation of STAT3 and LCK by HIV-1 proviruses in T cell lymphomas.

Retroviruses cause cancers in animals by integrating in or near oncogenes. Although HIV-1 infection increases the risk of cancer, most of the risk is associated with immunodeficiency and coinfection by oncogenic virus (Epstein-Barr virus, Kaposi sarcoma herpesvirus, and human papillomavirus). HIV-1 proviruses integrated in some oncogenes cause clonal expansion of infected T cells in vivo; however, the infected cells are not transformed, and it is generally believed that HIV-1 does not cause cancer directly. We show that HIV-1 proviruses integrated in the first introns of signal transducer and activator of transcription 3 (STAT3) and lymphocyte-specific protein tyrosine kinase (LCK) can play an important role in the development of T cell lymphomas. The development of these cancers appears to be a multistep process involving additional nonviral mutations, which could help explain why T cell lymphomas are rare in persons with HIV-1 infection.

Clonal Expansion of Infected CD4+ T Cells in People Living with HIV.

HIV infection is not curable with current antiretroviral therapy (ART) because a small fraction of CD4+ T cells infected prior to ART initiation persists. Understanding the nature of this latent reservoir and how it is created is essential to development of potentially curative strategies. The discovery that a large fraction of the persistently infected cells in individuals on suppressive ART are members of large clones greatly changed our view of the reservoir and how it arises. Rather than being the products of infection of resting cells, as was once thought, HIV persistence is largely or entirely a consequence of infection of cells that are either expanding or are destined to expand, primarily due to antigen-driven activation. Although most of the clones carry defective proviruses, some carry intact infectious proviruses; these clones comprise the majority of the reservoir. A large majority of both the defective and the intact infectious proviruses in clones of infected cells are transcriptionally silent; however, a small fraction expresses a few copies of unspliced HIV RNA. A much smaller fraction is responsible for production of low levels of infectious virus, which can rekindle infection when ART is stopped. Further understanding of the reservoir will be needed to clarify the mechanism(s) by which provirus expression is controlled in the clones of cells that constitute the reservoir.

Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining.

MmuPV1 is a useful model for studying papillomavirus-induced tumorigenesis. We used RNA-seq to look for chimeric RNAs that map to both MmuPV1 and host genomes. In tumor tissues, a higher proportion of total viral reads were virus-host chimeric junction reads (CJRs) (1.9‰ - 7‰) than in tumor-free tissues (0.6‰ - 1.3‰): most CJRs mapped to the viral E2/E4 region. Although most of the MmuPV1 integration sites were mapped to intergenic regions and introns throughout the mouse genome, integrations were seen more than once in several genes: Malat1, Krt1, Krt10, Fabp5, Pard3, and Grip1; these data were confirmed by rapid amplification of cDNA ends (RACE)-Single Molecule Real-Time (SMRT)-seq or targeted DNA-seq. Microhomology sequences were frequently seen at host-virus DNA junctions. MmuPV1 infection and integration affected the expression of host genes. We found that factors for DNA double-stranded break repair and microhomology-mediated end-joining (MMEJ), such as H2ax, Fen1, DNA polymerase Polθ, Cdk1, and Plk1, exhibited a step-wise increase and Mdc1 a decrease in expression in MmuPV1-infected tissues and MmuPV1 tumors relative to normal tissues. Increased expression of mitotic kinases CDK1 and PLK1 appears to be correlated with CtIP phosphorylation in MmuPV1 tumors, suggesting a role for MMEJ-mediated DNA joining in the MmuPV1 integration events that are associated with MmuPV1-induced progression of tumors.

Crystal Structure of a Retroviral Polyprotein: Prototype Foamy Virus Protease-Reverse Transcriptase (PR-RT).

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.

Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures.

Between 10 and 20 million people worldwide are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Despite causing life-threatening pathologies there is no therapeutic regimen for this deltaretrovirus. Here, we screened a library of integrase strand transfer inhibitor (INSTI) candidates built around several chemical scaffolds to determine their effectiveness in limiting HTLV-1 infection. Naphthyridines with substituents in position 6 emerged as the most potent compounds against HTLV-1, with XZ450 having highest efficacy in vitro. Using single-particle cryo-electron microscopy we visualised XZ450 as well as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the active site of the deltaretroviral intasome. The structures reveal subtle differences in the coordination environment of the Mg2+ ion pair involved in the interaction with the INSTIs. Our results elucidate the binding of INSTIs to the HTLV-1 intasome and support their use for pre-exposure prophylaxis and possibly future treatment of HTLV-1 infection.

Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR.

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.