Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural ...virologic control by sequencing viruses, T cell receptor genes, HIV integration sites, and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity.
Display omitted
•In HIV controllers, both TFH and non-TFH lymph node CD4 T cells contain HIV•Lymph node viruses in both TFH and non-TFH have attributes of active replication•Rare, recently infected cells that produce virus upon stimulation circulate in blood•Archival proviruses predominant in clonally expanded blood cells can be inducible
HIV persistence in people who can spontaneously control the infection involves different mechanisms within distinct anatomic and functional compartments.
Histone modifications regulate chromatin-dependent processes, yet the mechanisms by which they contribute to specific outcomes remain unclear. H3K4me3 is a prominent histone mark that is associated ...with active genes and promotes transcription through interactions with effector proteins that include initiation factor TFIID. We demonstrate that H3K4me3-TAF3 interactions direct global TFIID recruitment to active genes, some of which are p53 targets. Further analyses show that (1) H3K4me3 enhances p53-dependent transcription by stimulating preinitiation complex (PIC) formation; (2) H3K4me3, through TAF3 interactions, can act either independently or cooperatively with the TATA box to direct PIC formation and transcription; and (3) H3K4me3-TAF3/TFIID interactions regulate gene-selective functions of p53 in response to genotoxic stress. Our findings indicate a mechanism by which H3K4me3 directs PIC assembly for the rapid induction of specific p53 target genes.
Display omitted
► H3K4me3 facilitates global TFIID recruitment to the core promoters of active genes ► By stimulating PIC formation, H3K4me3 enhances p53-dependent activation ► H3K4me3 acts both independently of and cooperatively with the TATA box ► Interactions between H3K4me3 and TAF3/TFIID regulate gene-selective functions of p53
H3K4me3 plays a causal role in transcription by recruiting the TAF3 subunit of TFIID to promoters and facilitating the assembly of the transcriptional preinitiation complex. This activity is required for the activation of rapidly induced genes, including some p53 targets.
The major obstacle to curing HIV infection is the persistence of cells with intact proviruses that can produce replication-competent virus. This HIV reservoir is believed to exist primarily in CD4+ ...T-cells and is stable despite years of suppressive antiretroviral therapy. A potential mechanism for HIV persistence is clonal expansion of infected cells, but how often such clones carry replication-competent proviruses has been controversial. Here, we used single-genome sequencing to probe for identical HIV sequence matches among viruses recovered in different viral outgrowth cultures and between the sequences of outgrowth viruses and proviral or intracellular HIV RNA sequences in uncultured blood mononuclear cells from eight donors on suppressive ART with diverse proviral populations. All eight donors had viral outgrowth virus that was fully susceptible to their current ART drug regimen. Six of eight donors studied had identical near full-length HIV RNA sequences recovered from different viral outgrowth cultures, and one of the two remaining donors had identical partial viral sequence matches between outgrowth virus and intracellular HIV RNA. These findings provide evidence that clonal expansion of HIV-infected cells is an important mechanism of reservoir persistence that should be targeted to cure HIV infection.
Advances in technology have made it possible to analyze integration sites in cells from HIV-infected patients. A significant fraction of infected cells in patients on long-term therapy are clonally ...expanded; in some cases the integrated viral DNA contributes to the clonal expansion of the infected cells. Although the large majority (>95%) of the HIV proviruses in treated patients are defective, expanded clones can carry replication-competent proviruses, and cells from these clones can release infectious virus. As discussed in this Perspective, it is likely that cells that produce virus are strongly selected against in vivo, and cells with replication competent proviruses expand and survive because only a small fraction of the cells produce virus. These findings have implications for strategies that are intended to eliminate the reservoir of infected cells that has made it almost impossible to cure HIV-infected patients.
HIV remains largely incurable because the viral DNA integrates into long-lived cells where the provirus remains quiescent. Hughes and Coffin present their perspective on what controls the specificity of HIV integration, with particular emphasis on its importance in the generation and persistence of HIV-infected cells on long-term antiretroviral therapy.
Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but ...some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral-host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3' end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.
HIV-1 reverse transcription Hu, Wei-Shau; Hughes, Stephen H
Cold Spring Harbor perspectives in medicine,
10/2012, Letnik:
2, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Reverse transcription and integration are the defining features of the Retroviridae; the common name "retrovirus" derives from the fact that these viruses use a virally encoded enzyme, reverse ...transcriptase (RT), to convert their RNA genomes into DNA. Reverse transcription is an essential step in retroviral replication. This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the cellular and viral factors that can affect reverse transcription, and discusses fidelity and recombination, two processes in which reverse transcription plays an important role. In keeping with the theme of the collection, the emphasis is on HIV-1 and HIV-1 RT.
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.
Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV ...Integration targeting-se- quencing （HIT-Seq）, we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3＇ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disruption of histone mRNA processing, and polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Inte- grator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruit- ment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites.
Integrase strand transfer inhibitors (INSTIs) are the class of antiretroviral (ARV) drugs most recently approved by the FDA for the treatment of HIV-1 infections. INSTIs block the strand transfer ...reaction catalyzed by HIV-1 integrase (IN) and have been shown to potently inhibit infection by wild-type HIV-1. Of the three current FDA-approved INSTIs, Dolutegravir (DTG), has been the most effective, in part because treatment does not readily select for resistant mutants. However, recent studies showed that when INSTI-experienced patients are put on a DTG-salvage therapy, they have reduced response rates. Two new INSTIs, Cabotegravir (CAB) and Bictegravir (BIC), are currently in late-stage clinical trials.
Both CAB and BIC had much broader antiviral profiles than RAL and EVG against the INSTI-resistant single, double, and triple HIV-1 mutants used in this study. BIC was more effective than DTG against several INSTI-resistant mutants. Overall, in terms of their ability to inhibit a broad range of INSTI-resistant IN mutants, BIC was superior to DTG, and DTG was superior to CAB. Modeling the binding of CAB, BIC, and DTG within the active site of IN suggested that the "left side" of the INSTI pharmacophore (the side away from the viral DNA) was important in determining the ability of the compound to inhibit the IN mutants we tested.
Of the two INSTIs in late stage clinical trials, BIC appears to be better able to inhibit the replication of a broad range of IN mutants. BIC retained potency against several of the INSTI-resistant mutants that caused a decrease in susceptibility to DTG.
The rapid replication of HIV-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly ...challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work; so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways. A better understanding of the structure and function(s) of RT and of the mechanism(s) of inhibition can be used to generate better drugs; in particular, drugs that are effective against the current drug-resistant strains of HIV-1.
PDF
