Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction.

HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1BH10 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2EHO RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1BH10 RT (positions 342-351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of ß-sheets 17 and 18 and their connecting loop (residues 342-350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs.

Nucleocapsid Protein Precursors NCp9 and NCp15 Suppress ATP-Mediated Rescue of AZT-Terminated Primers by HIV-1 Reverse Transcriptase.

In HIV-1, development of resistance to AZT (3'-azido-3'-deoxythymidine) is mediated by the acquisition of thymidine analogue resistance mutations (TAMs) (i.e., M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q) in the viral reverse transcriptase (RT). Clinically relevant combinations of TAMs, such as M41L/T215Y or D67N/K70R/T215F/K219Q, enhance the ATP-mediated excision of AZT monophosphate (AZTMP) from the 3' end of the primer, allowing DNA synthesis to continue. Additionally, during HIV-1 maturation, the Gag polyprotein is cleaved to release a mature nucleocapsid protein (NCp7) and two intermediate precursors (NCp9 and NCp15). NC proteins interact with the viral genome and facilitate the reverse transcription process. Using wild-type and TAM-containing RTs, we showed that both NCp9 and NCp15 inhibited ATP-mediated rescue of AZTMP-terminated primers annealed to RNA templates but not DNA templates, while NCp7 had no effect on rescue activity. RNase H inactivation by introducing the active-site mutation E478Q led to the loss of the inhibitory effect shown by NCp9. NCp15 had a stimulatory effect on the RT's RNase H activity not observed with NCp7 and NCp9. However, analysis of RNase H cleavage patterns revealed that in the presence of NCp9, RNA/DNA complexes containing duplexes of 12 bp had reduced stability in comparison with those obtained in the absence of NC or with NCp7 or NCp15. These effects are expected to have a strong influence on the inhibitory action of NCp9 and NCp15 by affecting the efficiency of RNA-dependent DNA polymerization after unblocking DNA primers terminated with AZTMP and other nucleotide analogues.

SIX1 represses senescence and promotes SOX2-mediated cellular plasticity during tumorigenesis.

Six1 is a developmental transcriptional regulator frequently overexpressed in human tumors. Recent results show that SIX1 also acts as a repressor of cell senescence, an antiproliferative response with a key role in tumor suppression, among other physiological and pathological settings. Here, we set to study the impact of SIX1 gain of function in transformation and tumorigenesis of fibroblasts, in connection with senescence. Using transcriptomic, histological, and functional analyses in murine tumors and cells of fibroblast origin, we show that SIX1 has a strong pro-tumorigenic action in this model, linked to the repression of a senescence-related gene signature and the induction of an undifferentiated phenotype mediated, at least in part, by the regulation of the stemness factor Sox2. Moreover, functional analyses with human glioma cell lines also show that SIX1 controls SOX2 expression, senescence and self-renewal in this model. Collectively, our results support a general link of SIX1 with senescence and SOX2-mediated cell plasticity in tumors.