Retroviruses consume cellular deoxynucleoside triphosphates (dNTPs) to convert their RNA genomes into proviral DNA through reverse transcription. While all retroviruses replicate in dividing cells, lentiviruses uniquely replicate in nondividing cells such as macrophages. Importantly, dNTP levels in nondividing cells are extremely low, compared to dividing cells. Indeed, a recently discovered anti-HIV/SIV restriction factor, SAMHD1, which is a dNTP triphosphohydrolase, is responsible for the limited dNTP pool of the nondividing cells. Lentiviral reverse transcriptases (RT) uniquely stay functional even at the low dNTP concentrations of the nondividing cells. Interestingly, Vpx of HIV-2/SIVsm proteosomally degrades SAMHD1, which elevates cellular dNTP pools and accelerates lentiviral replication in nondividing cells. These Vpx-encoding lentiviruses rapidly replicate in nondividing cells by encoding both highly functional RTs and Vpx. Here, we discussed a series of mechanistic and virological studies that have contributed to conceptually linking cellular dNTP levels and the adaptation of lentiviral replication in nondividing cells.

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Background: SAMHD1 is an enzyme that maintains low dNTP concentrations in macrophages. Results: Depletion of SAMHD1 decreases HIV-1 sensitivity to nucleoside reverse transcriptase inhibitors (NRTIs) in macrophages, but does not significantly alter sensitivity in T cells. Conclusion: SAMHD1 expression levels in macrophages directly impact the efficacy of NRTIs by modulating cellular dNTP concentrations. Significance: SAMHD1 controls HIV-1 sensitivity to NRTIs.

Each segment of the influenza A virus (IAV) genome contains conserved sequences at the 5′- and 3′-terminal ends, which form the promoter region necessary for polymerase binding and initiation of RNA synthesis. Although several models of interaction have been proposed it remains unclear if these two short, partially complementary, and highly conserved sequences can form a stable RNA duplex at physiological temperatures. First, our time-resolved FRET analysis revealed that a 14-mer 3′-RNA and a 15-mer 5′-RNA associate in solution, even at 42 °C. We also found that a nonfunctional RNA promoter containing the 3′-G3U mutation, as well as a promoter containing the compensatory 3′-G3U/C8A mutations, was able to form a duplex as efficiently as wild type. Second, UV melting analysis demonstrated that the wild-type and mutant RNA duplexes have similar stabilities in solution. We also observed an increase in thermostability for a looped promoter structure. The absence of differences in the stability and binding kinetics between wild type and a nonfunctional sequence suggests that the IAV promoter can be functionally inactivated without losing the capability to form a stable RNA duplex. Finally, using uridine specific chemical probing combined with mass spectrometry, we confirmed that the 5′ and 3′ sequences form a duplex which protects both RNAs from chemical modification, consistent with the previously published panhandle structure. These data support that these short, conserved promoter sequences form a stable complex at physiological temperatures, and this complex likely is important for polymerase recognition and viral replication.

Background: Under limiting dNTP concentrations, HIV-1 RT incorporates rNTPs during DNA synthesis. Results: HIV-1 RT utilizes dNTP less efficiently around rNMPs, and mismatch extension fidelity is significantly reduced. Conclusion: Presence of an rNMP in DNA template slows HIV-1 RT-mediated DNA synthesis and reduces fidelity. Significance: This study provides insight into how rNMP incorporation during proviral DNA synthesis can affect HIV-1 replication kinetics and fidelity.

The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork.

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Background A crucial part of delirium care is determining if the delirium episode has resolved. Yet, there is no clear evidence or consensus on which assessments clinicians should use to assess for delirium recovery. Objective To evaluate current opinions from delirium specialists on assessment of delirium recovery. Design Online questionnaire-based survey distributed internationally to healthcare professionals involved in delirium care. Methods The survey covered methods for assessing recovery, the importance of different symptom domains for capturing recovery, and local guidance or pathways that recommend monitoring for delirium recovery. Results Responses from 199 clinicians were collected. Respondents were from the UK (51%), US (13%), Australia (9%), Canada (7%), Ireland (7%) and 16 other countries. Most respondents were doctors (52%) and nurses (27%). Clinicians worked mostly in geriatrics (52%), ICUs (21%) and acute assessment units (17%). Ninety-four percent of respondents indicated that they conduct repeat delirium assessments (i.e., on ≥2 occasions) to monitor delirium recovery. The symptom domains considered most important for capturing recovery were: arousal (92%), inattention (84%), motor disturbance (84%), and hallucinations and delusions (83%). The most used tool for assessing recovery was the 4 ’A’s Test (4AT, 51%), followed by the Confusion Assessment Method (CAM, 26%), the CAM for the ICU (CAM-ICU, 17%) and the Single Question in Delirium (SQiD, 11%). Twenty-eight percent used clinical features only. Less than half (45%) of clinicians reported having local guidance that recommends monitoring for delirium recovery. Conclusions The survey results suggest a lack of standardisation regarding tools and methods used for repeat delirium assessment, despite consensus surrounding the key domains for capturing delirium recovery. These findings emphasise the need for further research to establish best practice for assessing delirium recovery.