Blocking premature reverse transcription fails to rescue the HIV-1 nucleocapsid-mutant replication defect

<p>Abstract</p> <p>Background</p> <p>The nucleocapsid (NC) protein of HIV-1 is critical for viral replication. Mutational analyses have demonstrated its involvement in viral assembly, genome packaging, budding, maturation, reverse transcription, and integration. We previously reported that two conservative NC mutations, His23Cys and His44Cys, cause premature reverse transcription such that mutant virions contain approximately 1,000-fold more DNA than wild-type virus, and are replication defective. In addition, both mutants show a specific defect in integration after infection.</p> <p>Results</p> <p>In the present study we investigated whether blocking premature reverse transcription would relieve the infectivity defects, which we successfully performed by transfecting proviral plasmids into cells cultured in the presence of high levels of reverse transcriptase inhibitors. After subsequent removal of the inhibitors, the resulting viruses showed no significant difference in single-round infective titer compared to viruses where premature reverse transcription did occur; there was no rescue of the infectivity defects in the NC mutants upon reverse transcriptase inhibitor treatment. Surprisingly, time-course endogenous reverse transcription assays demonstrated that the kinetics for both the NC mutants were essentially identical to wild-type when premature reverse transcription was blocked. In contrast, after infection of CD4+ HeLa cells, it was observed that while the prevention of premature reverse transcription in the NC mutants resulted in lower quantities of initial reverse transcripts, the kinetics of reverse transcription were not restored to that of untreated wild-type HIV-1.</p> <p>Conclusions</p> <p>Premature reverse transcription is not the cause of the replication defect but is an independent side-effect of the NC mutations.</p

PLA Logistics and Sustainment: PLA Conference 2022

The US Army War College People’s Liberation Army Conference (PLA) Conference was held March 31 to April 2, 2022, at Carlisle Barracks, Pennsylvania. The conference focused on PLA logistics and sustainment. As the PLA continues to build and modernize its combat forces, it is important to examine if the capabilities meant to support combat operations are also being developed. Specific topics included: 1) China’s national-level logistics, including how China mobilizes national resources for the military and how it provides joint logistics support to the PLA Theater Commands; 2) the logistics capabilities of the different PLA services, especially the Army, Navy, and Air Forces; 3) PLA logistics in China’s remote regions, such as airpower projection in the Western Theater Command along the Indian border; and, 4) the PLA’s ability to sustain overseas operations at its base in Djibouti. Despite notable potential shortfalls and points of friction, the PLA has successfully sustained counterpiracy maritime operations for many years and conducted noncombatant evacuation operations well-distant from China. It is increasingly able to move forces across the vast distances of China and conduct large training exercises. Far more must be known about PLA sustainment and logistics before the hard questions about PLA operational reach and endurance can be answered.https://press.armywarcollege.edu/monographs/1954/thumbnail.jp

Drosophila S2 Cells Are Non-Permissive for Vaccinia Virus DNA Replication Following Entry via Low pH-Dependent Endocytosis and Early Transcription

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis

RISK AND SACRAMENT: BEING HUMAN IN A COVID‐19 WORLD

In this article we examine the changing relationship to risk as revealed by the COVID‐19 pandemic and the ways this has, and may in future, alter sacramental practice, considering the radical effects this could have on traditional Christian practice. We consider the cultural trends that may lie behind this developing approach to risk, examining this in the context of an emergent transhuman identity that is technologically moderated and seeks to overcome risks of human mortality