Proximity Ligation Assay to Detect the Proximity Between Host Proteins and Viral Proteins of HIV-1.

The study of host-pathogen interaction often requires interrogating the protein-protein interactions and examining post-translational modifications of the proteins. Traditional protein detection strategies are limited in their sensitivity, specificity, and multiplexing capabilities. The Proximity Ligation Assay (PLA), a versatile and powerful molecular technique, can overcome these limitations. PLA blends the specificity of antibodies, two antibodies detecting two different epitopes on the same or two different proteins, with the amplification efficiency of a polymerase to allow highly specific and sensitive detection of low-abundant proteins, protein-protein interactions, or protein modifications. In this protocol, we describe the application of PLA to detect the proximity between HIV-1 Tat with one of its cellular partners, p65, in an infected host cell. The protocol could be applied to any other context with slight modifications. Of note, PLA can only confirm the physical proximity between two epitopes or proteins; however, the proximity need not necessarily allude to the functional interaction between the two proteins.

The expression of HIV-1 tat in Lactococcus lactis.

Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being explored for recombinant protein expression. L. lactis, being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat. Finally, the recombinant Tat was functionally competent in transactivating the HIV-1 promoter in HLM-1 reporter cells. Our work provides a scaffold for future work on the expression of toxic proteins in Lactococcus lactis.

HIV-1 subtypes and latent reservoirs.

PURPOSE OF REVIEW: We explore the current status of research on HIV-1 subtype-specific variations and their impact on HIV-1 latency. We also briefly address the controversy surrounding the decision-making process governing the ON/OFF states of HIV-1 transcription, specifically focusing on the regulatory elements, the long terminal repeat (LTR), and Tat. Understanding the decision-making process is crucial for developing effective intervention strategies, such as the 'shock-and-kill' approach, to reactivate latent HIV-1. RECENT FINDINGS: Attention has been drawn to subtype-specific transcription factor binding site (TFBS) variations and the possible impact of these variations on viral latency. Further, diverse subtype-specific assays have been developed to quantify the latent viral reservoirs. One interesting observation is the relatively larger latent reservoirs in HIV-1B infection than those of other viral subtypes, which needs rigorous validation. The emergence of LTR-variant viral strains in HIV-1C demonstrating significantly higher levels of latency reversal has been reported. SUMMARY: Despite persistent and substantial efforts, latent HIV-1 remains a formidable challenge to a functional cure. Determined and continued commitment is needed to understand the ON/OFF decision-making process of HIV-1 latency, develop rigorous assays for accurately quantifying the latent reservoirs, and identify potent latency-reversing agents and cocktails targeting multiple latency stages. The review emphasizes the importance of including diverse viral subtypes in future latency research.