Consideration of multiple template switches is critical to the determination of the recombination rate on the HIV genome

Retroviral recombination drives viral diversity and facilitates the emergence of immune escape and drug resistant mutants that contribute to disease progression. Current estimates of retroviral recombination rates are based on indirect measurements that do not take into account the effects of multiple recombination events. In the presence of multiple template switches, any even number of template switches result in no observed recombination and any odd number is detected as a single recombination event. We demonstrate that ignoring multiple recombination events consistently underestimates the true recombination rate, especially over large genetic distances and high rates of recombination. Here, we present a novel approach to measure rates of recombination across different gene segments regardless of the effects of genetic distance and the overall rate of recombination. We apply these tools to a novel HIV-1 marker system, which mimics the recombination process between closely related genomes, analogous to those found within the quasispecies of an infected individual. We directly measure the recombination rate in gag, correcting for the effects of multiple template switches and background recombination. Furthermore, our analysis indicates that recombination rates are likely to vary across the viral genome. This system is applicable to other studies to accurately measure the recombination rate that is critical for the diversification of retroviruses.

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection.