During the early stages of HIV-1 replication the conical capsid composed of p24 ^CA protein dissociates from the rest of the cytoplasmic viral complex by a process called uncoating. Although proper uncoating is known to be required for HIV-1 infection, many questions remain about the timing and factors involved in the process. Here we have used two complementary assays to study the process of uncoating in HIV-1–infected cells, specifically looking at the timing of uncoating and its relationship to reverse transcription. We developed a fluorescent microscopy-based uncoating assay that detects the association of p24 ^CA with HIV-1 viral complexes in cells. We also used an owl monkey kidney (OMK) cell assay that is based on timed TRIM-CypA–mediated restriction of HIV-1 replication. Results from both assays indicate that uncoating is initiated within 1 h of viral fusion. In addition, treatment with the reverse transcriptase inhibitor nevirapine delayed uncoating in both assays. Analysis of reverse transcription products in OMK cells revealed that the generation of early reverse transcription products coincides with the timing of uncoating in these assays. Collectively, these results suggest that some aspect of reverse transcription has the ability to influence the kinetics of uncoating.

The primate TRIM5α proteins have recently been defined as cellular restriction factors, preventing primate infection by retroviruses from different species. For instance, rhesus TRIM5α (rhTRIM5α) restricts infection by HIV-1. Virtually all TRIM5α proteins block the early replication of retroviruses by preventing the accumulation of reverse transcription products, but the underlying mechanism remains unclear. In this article, we find that disrupting proteasome function alters rhTRIM5α localization and allows the normal generation of HIV-1 late reverse transcription products, even though HIV-1 infection and the generation of nuclear 1-LTR and 2-LTR viral cDNA forms remain impaired. This finding suggests rhTRIM5α restricts HIV infection in two distinct phases: ( i ) altering the normal passage of the reverse-transcribing viral genome to the nucleus and ( ii ) targeting the reverse transcription complex to be disrupted by the proteasome. Because proteasome inhibitor blocks the second phase, accumulation of a nonfunctional viral DNA genome can be readily observed. Defining each phase may reveal HIV-1 targets for future antiviral therapy in which dual blockade may be equally as effective as naturally occurring rhTRIM5α protein in preventing HIV-1 infection in vivo .

To reliably infect a primate model for human immunodeficiency virus (HIV), ∼10,000-fold more virus must be delivered vaginally than intravenously. However, the vaginal mechanisms that help protect against HIV are poorly understood. Here, we report that human cervicovaginal mucus (CVM), obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1,000-fold more slowly than it does in water. Lactobacilli acidify CVM to pH ∼4 by continuously producing lactic acid. At this acidic pH, we found that lactic acid, but not HCl, abolished the negative surface charge on HIV without lysing the virus membrane. In contrast, in CVM neutralized to pH 6 to 7, as occurs when semen temporarily neutralizes the vagina, HIV maintained its native surface charge and diffused only 15-fold more slowly than it would in water. Thus, methods that can maintain both a high lactic acid content and acidity for CVM during coitus may contribute to both vaginal and penile protection by trapping HIV before it can reach target cells. Our results reveal that CVM likely plays an important but currently unappreciated role in decreasing the rate of HIV sexual transmission.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, β, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.

Worldwide, HIV-1 infects millions of people annually, the majority of whom are women. To establish infection in the female reproductive tract (FRT), HIV-1 in male ejaculate must overcome numerous innate and adaptive immune factors, traverse the genital epithelium, and establish infection in underlying CD4⁺target cells. How the virus achieves this remains poorly defined. By utilizing a new technique, we define how HIV-1 interacts with different tissues of the FRT using human cervical explants andin vivoexposure in the rhesus macaque vaginal transmission model. Despite previous claims of the squamous epithelium being an efficient barrier to virus entry, we reveal that HIV-1 can penetrate both intact columnar and squamous epithelial barriers to depths where the virus can encounter potential target cells. In the squamous epithelium, we identify virus entry occurring through diffusive percolation, penetrating areas where cell junctions are absent. In the columnar epithelium, we illustrate that virus does not transverse barriers as well as previously thought due to mucus impediment. We also show a statistically significant correlation between the viral load of inocula and the ability of HIV-1 to pervade the squamous barrier. Overall, our results suggest a diffusive percolation mechanism for the initial events of HIV-1 entry. With these data, we also mathematically extrapolate the number of HIV-1 particles that penetrate the mucosa per coital act, providing a biological description of the mechanism for HIV-1 transmission during the acute and chronic stages of infection.