Yuntao Wu

Persistence of viral reservoirs prevents a cure or functional cure for HIV. Efforts to eradicate viral reservoirs have been constrained by the difficulties of identifying and eliminating rare, heterogeneous viral reservoir cells in vivo. Single-modality interventions such as “shock and kill” have faced persistent challenges despite decades of effort, highlighting the need for complementary strategies that more precisely target infected cells. In this review, I summarize our 20 years of effort in developing an HIV Rev-dependent lentiviral vector for selective targeting of viral reservoirs. This lentiviral vector expresses therapeutic genes under the control of HIV Tat and Rev, conferring high specificity. In vitro, superinfection of HIV-infected cells with the Rev-dependent particle can lead to the identification and selective killing of infected cells. In vivo, injection of Rev-dependent lentiviral particles into SIV-infected rhesus macaques stimulates antiviral immunity, leading to reduction of viral reservoirs and inhibition of viral rebound. We designate this Rev vector-based therapeutic approach as Rev-dependent HIV Immunotherapy (RHIT). The RHIT approach integrates three complementary reservoir-targeting strategies: (1) RHIT-mediated killing of reservoir cells via Rev-dependent therapeutic gene expression; (2) RHIT-mediated inhibition of active HIV replication via defective interfering particles; and (3) RHIT-induced anti-HIV immunity via induction of neutralizing antibodies and cell-mediated immune responses. In this review, I also discuss limitations and challenges for the future development of RHIT.

Nipah Virus (NiV) infection was first reported in Malaysia in 1998. The outbreak in pig farms caused 229 human cases of febrile encephalitis with 111 deaths (48% fatality). The viral pathogen was s...

The cortical actin cytoskeleton serves as a structural support and force-generating machinery in cells, but the actin meshwork can also act as a barrier, hindering the delivery of extracellular substances. To address the issue of the cortical actin barrier, we engineered synthetic libraries of human β-actin–derived peptides to promote actin dynamics. Here, we report the discovery of six major peptides, collectively termed actinators, that are biologically active in selective modulation of actin-related cellular activities. Using retro- and lentiviral vector transduction of CAR T cells as a model, we demonstrate that an actinator markedly enhances viral vector transduction 20- to 30-fold. An individual actinator can also selectively modulate cellular functions such as regulating surface receptors, enhancing T cell adhesion, and promoting the cellular uptake of exosomes. Molecular docking and protein phosphorylation studies confirmed that actinators can modulate actin-binding proteins like cofilin. Actinators represent a class of actin-derived bioactive peptides with diverse applications in gene and immune therapies, drug delivery, and disease treatment.

Detecting and measuring HIV reservoirs, neutralizing antibodies, and restriction factors are important for HIV cure research and the development of new the

Detecting and measuring HIV reservoirs, neutralizing antibodies, and restriction factors are important for HIV cure research and the development of new therapeutics and vaccines. Here we describe the development and validation of several HIV Rev-dependent indicator cell lines for these purposes. These reporter cells come from different T-lymphoblast cell lines, including MOLT-4-R5, SupT1-R5, CEM-SS, A3R5, and from the adherent TZM cell platform based on HeLa clone JC53. These cells express CD4, CXCR4, and various levels of CCR5. We compared these cell lines for responsiveness to both X4 and R5-tropic viruses, and confirmed that reporter expression in these cells is not affected by stimulation from mitogens but is responsive to HIV Tat and Rev, reducing non-specific reporter induction from the leaky LTR promoter. To validate the sensitivity of the Rev-dependent reporter cell systems, we conducted a viral dilution assay with three primary HIV-1 clade C swarms from an adult in Malawi. We also validated the systems for quantifying antibody neutralization and screening restriction factors; these systems are also sensitive for viral outgrowth assays for quantifying viral reservoirs in clinical and basic research settings. Given that the systems can measure HIV accurately in complex environments with mitogens or other substances, they can be used for versatile applications, such as quantifying latent reservoirs, testing inhibitory compounds, conducting neutralizing antibody assays, and identifying new restriction factors.

Alzheimer’s disease (AD) is a degenerative neurological disease characterized by various biological signatures, including synaptic dysfunction, β-amyloid plaques, hyperphosphorylated Tau, cofilin-actin rods, and Hirano bodies, all of which are linked to the actin cytoskeleton and its regulators. Additionally, the presence of herpes simplex virus type 1 (HSV-1) in the brains of AD patients has long been suggested as a contributing factor for AD. However, mechanisms by which HSV-1 accelerates AD pathogenesis remain poorly understood. Here we report that HSV-1 infection induces hyperphosphorylation of cofilin in the brains of 5×FAD mice. Cofilin is an actin depolymerizing factor, and its S3 phosphorylation inactivates cofilin’s activity to depolymerize actin filaments. These findings facilitate the understanding of impacts of HSV-1 infection on the development of Alzheimer’s disease and have implications in AD therapeutics.

PSGL-1 (P-selectin glycoprotein ligand-1) is a dimeric, mucin-like surface glycoprotein that interacts with selectins on the endothelium, facilitating leukocyte tethering and rolling during transmigration. Previous studies have demonstrated that PSGL-1 acts as an HIV restriction factor, blocking HIV infectivity by hindering particle attachment to target cells. A large part of PSGL-1’s extracellular region consists of a mucin-like domain, characterized by decameric repeats (DR), which comprise 14 to 16 DR tandems. Each DR is composed of 10 consensus amino acids (-A-T/M-E-A-Q-T-T-X-P/L-A/T-) and is notably rich in O-glycosylated threonines (30%) and prolines (10%). A proposed function of the DR is to elongate the protein backbone needed for selectin binding. However, the precise role of DR in PSGL-1’s antiviral mechanisms has yet to be fully elucidated. In this study, we performed DR deletion mutagenesis and molecular modeling, systematically deleting from one DR to all DRs, and quantified their effects on PSGL-1’s antiviral functions. Here, we demonstrate that DR is necessary for PSGL-1’s antiviral activity. Deleting DR did not affect virion incorporation of PSGL-1, but diminished PSGL-1’s ability to hinder virion attachment to target cells. We also discovered that individual single DR mutants retained 3.5 to 18% of the antiviral activity exhibited by full-length PSGL-1, and the basic antiviral activity of DR is cumulative; increasing the number of DRs correlates with heightened antiviral activity. Additionally, the intrinsic anti-HIV capability of DR was found to be transferrable; inserting the DR domain into the extracellular Ig-like domain of CD2 permitted the hybrid CD2-DR molecules to partially acquire the anti-HIV properties of PSGL-1. Further molecular modeling utilizing all-atom replica exchange molecular dynamics simulations highlighted a structure-function relationship between the anti-HIV potency of DR and the elongation of the DR backbone, as measured by DR backbone dihedral angles ( φ ) and the radial distributions of peptide atom number density ( r _max ). These results collectively suggest that DR serves as an essential antiviral unit within the framework of PSGL-1’s restriction mechanism against HIV.

Stem cells are highly resistant to viral infection compared to their differentiated progeny, and this resistance is associated with stem cell-specific restriction factors and intrinsic interferon stimulated genes (ISGs). In HIV infection, proviral DNA has been detected in certain bone marrow hematopoietic stem cells, yet widespread stem cell infection in vivo is restricted. Intriguingly, exposing bone marrow stem cells to HIV in vitro led to viral replication selectively only in the CD34 ^- population, but not in the CD34 ⁺ cells. The mechanism dictating this CD34-based HIV restriction remained a mystery, especially since HIV has a capacity to antagonize restriction factors and ISGs. CD34 is a common marker of hematopoietic stem and progenitor cells. Here, we report the intrinsic antiviral properties of CD34. Expression of CD34 in HIV-1 producer cells results in the loss of progeny virion infectivity. Conversely, removal of CD34 using CRISPR/Cas9 knockout or stem cell differentiation cytokines promotes HIV-1 replication in stem cells. These results suggest that in addition to restriction factors and intrinsic ISGs, CD34 serves as a host innate protection preventing retrovirus replication in stem cells. Mechanistically, CD34 does not block viral entry, integration, and release. Instead, it becomes incorporated onto progeny virions, which inactivates virus infectivity. These findings offer new insights into innate immunity in stem cells, and highlight intriguing retrovirus-host interactions in evolution.

SUMMARY: Nipah virus (NiV) is an emergent paramyxovirus that causes serious disease in humans and animals. Accurate quantification of neutralizing antibodies (nAB) against NiV is essential for vaccine development. The current standard, the plaque reduction neutralization test (PRNT), requires authentic infectious NiV and BSL-4 containment, taking 4-7 days to complete. In this study, we developed a hybrid alphavirus-Nipah virus pseudovirion (Ha-NiV) composed of a non-replicating Nipah virus virus-like particle (VLP) that encapsulates an RNA genome from a fast-expressing SFV (Semliki Forest Virus) alphaviral vector. Ha-NiV can infect NiV target cells but is replication incompetent, enabling rapid quantification of nAB (6-18 hours) in BSL-2 conditions. We validated Ha-NiV using sera from Nipah virus-infected African green monkeys and established a good correlation (R ² = 0.86) with PRNT, demonstrating comparable specificity and sensitivity. These results demonstrate that the Ha-NiV assay can serve as a novel platform for convenient and rapid quantification of nAB in BSL-2 settings.

As of 2023, there were 39.9 million people living with Human Immunodeficiency Virus type 1 (HIV-1). Although great strides have been made in treatment options for HIV-1, and our understanding of the HIV-1 life cycle has vastly improved since the start of this global health crisis, a functional cure remains elusive. One of the main barriers to a cure is latency, which allows the virus to persist despite combined antiretroviral therapy (cART). Recently, we have found that exosomes, which are small, membrane-enclosed particles released by virtually all cell types and known to mediate intercellular communication, caused an increase in RNA Polymerase II loading onto the HIV-1 promoter. This resulted in the production of both short- and long-length viral transcripts in infected cells under cART. This current study examines the effects of exosome-associated kinases on bystander cells. The phospho-kinase profiling of exosomes revealed differences in the kinase payload of exosomes derived from uninfected and HIV-1-infected cells, with CDK10, GSK3β, and MAPK8 having the largest concentration differences. These kinases were shown to be biologically active and capable of phosphorylating substrates, and they modulated changes in the cell cycle dynamics of exposed cells. Given the relevance of such effects for the immune response, our results implicate exosome-associated kinases as new possible key contributors to HIV-1 pathogenesis that affect bystander cells. These findings may guide new therapeutic avenues to improve the current antiretroviral treatment regimens.