Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

Identification of Host Proteins Involved in Hepatitis B Virus Genome Packaging.

A critical part of the hepatitis B virus (HBV) life cycle is the packaging of the pregenomic RNA (pgRNA) into nucleocapsids. While this process is known to involve several viral elements, much less is known about the identities and roles of host proteins in this process. To better understand the role of host proteins, we isolated pgRNA and characterized its protein interactome in cells expressing either packaging-competent or packaging-incompetent HBV genomes. We identified over 250 host proteins preferentially associated with pgRNA from the packaging-competent version of the virus. These included proteins already known to support capsid formation, enhance viral gene expression, catalyze nucleocapsid dephosphorylation, and bind to the viral genome, demonstrating the ability of the approach to effectively reveal functionally significant host-virus interactors. Three of these host proteins, AURKA, YTHDF2, and ATR, were selected for follow-up analysis. RNA immunoprecipitation qPCR (RIP-qPCR) confirmed pgRNA-protein association in cells, and siRNA knockdown of the proteins showed decreased encapsidation efficiency. This study provides a template for the use of comparative RNA-protein interactome analysis in conjunction with virus engineering to reveal functionally significant host-virus interactions.

Revealing Retroviral Life Cycles Using Visible Viruses and Live Cell Imaging.

Viruses exploit key host cell factors to accomplish each individual stage of the viral replication cycle. To understand viral pathogenesis and speed the development of new antiviral strategies, high-resolution visualization of virus-host interactions is needed to define where and when these events occur within cells. Here, we review state-of-the-art live cell imaging techniques for tracking individual stages of viral life cycles, focusing predominantly on retroviruses and especially human immunodeficiency virus type 1, which is most extensively studied. We describe how visible viruses can be engineered for live cell imaging and how nonmodified viruses can, in some instances, be tracked and studied indirectly using cell biosensor systems. We summarize the ways in which live cell imaging has been used to dissect the retroviral life cycle. Finally, we discuss select challenges for the future including the need for better labeling strategies, increased resolution, and multivariate systems that will allow for the study of full viral replication cycles.

HIV-1 Vif disrupts phosphatase feedback regulation at the kinetochore, leading to a pronounced pseudo-metaphase arrest.

The human immunodeficiency virus type 1 (HIV-1) Virion Infectivity Factor (Vif) targets and degrades cellular APOBEC3 proteins, key regulators of intrinsic and innate antiretroviral immune responses, thereby facilitating HIV-1 infection. While Vif's role in degrading APOBEC3G is well-studied, Vif is also known to cause cell cycle arrest but the detailed nature of Vif's effects on the cell cycle has yet to be delineated. In this study, we employed high-temporal single-cell live imaging and super-resolution microscopy to monitor individual cells during Vif-induced cell cycle arrest. Our findings reveal that Vif does not affect the G2/M boundary as previously thought. Instead, Vif triggers a unique and robust pseudo-metaphase arrest, which is markedly distinct from the mild prometaphase arrest induced by the HIV-1 accessory protein, Vpr, known for modulating the cell cycle. During Vif-mediated arrest, chromosomes align properly to form a metaphase plate but later disassemble, resulting in polar chromosomes. Notably, unlike Vpr, Vif significantly reduces the levels of both Phosphatase 1 (PP1) and 2 (PP2) at kinetochores, which are key regulators of chromosome-microtubule interactions. These results reveal a novel function of Vif in kinetochore regulation that governs the spatial organization of chromosomes during mitosis.