Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome.

Retroviruses exploit host proteins to assemble and release virions from infected cells. Previously, most studies focused on interacting partners of retroviral Gag proteins that localize to the cytoplasm or plasma membrane. Given that several full-length Gag proteins have been found in the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings involving previously unknown host processes.

Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome.

Retroviruses exploit a variety of host proteins to assemble and release virions from infected cells. To date, most studies that examined possible interacting partners of retroviral Gag proteins focused on host proteins that localize primarily to the cytoplasm or plasma membrane. Given the recent findings that several full-length Gag proteins localize to the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings that reveal previously unknown host processes.

HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.

The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression.

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55 (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size.

The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol.

The Rous sarcoma virus Gag polyprotein forms biomolecular condensates driven by intrinsically-disordered regions.

Biomolecular condensates (BMCs) play important roles in cellular structures including transcription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol.

HIV-1 Gag colocalizes with euchromatin histone marks at the nuclear periphery.

Unlabelled: The retroviral Gag protein of human immunodeficiency virus type 1 (HIV-1) plays a central role in the selection of unspliced viral genomic RNA for packaging into new virions. Previously, we demonstrated that full-length HIV-1 Gag undergoes nuclear trafficking where it associates with unspliced viral RNA (vRNA) at transcription sites. To further explore the kinetics of HIV-1 Gag nuclear localization, we used biochemical and imaging techniques to examine the timing of HIV-1 entry into the nucleus.

Liquid-liquid phase separation of nucleocapsid proteins during SARS-CoV-2 and HIV-1 replication.

The leap of retroviruses and coronaviruses from animal hosts to humans has led to two ongoing pandemics and tens of millions of deaths worldwide. Retrovirus and coronavirus nucleocapsid proteins have been studied extensively as potential drug targets due to their central roles in virus replication, among which is their capacity to bind their respective genomic RNAs for packaging into nascent virions. This review focuses on fundamental studies of these nucleocapsid proteins and how their intrinsic abilities to condense through liquid-liquid phase separation (LLPS) contribute to viral replication.

Clinician-Scientist Faculty Mentoring Program (FAME) - A New Inclusive Training Model at Penn State Increases Scholarly Productivity and Extramural Grant Funding.

Purpose: Clinician-scientists have a high attrition rate at the junior-faculty level, before they gain independent funding. We identified the lack of skill set, clinician-scientist community and collaboration between clinician-scientists and clinicians with predominantly clinical duties, as key problems in our medium-size college of medicine.

Methods: We designed a novel two-year educational program, the Clinician-scientist Faculty Mentoring program (FAME) specifically to target junior clinician-scientists.

Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

Background: The generation of over 69 spliced HIV-1 mRNAs from one primary transcript by alternative RNA splicing emphasizes the central role that RNA processing plays in HIV-1 replication. Control is mediated in part through the action of host SR proteins whose activity is regulated by multiple SR kinases (CLK1-4, SRPKs).

Methods: Both shRNA depletion and small molecule inhibitors of host SR kinases were used in T cell lines and primary cells to evaluate the role of these factors in the regulation of HIV-1 gene expression.

SARS-CoV-2 vaccine effectiveness against infection, symptomatic and severe COVID-19: a systematic review and meta-analysis.

Background: The temporal evolution of SARS-CoV-2 vaccine efficacy and effectiveness (VE) against infection, symptomatic, and severe COVID-19 is incompletely defined. The temporal evolution of VE could be dependent on age, vaccine types, variants of the virus, and geographic region. We aimed to conduct a systematic review and meta-analysis of the duration of VE against SARS-CoV-2 infection, symptomatic COVID-19 and severe COVID-19.

Monoclonal Antibodies to S and N SARS-CoV-2 Proteins as Probes to Assess Structural and Antigenic Properties of Coronaviruses.

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses.

Diabetes, Drug Treatment, and Mortality in COVID-19: A Multinational Retrospective Cohort Study.

Patients with type 2 diabetes mellitus (T2DM) are at increased risk of severe coronavirus disease 2019 (COVID-19) outcomes possibly because of dysregulated inflammatory responses. Glucose-regulating medications, such as glucagon-like peptide 1 receptor (GLP-1R) agonists, dipeptidyl peptidase 4 (DPP-4) inhibitors, and pioglitazone, are known to have anti-inflammatory effects that may improve outcomes in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In a multinational retrospective cohort study, we used the TriNetX COVID-19 Research Network of 56 large health care organizations to examine these medications in relation to the incidence of hospital admissions, respiratory complications, and mortality within 28 days after a COVID-19 diagnosis.

Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane.

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers.

Building a System to Engage and Sustain Research Careers for Physicians.

There are increasing needs for physician-investigators to translate the rapid expansion of knowledge, technology/interventions, and big data into the clinical realm at a time of increasing age-related disabilities and communicable diseases. Yet, the number of physician-investigators has continued to decline, and only a small number of medical school graduates in the United States are actively engaged in research. This problem may be particularly pronounced in small- and medium-sized academic institutions due to more limited educational and mentoring infrastructure.

HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites.

The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs.

TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain.

Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLSs) in Gag.

Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity.

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a "Ψ" packaging signal located in the gRNA 5'-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood.

Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus.

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L.

RNA-Binding Domains of Heterologous Viral Proteins Substituted for Basic Residues in the RSV Gag NC Domain Restore Specific Packaging of Genomic RNA.

The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach.

A non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the HIV-1 Pr55 polyprotein alters nucleic acid binding properties.

HIV Gag (Pr55), a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (HIV), is an active target of antiretroviral inhibitor development. However, highly pure, stable, recombinant Pr55 has been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. Stability has been improved by using a construct that omits the p6 domain (Δp6).

Orchestrating the Selection and Packaging of Genomic RNA by Retroviruses: An Ensemble of Viral and Host Factors.

Infectious retrovirus particles contain two copies of unspliced viral RNA that serve as the viral genome. Unspliced retroviral RNA is transcribed in the nucleus by the host RNA polymerase II and has three potential fates: (1) it can be spliced into subgenomic messenger RNAs (mRNAs) for the translation of viral proteins; or it can remain unspliced to serve as either (2) the mRNA for the translation of Gag and Gag-Pol; or (3) the genomic RNA (gRNA) that is packaged into virions. The Gag structural protein recognizes and binds the unspliced viral RNA to select it as a genome, which is selected in preference to spliced viral RNAs and cellular RNAs.

Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag.

Retroviruses specifically package full-length, dimeric genomic RNA (gRNA) even in the presence of a vast excess of cellular RNA. The "psi" (Ψ) element within the 5'-untranslated region (5'UTR) of gRNA is critical for packaging through interaction with the nucleocapsid (NC) domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different.