We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

• Keywords
• DEAH-box RNA helicase, DHX15, G-patch, gRNA packaging, retrovirus,
• MeSH
• Cell Nucleus metabolism   virology   MeSH
• DEAD-box RNA Helicases metabolism   genetics   MeSH
• Genome, Viral MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus * genetics   metabolism   physiology   MeSH
• Virus Replication genetics   physiology   MeSH
• RNA, Viral * metabolism   genetics   MeSH
• RNA Helicases metabolism   genetics   MeSH
• Virus Assembly * genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DEAD-box RNA Helicases MeSH
• DHX15 protein, human MeSH Browser
• RNA, Viral * MeSH
• RNA Helicases MeSH

Viral proteases are indispensable for successful virion maturation, thus making them a prominent drug target. Their enzyme activity is tightly spatiotemporally regulated by expression in the precursor form with little or no activity, followed by activation via autoprocessing. These cleavage events are frequently triggered upon transportation to a specific compartment inside the host cell. Typically, precursor oligomerization or the presence of a co-factor is needed for activation. A detailed understanding of these mechanisms will allow ligands with non-canonical mechanisms of action to be designed, which would specifically modulate the initial irreversible steps of viral protease autoactivation. Binding sites exclusive to the precursor, including binding sites beyond the protease domain, can be exploited. Both inhibition and up-regulation of the proteolytic activity of viral proteases can be detrimental for the virus. All these possibilities are discussed using examples of medically relevant viruses including herpesviruses, adenoviruses, retroviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, and coronaviruses.

• Keywords
• Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), activation, adenoviruses, autoprocessing, flaviviruses, herpesviruses, precursor, protease,
• MeSH
• Antiviral Agents pharmacology   MeSH
• Flavivirus drug effects   metabolism   MeSH
• Herpesviridae drug effects   metabolism   MeSH
• HIV-1 drug effects   MeSH
• Viral Protease Inhibitors pharmacology   MeSH
• Humans MeSH
• Adenoviruses, Human drug effects   metabolism   MeSH
• SARS-CoV-2 drug effects   metabolism   MeSH
• Virus Diseases drug therapy   MeSH
• Viral Proteases biosynthesis   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Antiviral Agents MeSH
• Viral Protease Inhibitors MeSH
• Viral Proteases MeSH

Mason-Pfizer monkey virus protease (PR) was crystallized in complex with two pepstatin-based inhibitors in P1 space group. In both crystal structures, the extended flap loops that lock the inhibitor/substrate over the active site, are visible in the electron density either completely or with only small gaps, providing the first observation of the conformation of the flap loops in dimeric complex form of this retropepsin. The H-bond network in the active site (with D26N mutation) differs from that reported for the P21 crystal structures and is similar to a rarely occurring system in HIV-1 PR.

• Keywords
• M-PMV, Mason-Pfizer monkey virus, active site architecture, aspartic protease, dimer, flap structure, inhibitor, retropepsin, retrovirus,
• MeSH
• Protease Inhibitors chemistry   MeSH
• Mason-Pfizer monkey virus enzymology   genetics   MeSH
• Mutation, Missense MeSH
• Pepstatins chemistry   MeSH
• Peptide Hydrolases chemistry   genetics   MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Substitution MeSH
• Viral Proteins antagonists & inhibitors   chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protease Inhibitors MeSH
• pepstatin MeSH Browser
• Pepstatins MeSH
• Peptide Hydrolases MeSH
• Viral Proteins MeSH

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

• MeSH
• Anilino Naphthalenesulfonates metabolism   MeSH
• Atmospheric Pressure MeSH
• Darunavir metabolism   MeSH
• Dimerization MeSH
• Spectrometry, Fluorescence MeSH
• HIV Protease chemistry   metabolism   MeSH
• HIV Protease Inhibitors metabolism   MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Protein Folding * MeSH
• Protein Stability drug effects   MeSH
• Thermodynamics MeSH
• Tryptophan metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-anilino-8-naphthalenesulfonate MeSH Browser
• Anilino Naphthalenesulfonates MeSH
• Darunavir MeSH
• HIV Protease MeSH
• HIV Protease Inhibitors MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Browser
• Tryptophan MeSH