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.

• Klíčová slova
• DEAH-box RNA helicase, DHX15, G-patch, gRNA packaging, retrovirus,
• MeSH
• buněčné jádro metabolismus   virologie   MeSH
• DEAD-box RNA-helikasy metabolismus   genetika   MeSH
• genom virový MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus * genetika   metabolismus   fyziologie   MeSH
• replikace viru genetika   fyziologie   MeSH
• RNA virová * metabolismus   genetika   MeSH
• RNA-helikasy metabolismus   genetika   MeSH
• sestavení viru * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• DHX15 protein, human MeSH Prohlížeč
• RNA virová * MeSH
• RNA-helikasy MeSH

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

• Klíčová slova
• betaretrovirus, infectious disease, matrix protein, maturation, microbiology, myristoyl switch, protease, viruses,
• MeSH
• buněčná membrána MeSH
• endopeptidasy MeSH
• genové produkty gag chemie   MeSH
• Masonův-Pfizerův opičí virus * chemie   fyziologie   MeSH
• proteiny MeSH
• sestavení viru MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endopeptidasy MeSH
• genové produkty gag MeSH
• proteiny 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.

• Klíčová slova
• Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), activation, adenoviruses, autoprocessing, flaviviruses, herpesviruses, precursor, protease,
• MeSH
• antivirové látky farmakologie   MeSH
• Flavivirus účinky léků   metabolismus   MeSH
• Herpesviridae účinky léků   metabolismus   MeSH
• HIV-1 účinky léků   MeSH
• inhibitory virových proteáz farmakologie   MeSH
• lidé MeSH
• lidské adenoviry účinky léků   metabolismus   MeSH
• SARS-CoV-2 účinky léků   metabolismus   MeSH
• virové nemoci farmakoterapie   MeSH
• virové proteasy biosyntéza   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• antivirové látky MeSH
• inhibitory virových proteáz MeSH
• virové proteasy MeSH

Mason-Pfizer monkey virus (M-PMV), like some other betaretroviruses, encodes a G-patch domain (GPD). This glycine-rich domain, which has been predicted to be an RNA binding module, is invariably localized at the 3' end of the pro gene upstream of the pro-pol ribosomal frameshift sequence of genomic RNAs of betaretroviruses. Following two ribosomal frameshift events and the translation of viral mRNA, the GPD is present in both Gag-Pro and Gag-Pro-Pol polyproteins. During the maturation of the Gag-Pro polyprotein, the GPD transiently remains a C-terminal part of the protease (PR), from which it is then detached by PR itself. The destiny of the Gag-Pro-Pol-encoded GPD remains to be determined. The function of the GPD in the retroviral life cycle is unknown. To elucidate the role of the GPD in the M-PMV replication cycle, alanine-scanning mutational analysis of its most highly conserved residues was performed. A series of individual mutations as well as the deletion of the entire GPD had no effect on M-PMV assembly, polyprotein processing, and RNA incorporation. However, a reduction of the reverse transcriptase (RT) activity, resulting in a drop in M-PMV infectivity, was determined for all GPD mutants. Immunoprecipitation experiments suggested that the GPD is a part of RT and participates in its function. These data indicate that the M-PMV GPD functions as a part of reverse transcriptase rather than protease.

• MeSH
• buněčné linie MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus chemie   enzymologie   genetika   MeSH
• polyproteiny chemie   genetika   metabolismus   MeSH
• reverzní transkriptasa chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• polyproteiny MeSH
• reverzní transkriptasa MeSH
• virové proteiny MeSH

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

• MeSH
• acyltransferasy chemie   genetika   izolace a purifikace   MeSH
• Escherichia coli enzymologie   genetika   MeSH
• exprese genu MeSH
• kyselina myristová chemie   MeSH
• Masonův-Pfizerův opičí virus chemie   genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny virové matrix chemie   genetika   izolace a purifikace   MeSH
• rekombinantní fúzní proteiny chemie   genetika   izolace a purifikace   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acyltransferasy MeSH
• glycylpeptide N-tetradecanoyltransferase MeSH Prohlížeč
• kyselina myristová MeSH
• proteiny virové matrix MeSH
• rekombinantní fúzní proteiny MeSH

All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be approximately 10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.

• MeSH
• algoritmy MeSH
• aspartátové endopeptidasy chemie   genetika   metabolismus   MeSH
• bodová mutace genetika   MeSH
• dimerizace MeSH
• fluorescenční barviva metabolismus   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• molekulární modely MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Retroviridae - proteiny chemie   genetika   metabolismus   MeSH
• serin chemie   genetika   MeSH
• stabilita enzymů genetika   MeSH
• substrátová specifita MeSH
• threonin chemie   genetika   MeSH
• vazebná místa genetika   MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• fluorescenční barviva MeSH
• HIV-proteasa MeSH
• protease p15 MeSH Prohlížeč
• rekombinantní proteiny MeSH
• Retroviridae - proteiny MeSH
• serin MeSH
• threonin MeSH