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Activation of the Mason-Pfizer monkey virus protease within immature capsids in vitro.

ABSTRACT: For all retroviruses, the completion of the viral budding process correlates with the activation of the viral protease by an unknown mechanism, and, as the structural (Gag) polyproteins are cleaved by the viral protease, maturation of the immature virus-like particle into an infectious virion. Unlike most retroviruses, the Mason-Pfizer monkey virus Gag polyproteins assemble into immature capsids within the cytoplasm of the cell before the viral budding event. The results reported here describe a unique experimental system in which Mason-Pfizer monkey virus immature capsids are removed from the cell, and the protease is activated in vitro by the addition of a reducing agent. The cleavage of the protease from the precursor form is a primary event, which proceeds with a half time of 14 min, and is followed by authentic processing of the Gag polyproteins. Activity of the viral protease in vitro depends on pH, with an increase in catalytic rates at acidic and neutral pH. The initiation of protease activity within immature capsids in vitro demonstrates that viral protease activity is sensitive to oxidation-reduction conditions, and that the viral protease can be activated in the absence of viral budding.

SUBMITTER: Parker SD

PROVIDER: S-EPMC64733 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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Activation of the Mason-Pfizer monkey virus protease within immature capsids in vitro.

Parker S D SD Hunter E E

Proceedings of the National Academy of Sciences of the United States of America 20011127 25

For all retroviruses, the completion of the viral budding process correlates with the activation of the viral protease by an unknown mechanism, and, as the structural (Gag) polyproteins are cleaved by the viral protease, maturation of the immature virus-like particle into an infectious virion. Unlike most retroviruses, the Mason-Pfizer monkey virus Gag polyproteins assemble into immature capsids within the cytoplasm of the cell before the viral budding event. The results reported here describe a ...[more]

PMID: 11724937

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Project description:The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-Å cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly.Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

Project description:Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

Dataset Information

Activation of the Mason-Pfizer monkey virus protease within immature capsids in vitro.

Publications

Activation of the Mason-Pfizer monkey virus protease within immature capsids in vitro.

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