BACKGROUND: Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated. RESULTS: Here, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release. CONCLUSIONS: Based on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.

• MeSH
• biologické modely MeSH
• buněčné linie MeSH
• krysa rodu Rattus MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu * MeSH
• posttranslační úpravy proteinů * MeSH
• proteiny virové matrix chemie   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• virus myšího tumoru prsní žlázy chemie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny virové matrix MeSH

The mouse mammary tumor virus (MMTV) Gag contains the unique domains pp21, p3, p8, and n. We investigated the contribution of these domains to particle assembly and found that the region spanning the p8 and n domains is critical for shape determination and assembly. Deletion of pp21 and p3 reduced the number of released particles, but deletion of the n domain resulted in frequent formation of aberrant particles, while deletion of p8 severely impaired assembly. Further investigation of p8 revealed that both the basic and the proline-rich motifs within p8 contribute to MMTV assembly.

• MeSH
• genové produkty gag fyziologie   MeSH
• myši MeSH
• sestavení viru * MeSH
• terciární struktura proteinů MeSH
• virové plášťové proteiny MeSH
• virus myšího tumoru prsní žlázy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag MeSH
• virové plášťové proteiny MeSH

Mason-Pfizer monkey virus (M-PMV) Gag protein contains a domain p12 that is unique to this virus (simian retrovirus-3) and its close relatives. The alpha-helical N-terminal half of p12, which contains a leucine zipper-like region, forms ordered structures in E. coli and the C-terminal half can form SDS-resistant oligomers in vitro. Together these properties suggest that p12 is a strong protein-protein interaction domain that facilitates Gag-Gag oligomerization. We have analyzed the oligomerization potential of a panel of p12 mutants, including versions containing substituted dimer, trimer, and tetramer leucine zippers, expressed in bacteria and in the context of the Gag precursor expressed in vitro and in cells. Purified recombinant p12 and its mutants could form various oligomers as shown by chemical cross-linking experiments. Within Gag these same mutants could assemble when overexpressed in cells. In contrast, all the mutants, including the leucine zipper mutants, were assembly defective in a cell-free system. These data highlight the importance of a region containing alternating leucines and isoleucines within p12, but also indicate that this domain's scaffold-like function is more complex than small number oligomerization.

• MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• genové produkty gag genetika   metabolismus   MeSH
• leucinové zipy MeSH
• Masonův-Pfizerův opičí virus genetika   fyziologie   ultrastruktura   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• terciární struktura proteinů MeSH
• transmisní elektronová mikroskopie MeSH
• vazba proteinů MeSH
• virion ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• genové produkty gag MeSH
• p12 protein, Mason-Pfizer monkey virus MeSH Prohlížeč
• rekombinantní proteiny MeSH