The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.
- MeSH
- AIDS opičí virologie MeSH
- buněčná membrána metabolismus virologie MeSH
- endozomy metabolismus virologie MeSH
- genové produkty env genetika metabolismus MeSH
- Masonův-Pfizerův opičí virus genetika fyziologie MeSH
- sestavení viru MeSH
- transport proteinů MeSH
- transportní vezikuly metabolismus virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
HIV infekce u lidí a SIV infekce u makaků Rhesus (RM) se vyznačuje zvýšenou apoptózou a aktivací vyvolanou buněčnou smrtí (AICD) CD4 lymfocytů. Avšak některé druhy primátů, jako například afričtí mangabejové (SM), jsou přirozeně infikováni SIV a nerozvine se u nich symptomatické onemocnění. Tato studie ukazuje, že lymfocyty RM infikovaných SIV vykazují sníženou vnitrobuněčnou expresi GSK3beta a současně zvýšenou expresi a fosforylaci (Thr308) kinázy Akt spojenou se stimulací receptorů CD3 a CD28. Tyto rozdíly jsou specificky měřitelné pouze v některých definovaných subpopulacích CD4 lymfocytů. Je to jeden z mála příkladů monofosforylace Akt a tato signalizace může představovat jeden z mechanismů vedoucích k rozdílům v AICD a případně i rezistenci k infekci SIV.
Increased CD4+ T cell apoptosis and activation induced cell death (AICD) as a result of HIV infection in humans and SIV infection in Rhesus macaques (RM) is indicative of disease. Some non-human primate species naturally infected by SIV, such as African sooty mangabeys (SM), do not succumb to SIV despite high viral loads. Previously, we showed that mRNA levels of GSK-3β a kinase involved in T cell signaling, are significantly decreased in SIV+ RM compared to SIV+ SM. The current study confirms that expression of GSK-3β is decreased at the protein level in SIV+ RM. In addition, CD4+ T cells from SIV+ RM, but not other animals show an increase in both total Akt, a kinase directly interacting with GSK-3β and p-AktThr308 in response to stimulation via CD3/CD28, which is associated with an increase in apoptosis. Furthermore, the differences between the uninfected and pathogenically or non-pathogenically infected animals are not only species specific, but also T cell subset specific and that these trends correlate with AICD. This is one of few studies indicating the activity of Akt can be specific to only one phosphorylation site and may be linked to the differences in AICD and resistance to the lentivirus induced disease.
- MeSH
- AIDS opičí * imunologie MeSH
- antigeny CD28 MeSH
- antigeny CD3 MeSH
- apoptóza MeSH
- CD4-pozitivní T-lymfocyty * MeSH
- Cercocebus atys MeSH
- fosforylace * MeSH
- imunoblotting MeSH
- kinasa 3 glykogensynthasy MeSH
- kultivované buňky MeSH
- Macaca mulatta MeSH
- nemoci opic MeSH
- protein-serin-threoninkinasy MeSH
- průtoková cytometrie MeSH
- signální transdukce MeSH
- virus opičí imunodeficience patogenita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles.
- MeSH
- AIDS opičí metabolismus virologie MeSH
- buněčná membrána virologie MeSH
- Cercopithecus aethiops MeSH
- genové produkty env genetika metabolismus MeSH
- genové produkty gag genetika metabolismus MeSH
- Macaca mulatta MeSH
- Masonův-Pfizerův opičí virus genetika metabolismus MeSH
- mikrotubuly metabolismus virologie MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
BACKGROUND: Formation of a mature core is a crucial event for infectivity of retroviruses such as Mason-Pfizer monkey virus (M-PMV). The process is triggered by proteolytic cleavage of the polyprotein precursor Gag, which releases matrix, capsid (CA), and nucleocapsid proteins. Once released, CA assembles to form a mature core - a hexameric lattice protein shell that protects retroviral genomic RNA. Subtle conformational changes within CA induce the transition from the immature lattice to the mature lattice. Upon release from the precursor, the initially unstructured N-terminus of CA is refolded to form a β-hairpin stabilized by a salt bridge between the N-terminal proline and conserved aspartate. Although the crucial role of the β-hairpin in the mature core assembly has been confirmed, its precise structural function remains poorly understood. RESULTS: Based on a previous NMR analysis of the N-terminal part of M-PMV CA, which suggested the role of additional interactions besides the proline-aspartate salt bridge in stabilization of the β-hairpin, we introduced a series of mutations into the CA sequence. The effect of the mutations on virus assembly and infectivity was analyzed. In addition, the structural consequences of selected mutations were determined by NMR spectroscopy. We identified a network of interactions critical for proper formation of the M-PMV core. This network involves residue R14, located in the N-terminal β-hairpin; residue W52 in the loop connecting helices 2 and 3; and residues Q113, Q115, and Y116 in helix 5. CONCLUSION: Combining functional and structural analyses, we identified a network of supportive interactions that stabilize the β-hairpin in mature M-PMV CA.
- MeSH
- AIDS opičí genetika metabolismus MeSH
- buněčné linie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- sekundární struktura proteinů genetika MeSH
- sekvence aminokyselin MeSH
- sestavení viru genetika MeSH
- virion genetika metabolismus MeSH
- virové plášťové proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Most retroviruses employ a frameshift mechanism during polyprotein synthesis to balance appropriate ratios of structural proteins and enzymes. To investigate the requirements for individual precursors in retrovirus assembly, we modified the polyprotein repertoire of Mason-Pfizer monkey virus (M-PMV) by mutating the frameshift sites to imitate the polyprotein organization of Rous sarcoma virus (Gag-Pro and Gag-Pro-Pol) or Human immunodeficiency virus (Gag and Gag-Pro-Pol). For the "Rous-like" virus, assembly was impaired with no incorporation of Gag-Pro-Pol into particles and for the "HIV-like" virus an altered morphogenesis was observed. A mutant expressing Gag and Gag-Pro polyproteins and lacking Gag-Pro-Pol assembled intracellular particles at a level similar to the wild-type. Gag-Pro-Pol polyprotein alone neither formed immature particles nor processed the precursor. All the mutants were non-infectious except the "HIV-like", which retained fractional infectivity.
- MeSH
- AIDS opičí virologie MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- financování organizované MeSH
- genové produkty gag genetika MeSH
- genové produkty pol genetika MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus genetika patogenita MeSH
- messenger RNA genetika MeSH
- posunová mutace MeSH
- proteosyntéza MeSH
- RNA virová genetika MeSH
- transfekce MeSH
- virion genetika patogenita MeSH
- virové proteiny genetika MeSH
- virus Rousova sarkomu genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
