[PubMed] [Google Scholar] 46. other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses. All positive-strand RNA viruses replicate their genomes in association with cellular membranes. The formation and activity of the membrane-bound replication complexes (RCs) can result in extensive alteration of membrane Ozagrel(OKY-046) structures (11, 40, 48). Different viruses use different cytoplasmic membrane compartments as platforms for replication. Currently, there is only a limited understanding of how the virus-encoded and cellular proteins coordinate the formation of the replication-induced membrane structures. We address the mechanisms of membrane-bound replication with alphaviruses, particularly Semliki Forest virus (SFV). The alphaviruses comprise several human and animal pathogens, including the encephalitogenic alphaviruses (e.g., Western, Eastern, and Venezuelan equine encephalitis viruses) as well as the recently reemerging chikungunya virus, which belongs to the SFV clade of alphaviruses. During the past 5 years, chikungunya virus has caused more than 2 million infections and 500 deaths, and a new strain has spread throughout the areas surrounding the Indian Ocean (50). The alphaviruses use mosquitoes as intermediate hosts and transmission vectors, and at present no vaccines or antivirals are available to control these infections. The cytoplasmic replication of alphaviruses depends on the four viral nonstructural (ns) proteins, nsP1 to nsP4, which are all essential and act as a membrane-bound replication complex. The nsPs are translated from the viral positive-sense RNA genome as one large polyprotein. Cleavages catalyzed by the nsP2 moiety result in the release of the individual proteins. A large fraction of the synthesized nsPs is involved in genome replication and associates with membranes, but a sizable fraction dissociates and is distributed in different cellular compartments: nsP1 binds to the inner surface of the plasma membrane (PM); nsP2 is translocated into the nucleus; nsP3 seems to form aggregates Mouse monoclonal to ERBB2 in the cytoplasm; and most of the extra nsP4, the core RNA polymerase, is degraded by the proteasome. While the major enzymatic functions of the individual nsPs have been elucidated (21), little is known of how they function together in the replication machinery. As in other positive-strand RNA viruses, the RCs of alphaviruses are associated with altered intracellular membranes, which were first described in the late 1960s and early 1970s (13, 14, 18). In these early studies, it was shown Ozagrel(OKY-046) that virus replication induces bulb-shaped membrane invaginations with a diameter of 50 nm, which were called spherules. The spherules Ozagrel(OKY-046) were found on the limiting membranes of large cytoplasmic vacuoles, which were named virus-induced (CPV-I). On rare occasions, the spherules were seen also at the PM. By electron microscopic (EM) autoradiography, it was also shown that the spherules both at the CPV-I and at the PM could be sites of RNA synthesis (18). Subsequently, Froshauer et al. (15) showed that CPV-I are positive for endosomal and lysosomal markers. Moreover, using EM, they showed that the inside of the spherule is connected to the cytoplasm by a pore from which electron-dense material (which the authors suggest to be the newly synthesized RNA) seems to diffuse into the cytoplasm. During the past decade, our group has addressed the biogenesis of the CPV-I. We demonstrated that the formation of the spherules did not require structural proteins (44) and, more recently, that all four nsPs were associated with the spherules together with newly formed RNA (labeled by bromouridine), strongly suggesting that they were the actual units of RNA replication (RCs) (28). We also suggested as one possibility that the spherules could first arise at the PM; subsequent endocytosis of the spherules could account for the formation of the CPV-I (28, 44). Of the four nsPs, only nsP1 has affinity for membranes, and when expressed alone, it is specifically targeted to the inner surface of the PM (45). NsP1 is a monotopic membrane protein; its affinity for membranes is dictated by an amphipathic alpha helix, located in the central region of the protein (4, 31). NsP1 has a specific affinity for negatively charged phospholipids, which could potentially account for its prevalent localization to the PM, where such lipids are enriched. Later we showed that the membrane binding of nsP1 through the.