Properly Folded Nonstructural Polyprotein Directs the Semliki Forest Virus Replication Complex to the Endosomal Compartment

Abstract

The late RNA synthesis in alphavirus-infected cells, generating plus-strand RNAs, takes place on cytoplasmic vacuoles (CPVs), which are modified endosomes and lysosomes. The cytosolic surface of CPVs consists of regular membrane invaginations or spherules, which are the sites of RNA synthesis (P. Kujala, A. Ikähei-monen, N. Ehsani, H. Vihinen, P. Auvinen, and L. Kääriäinen J. Virol. 75:3873-3884, 2001). To understand how CPVs arise, we have expressed the individual Semliki Forest virus (SFV) nonstructural proteins nsP1 to nsP4 in different combinations, as well as their precursor polyprotein P1234 and its cleavage intermediates. A complex of nsPs was obtained from P123 or P1234, indicating that the precursor stage is essential for the assembly of the polymerase complex. To prevent the processing of the polyprotein and its cleavage intermediates, constructs with the mutation C478A (designated with a superscript CA) in the active site of the protease domain of nsP2 were used. Uncleaved polyproteins containing nsP1 were membrane bound and palmitoylated, and those containing nsP3 were phosphorylated, reflecting properties of authentic nsP1 and nsP3, respectively. Similarly, polyproteins containing nsP1 or nsP2 had enzymatic activities specific for the individual proteins, indicating that they were correctly folded in the precursor state. Uncleaved P12 CA was localized almost exclusively to the plasma membrane and filopodia, like nsP1 alone, whereas P12 CA 3 and P12 CA 34 were found on cytoplasmic vesicles, some of which contained late endosomal markers. In immunoelectron microscopy these vesicles resembled CPVs in SFV-infected cells. Our results indicate that the nsP1 domain alone is responsible for the membrane association of the nonstructural polyprotein, whereas the nsP1 domain together with the nsP3 domain targets it to the intracellular vesicles.The alphaviruses replicate in the cytoplasm of both invertebrate and vertebrate cells. The virus enters the cell by adsorptive endocytosis, followed by fusion of the virus envelope with endosomal membranes (34). The virus nucleocapsid is disassembled by ribosomes, which have affinity for the capsid protein (52, 63). The capped positive-strand RNA genome of about 11.5 kb is then translated to yield a polyprotein, P1234, of about 2,500 amino acids (aa), the precursor of nonstructural proteins nsP1 to nsP4. The parental 42S RNA genome is copied to complementary minus-strand RNA by a short-lived RNA polymerase consisting of the catalytic subunit nsP4 and polyprotein P123, the initial cleavage products of P1234 (31, 50). By inhibition of cleavage between nsP2 and nsP3, minusstrand RNA synthesis was demonstrated also to occur by the nsP1-P23-nsP4 combination (30).Early work with Semliki Forest virus (SFV) and Sindbis virus showed that the parental RNA was converted into an RNaseresistant, membrane-associated form soon after infection, suggesting that the synthesis of minus strands takes place in association with membranes (8,12,17). The cellular structures associated with the...

Regulation of the Sequential Processing of Semliki Forest Virus Replicase Polyprotein

“…8A) (15,16,25). In this study we have analyzed the proteolytic processing pathway of the ns polyprotein, which regulates the synthesis of the short-lived complementary RNA, as well as membrane association and targeting of the replication complex (18) . FIG.…”

“…We propose that the slow cleavage of site 1/2 enables the folding of the polyprotein so that protein-protein interactions between the components can take place before proteolytic processing (18). We further propose that the active membrane-associated replication complexes can form only through this polyprotein pathway, which includes as an obligatory step the synthesis of complementary RNA strand.…”

“…P12 is cleaved in cis giving rise to nsP1, which is targeted to the plasma membrane (37), and nsP2, which is transported to the nucleus (38). P34 is cleaved by free nsP2 resulting in the aggregation of nsP3 in the cytoplasm (18), whereas nsP4 is rapidly degraded by the ubiquitin pathway ( Fig. 8B) (20,39).…”

“…P12 is cleaved in cis and P34 in trans as shown in this article. NsP1 is directed to the plasma membrane, nsP2 to the nucleus, nsP3 to cytoplasmic aggregates, and nsP4 to the proteasomes (5,10,18,22,24,37,38).…”

“…This is because processing of P123 into its individual subunits changes the specialization of the polymerase complex toward the synthesis of full-length and subgenomic plus-sense RNAs on the minus-strand template. The polyprotein precursor is also indispensable for proper assembly, targeting, and membrane association of the replication complex (18). Given such a rapid timetable and exact requirements, the processing of P1234 must be coordinated precisely.…”