Formation of nsP3-specific protein complexes during Sindbis virus replication.
Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. Their replication proceeds in the cytoplasm of infected cells and does not directly depend on nuclei. Alphaviruses encode only four nonstructural proteins that are required for the replication of viral genome and transcription of the subgenomic RNA. However, the replicative enzyme complexes (RCs) appear to include cellular proteins and assemble on cellular organelles. We have developed a set of recombinant Sindbis (SIN) viruses with green fluorescent protein (GFP) insertions in one of the nonstructural proteins, nsP3, to further understand the RCs' genesis and structure. We studied the assembly of nsP3/GFP-containing protein complexes at different stages of infection and isolated a combination of cellular proteins that are associated with SIN nsP3. We demonstrated the following. (i) SIN nsP3 can tolerate the insertion of GFP into different fragments of the coding sequence; the designed recombinant viruses are viable, and their replication leads to the assembly of nsP3/GFP chimeric proteins into gradually developing, higher-order structures differently organized at early and late times postinfection. (ii) At late times postinfection, nsP3 is assembled into complexes of similar sizes, which appear to be bound to cytoskeleton filaments and can aggregate into larger structures. (iii) Protein complexes that are associated with nsP3/GFP contain a high concentration of cytoskeleton proteins, chaperones, elongation factor 1A, heterogeneous nuclear ribonucleoproteins, 14-3-3 proteins, and some of the ribosomal proteins. These proteins are proposed to be essential for SIN RC formation and/or functioning.
Frolova
Elena
E
Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1019, USA.
Gorchakov
Rodion
R
Garmashova
Natalia
N
Atasheva
Svetlana
S
Vergara
Leoncio A
LA
Frolov
Ilya
I
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012845:Sindbis Virus / Q000502:physiology*; D014746:Vimentin / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
D050883:HSC70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D014746:Vimentin; D017361:Viral Nonstructural Proteins; C089383:nsp3 protein, alphavirus; D049452:Green Fluorescent Proteins