title: Functional Sindbis virus replicative complexes are formed at the plasma membrane.
abstract: Formation of virus-specific replicative complexes (RCs) in infected cells is one of the most intriguing and important processes that determine virus replication and ultimately their pathogenesis on the molecular and cellular levels. Alphavirus replication was known to lead to formation of so-called type 1 cytopathic vacuoles (CPV1s), whose distinguishing feature is the presence of numerous membrane invaginations (spherules) and accumulation of viral nonstructural proteins (nsPs) at the cytoplasmic necks of these spherules. These CPV1s, modified endosomes and lysosomes, were proposed as the sites of viral RNA synthesis. However, our recent studies have demonstrated that Sindbis virus (SINV)-specific, double-stranded RNA (dsRNA)- and nonstructural protein (nsP)-containing RCs are initially formed at the plasma membrane. In this new study, we present extensive evidence that (i) in cells of vertebrate origin, at early times postinfection, viral nsPs colocalize with spherules at the plasma membrane; (ii) viral dsRNA intermediates are packed into membrane spherules and are located in their cavities on the external surface of the plasma membrane; (iii) formation of the membrane spherules is induced by the partially processed nonstructural polyprotein P123 and nsP4, but synthesis of dsRNA is an essential prerequisite of their formation; (iv) plasma membrane-associated dsRNA and protein structures are the active sites of single-stranded RNA (ssRNA) synthesis; (v) at late times postinfection, only a small fraction of SINV nsP-containing complexes are relocalized into the cytoplasm on the endosome membrane. (vi) pharmacological drugs inhibiting different endocytotic pathways have either only minor or no negative effects on SINV RNA replication; and (vii) in mosquito cells, at any times postinfection, dsRNA/nsP complexes and spherules are associated with both endosomal/lysosomal and plasma membranes, suggesting that mechanisms of RC formation may differ in cells of insect and vertebrate origins.
authors.affiliation: Department of Microbiology, University of Alabama, Birmingham, AL 35294-2170, USA. ivfrolov@UAB.edu
authors.forename: Elena I
authors.initials: EI
authors.lastname: Frolova
authors.forename: Rodion
authors.initials: R
authors.lastname: Gorchakov
authors.forename: Larisa
authors.initials: L
authors.lastname: Pereboeva
authors.forename: Svetlana
authors.initials: S
authors.lastname: Atasheva
authors.forename: Ilya
authors.initials: I
authors.lastname: Frolov
mesh_terms: D018354:Alphavirus Infections / Q000378:metabolism / Q000821:virology*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000821:virology*; D006224:Cricetinae; D009033:Culicidae; D051379:Mice; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D017361:Viral Nonstructural Proteins
references.citation: J Virol. 1991 Aug;65(8):4107-13
references.pmid: 2072446
references.citation: Methods Mol Biol. 2008;440:15-33
references.pmid: 18369934
references.citation: J Virol. 2003 Feb;77(3):1691-702
references.pmid: 12525603
references.citation: J Virol. 2007 Dec;81(24):13552-65
references.pmid: 17913819
references.citation: Virology. 1984 Feb;133(1):92-110
references.pmid: 6322438
references.citation: J Virol. 1994 Mar;68(3):1874-85
references.pmid: 8107248
references.citation: J Virol. 1993 Apr;67(4):1916-26
references.pmid: 8445717
references.citation: RNA. 2001 Nov;7(11):1638-51
references.pmid: 11720292
references.citation: Virology. 2009 Apr 25;387(1):211-21
references.pmid: 19278709
references.citation: J Virol. 2008 Oct;82(20):10088-101
references.pmid: 18684830
references.citation: Proc Natl Acad Sci U S A. 1983 Jun;80(11):3279-83
references.pmid: 6304704
references.citation: Microbiol Rev. 1994 Sep;58(3):491-562
references.pmid: 7968923
references.citation: J Biol Chem. 2006 Oct 6;281(40):30269-78
references.pmid: 16895903
references.citation: Methods Enzymol. 2008;438:77-93
references.pmid: 18413242
references.citation: J Virol. 2004 Sep;78(17):9499-511
references.pmid: 15308742
references.citation: Nat Methods. 2006 Dec;3(12):995-1000
references.pmid: 17072308
references.citation: J Virol. 1972 Sep;10(3):504-15
references.pmid: 4342056
references.citation: J Virol. 2009 Sep;83(17):8553-64
references.pmid: 19515769
references.citation: J Virol. 2006 Jun;80(12):5686-96
references.pmid: 16731907
references.citation: J Virol. 1995 Dec;69(12):7391-401
references.pmid: 7494243
references.citation: J Virol. 2002 Nov;76(22):11254-64
references.pmid: 12388685
references.citation: J Virol. 1972 Sep;10(3):492-503
references.pmid: 4672392
references.citation: J Virol. 1991 Aug;65(8):4017-25
references.pmid: 2072444
references.citation: J Virol. 2009 Sep;83(18):9031-44
references.pmid: 19570872
references.citation: J Virol. 2001 Apr;75(8):3873-84
references.pmid: 11264376
references.citation: J Virol. 1981 Aug;39(2):348-58
references.pmid: 7277580
references.citation: J Biol Chem. 2004 Aug 20;279(34):35557-63
references.pmid: 15205456
references.citation: Virology. 2007 Jun 5;362(2):320-30
references.pmid: 17292435
references.citation: J Virol. 1968 Nov;2(11):1326-38
references.pmid: 5750316
references.citation: J Virol. 1984 Mar;49(3):865-72
references.pmid: 6699940
references.citation: J Virol. 2008 Jul;82(13):6218-31
references.pmid: 18417571
references.citation: J Virol. 2007 May;81(10):5046-57
references.pmid: 17329335
references.citation: J Virol. 1999 May;73(5):3854-65
references.pmid: 10196280
references.citation: Virology. 2006 Jan 5;344(1):151-7
references.pmid: 16364746
references.citation: J Virol. 1996 Aug;70(8):5153-64
references.pmid: 8764023
references.citation: Mol Cell. 2002 Mar;9(3):505-14
references.pmid: 11931759
references.citation: Cell. 2009 Jun 26;137(7):1308-19
references.pmid: 19563761
references.citation: J Virol. 1990 Jun;64(6):3001-11
references.pmid: 2159558
references.citation: PLoS Pathog. 2009 Dec;5(12):e1000705
references.pmid: 20041173
references.citation: J Cell Biol. 1988 Dec;107(6 Pt 1):2075-86
references.pmid: 2904446
references.citation: J Virol. 2010 Aug;84(15):7543-57
references.pmid: 20484502
references.citation: J Muscle Res Cell Motil. 2004;25(4-5):337-41
references.pmid: 15548862
references.citation: Nat Chem Biol. 2006 Jul;2(7):355-6
references.pmid: 16783339
references.citation: J Virol. 1987 Dec;61(12):3809-19
references.pmid: 3479621
references.citation: J Neurol Sci. 1981 Mar;49(3):397-418
references.pmid: 7217991
references.citation: J Virol. 2006 Apr;80(8):4122-34
references.pmid: 16571828
references.citation: J Virol. 2007 Mar;81(5):2472-84
references.pmid: 17108023
references.citation: J Virol. 1991 Mar;65(3):1496-506
references.pmid: 1847467
references.citation: J Gen Virol. 2007 Jul;88(Pt 7):1977-1985
references.pmid: 17554031
references.citation: PLoS Biol. 2007 Sep;5(9):e220
references.pmid: 17696647
references.citation: Arch Virol Suppl. 1994;9:213-20
references.pmid: 7518272
references.citation: Adv Exp Med Biol. 1996;416:321-6
references.pmid: 9131167
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 3;101(31):11263-8
references.pmid: 15280537
references.citation: J Virol. 2010 Jul;84(13):6720-32
references.pmid: 20392851