Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes.
We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.
Brunner
Jo Ellen
JE
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Med. Sci. B240, Irvine, CA 92697, USA.
Nguyen
Joseph H C
JH
Roehl
Holger H
HH
Ho
Tri V
TV
Swiderek
Kristine M
KM
Semler
Bert L
BL
D000086042:3C Viral Proteases; D019943:Amino Acid Substitution; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006367:HeLa Cells; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000378:metabolism*; D006801:Humans; D017955:Poliovirus / Q000502:physiology*; D011485:Protein Binding; D012367:RNA, Viral / Q000096:biosynthesis*; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases
Virology. 2000 Sep 30;275(2):244-8
10998323
J Virol. 2000 Sep;74(17):8127-34
10933723
Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2249-54
9122180
Nature. 1970 Aug 15;227(5259):680-5
5432063
J Virol. 2000 Jan;74(1):334-43
10590122
Virology. 1994 Feb 15;199(1):176-87
8116241
Nucleic Acids Res. 1999 Mar 15;27(6):1457-63
10037806
Science. 1991 Dec 13;254(5038):1647-51
1661029
J Virol. 1998 Sep;72(9):7191-200
9696813
Methods Enzymol. 1996;275:35-57
9026649
Cell. 1990 Oct 19;63(2):369-80
2170027
J Biol Chem. 1996 Mar 29;271(13):7654-8
8631802
J Virol. 2002 Sep;76(17):8787-96
12163599
FEBS Lett. 2002 Jul 17;523(1-3):73-8
12123807
EMBO J. 1991 Nov;10(11):3447-56
1833186
RNA. 1996 Nov;2(11):1139-52
8903344
J Biol Chem. 1994 Feb 18;269(7):5377-83
8106521
J Virol. 1997 Aug;71(8):6243-6
9223526
Nat Rev Mol Cell Biol. 2002 Mar;3(3):195-205
11994740
Virology. 2001 Feb 1;280(1):41-51
11162817
J Biol Chem. 1994 Sep 16;269(37):23074-8
8083209
J Virol. 1991 Jun;65(6):3384-7
1851886
EMBO J. 1992 Sep;11(9):3289-95
1380452
Virology. 1998 Apr 25;244(1):1-12
9581772
J Virol. 1995 May;69(5):2954-61
7707521
RNA. 1997 Oct;3(10):1124-34
9326487
Annu Rev Biophys Biomol Struct. 1998;27:407-45
9646873
EMBO J. 1993 Sep;12(9):3587-98
8253083
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11115-20
8855318
J Virol. 1995 Oct;69(10):6367-75
7666538
J Virol. 1997 Dec;71(12):9570-8
9371621
Nucleic Acids Res. 1992 Apr 11;20(7):1739-45
1315956
J Virol. 1997 Nov;71(11):8482-9
9343205
Nucleic Acids Res. 1998 Jul 15;26(14):3410-7
9649627
J Virol. 2003 Apr;77(8):4739-50
12663781
J Virol. 1997 Jan;71(1):578-85
8985386
Proc Natl Acad Sci U S A. 1989 Dec;86(24):9788-92
2557628
Anal Biochem. 1995 Jan 1;224(1):451-5
7710111
Virology. 1979 Sep;97(2):396-405
224589
J Mol Biol. 2001 Jan 26;305(4):829-38
11162095
J Am Soc Mass Spectrom. 1995 Jul;6(7):571-7
24214354
RNA. 1997 Aug;3(8):882-92
9257647
EMBO J. 2001 Jan 15;20(1-2):240-9
11226174
Annu Rev Biochem. 1993;62:289-321
8352591
J Virol. 2003 May;77(9):5136-44
12692216
J Virol. 1998 Nov;72(11):8782-8
9765422
J Biol Chem. 1993 Apr 25;268(12):8881-7
8473331
Mol Cell. 2001 Mar;7(3):581-91
11463383
J Biol Chem. 2001 Mar 9;276(10):7681-8
11113151
J Virol. 2001 Jun;75(11):5009-17
11333880
J Biol Chem. 1995 Apr 28;270(17):10105-12
7730315