Helicase and Capping Enzyme Active Site Mutations in Brome Mosaic Virus Protein 1a Cause Defects in Template Recruitment, Negative-Strand RNA Synthesis, and Viral RNA Capping

Journal of Virology - Tập 74 Số 19 - Trang 8803-8811 - 2000
Tero Ahola1, Johan A. den Boon1, Paul Ahlquist1
1Institute for Molecular Virology and Howard Hughes Medical Institute, University of Wisconsin—Madison, Madison, Wisconsin 53706

Tóm tắt

ABSTRACT Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae , which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1 , encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.

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Tài liệu tham khảo

10.1016/S0959-437X(05)80325-9

10.1128/mcb.4.12.2876-2882.1984

10.1128/JVI.73.12.10061-10069.1999

10.1073/pnas.92.2.507

10.1128/jvi.71.1.392-397.1997

10.1093/emboj/18.11.3164

Ausubel F. M. Brent R. Kingston R. E. Moore D. D. Seidman J. G. Smith J. A. Struhl K. Current protocols in molecular biology. 1987 John Wiley & Sons New York N.Y

10.1128/jvi.71.11.8482-8489.1997

10.1128/JVI.74.9.4310-4318.2000

10.1128/jvi.70.5.2706-2719.1996

10.1016/0966-842X(94)90342-5

10.1073/pnas.080072997

10.1093/nar/27.16.3310

10.1128/jvi.61.5.1457-1465.1987

10.1016/S0014-5793(99)00321-X

Gorbalenya A. E. Koonin E. V. Helicases: amino acid sequence comparisons and structure-function relationships.Curr. Opin. Cell Biol. 3 1993 419 429

10.1128/JVI.73.11.9196-9205.1999

10.1074/jbc.272.7.4623

10.1006/viro.1996.0125

10.1128/JVI.74.4.1794-1800.2000

10.1002/yea.320020304

10.1128/mcb.13.8.4826-4835.1993

10.1073/pnas.94.25.13810

10.1128/jvi.71.10.7781-7790.1997

10.1128/jb.153.1.163-168.1983

10.1073/pnas.95.5.2227

10.1016/0092-8674(93)90584-D

10.1128/jvi.70.11.8169-8174.1996

10.1006/viro.1999.9763

10.3109/10409239309078440

10.1128/jvi.64.12.6110-6120.1990

10.1128/jvi.68.11.7418-7425.1994

10.1016/0378-1119(90)90417-P

10.1101/gad.5.12a.2303

10.1128/JVI.72.12.10093-10099.1998

10.1016/S0014-5793(99)00856-X

10.1016/0022-2836(86)90332-3

10.1128/MCB.15.4.2145

Newman T. C. Ohme-Takagi M. Taylor C. B. Green P. J. DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobacco.Plant Cell 5 1993 701 714

10.1128/jvi.71.10.7526-7532.1997

10.1128/jvi.65.7.3693-3703.1991

10.1074/jbc.274.11.6992

10.1073/pnas.92.11.4892

10.1128/jvi.70.12.8908-8916.1996

10.1128/JVI.73.12.10303-10309.1999

10.1006/viro.1996.0204

10.1128/jvi.68.9.5804-5810.1994

10.1016/0042-6822(89)90553-9

10.1128/JVI.73.4.2622-2632.1999

10.1128/jvi.64.1.69-77.1990

10.1006/viro.1996.0147

10.1016/0042-6822(82)90261-6

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Journal of Virology

Tập 74 Số 19

8803-8811

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