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Journal of Virology, March 2001, p. 2213-2223, Vol. 75, No. 5
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.5.2213-2223.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Increased Readthrough Transcription across the Simian Virus 5 M-F Gene Junction Leads to Growth Defects and a Global Inhibition of Viral mRNA Synthesis

Griffith D. Parks,* Kimberly R. Ward, and John C. Rassadagger

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1064

Received 19 September 2000/Accepted 17 November 2000

Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50- to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ~1.5- to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5' end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1064. Phone: (336) 716-9083. Fax: (336) 716-9928. E-mail: gparks{at}wfubmc.edu.

dagger Present address: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Journal of Virology, March 2001, p. 2213-2223, Vol. 75, No. 5
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.5.2213-2223.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.