Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs

Asit K Pattnaik, Gail W. Wertz

Research output: Contribution to journalArticle

98 Citations (Scopus)

Abstract

Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8- to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.

Original languageEnglish (US)
Pages (from-to)2948-2957
Number of pages10
JournalJournal of virology
Volume64
Issue number6
StatePublished - Dec 1 1990

Fingerprint

Defective Viruses
Vesiculovirus
Vesicular Stomatitis
viral proteins
Viral Proteins
Complementary DNA
RNA
Viruses
cells
proteins
Proteins
transfection
DNA-directed RNA polymerase
Transfection
Plasmids
plasmids
Nucleocapsid Proteins
nucleocapsid proteins
Vaccinia virus
Viral Genome

ASJC Scopus subject areas

  • Immunology

Cite this

@article{83fa31190bfc41928c546dd9830abe31,
title = "Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs",
abstract = "Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8- to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.",
author = "Pattnaik, {Asit K} and Wertz, {Gail W.}",
year = "1990",
month = "12",
day = "1",
language = "English (US)",
volume = "64",
pages = "2948--2957",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "6",

}

TY - JOUR

T1 - Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs

AU - Pattnaik, Asit K

AU - Wertz, Gail W.

PY - 1990/12/1

Y1 - 1990/12/1

N2 - Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8- to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.

AB - Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8- to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.

UR - http://www.scopus.com/inward/record.url?scp=0025311446&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025311446&partnerID=8YFLogxK

M3 - Article

VL - 64

SP - 2948

EP - 2957

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 6

ER -