In vitro assembly of a functional nucleocapsid from the negative-stranded genome RNA of a defective interfering particle of vesicular stomatitis virus.

The genome of human parainfluenza virus type 3 (PIV3) is a single negative-sense RNA strand (vRNA) that is 15,463 nucleotides in length. A cDNA was constructed to encode an 898-nucleotide, internally deleted version of PIV3 vRNA, P1V3-CAT vRNA, in which the viral genes were replaced with the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. The CAT gene was flanked in turn by sequences representing (i) nontranslated sequences of the first and last genes in the PIV3 genome, (ii) PIV3 gene-start and gene-end sequences, which are presumed to be transcription signals, and (iii) 3' extracistronic (leader) and 5' extracistronic (trailer) terminal regions of PIV3 vRNA. A second cDNA was constructed to encode the exact complement of PIV3-CAT vRNA; this positive-sense RNA, P1V3-CAT vcRNA, would correspond to the predicted replicative intermediate of PIV3-CAT vRNA. When synthesized in vitro by runoff transcription with T7 RNA polymerase and transfected separately into PIV3-infected cells, both PIV3-CAT vRNA and vcRNA were rescued with similar efficiencies; that is, they were expressed to yield CAT and were packaged into particles that could be used to infect fresh cells. Rescue of PIV3-CAT vRNA was strictly dependent on complementation by PIV3; PIV3 could not be replaced by respiratory syncytial virus or, unexpectedly, by a bovine strain of PIV3. Passage was blocked by prior incubation with neutralizing monoclonal antibodies specific to the PIV3 attachment protein. Also, during nine serial passages, the expression of CAT by PIV3-CAT vRNA increased more than 3,000-fold. These results indicated that the 3'-terminal 111 nucleotides and the 5'-terminal 115 nucleotides of PIV3 vRNA, which are present in PIV3-CAT vRNA, contained all of the cis-acting RNA sequences required for replication, gene expression, and transmission.

mentioning

confidence: 99%

Rescue of synthetic analogs of genomic RNA and replicative-intermediate RNA of human parainfluenza virus type 3

Dimock

Collins

1993

“…Several in vitro systems were developed in past years to study the requirements for VSV RNA replication (10,11,21,33,39). A permeable-cell system from VSV-infected cells (10) supports transcription and replication of full-length VSV RNAs, which are assembled into nucleocapsid structures. Cell extracts prepared from VSV-infected cells (21,39) also support transcription as well as replication and assembly of both full-length positiveand negative-sense RNAs.…”

mentioning

confidence: 99%

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

Pattnaik

Wertz

1990

127

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 8to 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.

“…RNPs have been reconstituted from naked RNA of vesicular stomatitis virus defective interfering particles by using infected cell extracts as the protein source. These RNPs were then replicated when added back to infected cells (18). With regard to influenza viruses, it was recently reported that naked RNA purified from virus was used to reconstitute RNPs.…”

mentioning

confidence: 99%

Promoter analysis of influenza virus RNA polymerase

Parvin

Palese

Honda

et al. 1989

166

101

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonucleaseprimed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.