Regulation of Sendai virus transcription: evidence for a single promoter in vivo

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.

Section: Figsupporting

confidence: 65%

Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3

Galinski

Banerjee

1990

“…Dead mice are marked by †. Wild-type 10 8 5/5 NT b 10 7 5/5 3/5 10 6 1/4 1.78 ϫ 10 6 2/5 3.16 ϫ 10 6 10 5 0/5 NT 10 4 0/5 0/5 SeVmSf 10 8 5/5 NT 10 7 5/5 5/5 10 6 5/5 7.94 ϫ 10 4 5/5 7.94 ϫ 10 4 10 5 3/5 3/5 10 4 0/5 0/5…”

Section: Discussionmentioning

confidence: 99%

“…There is only a single promoter at the 3Ј end of the viral RNA polymerase, comprising the P and L proteins (11). By recognizing the short conserved end (E) and restart (S) signals at each gene boundary, the polymerase gives rise to leader RNA and each mRNA (7). There is a trinucleotide intergenic sequence between the E and S signals, which is not transcribed (9,31).…”

mentioning

confidence: 99%

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Sendai Virus Gene Start Signals Are Not Equivalent in Reinitiation Capacity: Moderation at the Fusion Protein Gene

Kato

Kiyotani

Hasan

et al. 1999

In paramyxovirus transcription, viral RNA polymerase synthesizes each monocistronic mRNA by recognizing the gene start (S) and end (E) signals flanking each gene. These signal sequences are well conserved in the virus family; nevertheless, they do exhibit some variations even within a virus species. In Sendai virus (SeV) Z strain, the E signals are identical for all six genes but there are four (N, P/M/HN, F, and L) different S signals with one or two nucleotide variations. The significance of these variations for in vitro and in vivo replication has been unknown. We addressed this issue by SeV reverse genetics. The luciferase gene was placed between the N and P gene so that recombinant SeVs expressed luciferase under the control of each of the four different S signals. The S signal for the F gene was found to drive a lower level of transcription than that of the other three, which exhibited comparable reinitiation capacities. The polar attenuation of SeV transcription thus appeared to be not linear but biphasic. Then, a mutant SeV whose F gene S signal was replaced with that used for the P, M, and HN genes was created, and its replication capability was examined. The mutant produced a larger amount of F protein and downstream gene-encoded proteins and replicated faster than wild-type SeV in cultured cells and in embryonated eggs. Compared with the wild type, the mutant virus also replicated faster in mice and was more virulent, requiring a dose 20 times lower to kill 50% of mice. On the other hand, the unique F start sequence as well as the other start sequences are perfectly conserved in all SeV isolates sequenced to date, including highly virulent fresh isolates as well as egg-adapted strains, with a virulence several magnitudes lower than that of the fresh isolates. This moderation of transcription at the F gene may therefore be relevant to viral fitness in nature.

“…IrTadiation of NDV inactivates infectivity with single-hit kinetics (5,7,32,39) and blocks mRNA transcription (7). Recent irradiation studies have shown that the NDV genes are sequentially transcribed from a single promoter (9), as previously shown for Sendai virus (18) and vesicular stomatitis virus (VSV) (1,2). Genes for the three proteins associated with viral nucleocapsids (6,10,40), and thought to be involved in NDV-specific RNA synthesis, appear widely separated on the genome; those for the NP and P proteins are least sensitive to irradiation and thus closest to the promoter, whereas that for the L protein is as sensitive as infectivity and thus furthest from the promoter (9).…”

mentioning

confidence: 82%

UV irradiation analysis of complementation between, and replication of, RNA-negative temperature-sensitive mutants of Newcastle disease virus

Peeples¹,

Bratt²

1982

Random UV irradiation-induced lesions destroy the infectivity of Newcastle disease virus (NDV) by blocking downstream transcription from the single viral promoter. The nucleocapsid-associated polypeptides most likely to be involved in RNA synthesis are located at the extreme ends of the genome: NP and P are promoter proximal genes, and L is the most distal gene. We attempted to order the two temperature-sensitive (ts) RNA-negative (RNA-) mutant groups of NDV by determining the UV target sizes for the complementing abilities of mutants Al and El. After UV irradiation, El was unable to complement Al, a result compatible with the A mutation lying in the L gene. In contrast, after UV irradiation, Al was able to complement El for both virus production and viral protein synthesis, with a target size most consistent with the E mutation lying in the P gene. UVirradiated virus was unable to replicate as indicated by its absence in the yields of multiply infected cells, either as infectious virus or as particles with complementing activity. After irradiation, ts mutant BlAP, with a non-ts mutation affecting the electrophoretic mobility of the P protein, complemented El in a manner similar to Al, but it did not amplify the expression of AP in infected cells. This too is consistent with irradiated virus being unable to replicate despite the presence of the components needed for replication of El. At high UV doses, Al was able to complement El in a different, UV-resistant manner, probably by direct donation of input polypeptides. Multiplicity reactivation has previously been observed at high-multiplicity infection by UV-irradiated paramyxoviruses. In this case, virions which are noninfectious because they lack a protein component may be activated by a protein from irradiated virions. Peeples, L. L. Rasenas, and M. A. Bratt, submitted for publication). As yet, no definite as-965 on July 7, 2020 by guest