Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Jul;71(7):4944–4953. doi: 10.1128/jvi.71.7.4944-4953.1997

Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA.

L Kuo 1, R Fearns 1, P L Collins 1
PMCID: PMC191725  PMID: 9188557

Abstract

The gene start (GS) and gene end (GE) transcription signals of human respiratory syncytial virus (RSV) strain A2 were analyzed in helper-dependent monocistronic and dicistronic minireplicons which were complemented by a standard RSV strain. The GS signal, which is the start site for mRNA synthesis, is highly conserved for the first nine genes: 3'-CCCCGUUUA(U/C) (negative sense). This conserved version of the signal was analyzed by "saturation" mutagenesis, in which all 10 positions, as well as one downstream and one upstream position, were changed one at a time into each of the other three nucleotides. Most of the positions appear to contribute to the signal: positions 1, 3, 6, 7, and, in particular, 9 were the most sensitive, whereas position 5 was relatively insensitive. The effect of nucleotide substitution in the first position of the signal was examined further by cDNA cloning and sequence analysis of the residual mRNA which was produced. For the two mutants examined (1C to U, and 1C to A), the site of initiation was unchanged. However, the mRNAs were dimorphic with regard to the assignment of the 5'-terminal nucleotide: two-thirds contained the predicted mutant substitution, and one-third contained the parental assignment. Intracellular minigenome contained only the mutant assignment, indicating that the heterogeneity was at the level of transcription by the RSV polymerase. This suggests that the templated mutant assignment at position 1 can sometimes be overridden by an innate preference for the parental assignment, a phenomenon which we dubbed quasi-templated initiation. The GS signal of the L gene, encoding the 10th RSV mRNA, contains three differences (3'-CCCUGUUUUA) compared to the conserved version. It was shown to be equal in efficiency to the conserved version. This was unexpected, since the saturation mutagenesis described above indicated that U in place of A at position 9 should be highly inhibitory. Instead, the A at position 10 of the L GS signal was found to be critical for activity, indicating that an essential A residue indeed was present in both versions of the GS signal but that its spacing differed. The GE signal, which directs termination and polyadenylation, has more sequence diversity in nature than does the GS signal. The naturally occurring GE signals of strain A2 were compared by their individual incorporation into a dicistronic minigenome. They were similar in the ability to produce translatable mRNA except in the cases of NS1 and NS2, which were approximately 60% as efficient.

Full Text

The Full Text of this article is available as a PDF (800.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barik S. The structure of the 5' terminal cap of the respiratory syncytial virus mRNA. J Gen Virol. 1993 Mar;74(Pt 3):485–490. doi: 10.1099/0022-1317-74-3-485. [DOI] [PubMed] [Google Scholar]
  2. Bukreyev A., Camargo E., Collins P. L. Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene. J Virol. 1996 Oct;70(10):6634–6641. doi: 10.1128/jvi.70.10.6634-6641.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chambers P., Matthews D. A., Pringle C. R., Easton A. J. The nucleotide sequences of intergenic regions between nine genes of pneumonia virus of mice establish the physical order of these genes in the viral genome. Virus Res. 1991 Mar;18(2-3):263–270. doi: 10.1016/0168-1702(91)90023-o. [DOI] [PubMed] [Google Scholar]
  4. Collins P. L., Dickens L. E., Buckler-White A., Olmsted R. A., Spriggs M. K., Camargo E., Coelingh K. V. Nucleotide sequences for the gene junctions of human respiratory syncytial virus reveal distinctive features of intergenic structure and gene order. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4594–4598. doi: 10.1073/pnas.83.13.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins P. L., Hill M. G., Camargo E., Grosfeld H., Chanock R. M., Murphy B. R. Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11563–11567. doi: 10.1073/pnas.92.25.11563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Collins P. L., Hill M. G., Cristina J., Grosfeld H. Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):81–85. doi: 10.1073/pnas.93.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collins P. L., Huang Y. T., Wertz G. W. Nucleotide sequence of the gene encoding the fusion (F) glycoprotein of human respiratory syncytial virus. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7683–7687. doi: 10.1073/pnas.81.24.7683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Collins P. L., Mink M. A., Stec D. S. Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9663–9667. doi: 10.1073/pnas.88.21.9663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collins P. L., Olmsted R. A., Spriggs M. K., Johnson P. R., Buckler-White A. J. Gene overlap and site-specific attenuation of transcription of the viral polymerase L gene of human respiratory syncytial virus. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5134–5138. doi: 10.1073/pnas.84.15.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Collins P. L., Wertz G. W. cDNA cloning and transcriptional mapping of nine polyadenylylated RNAs encoded by the genome of human respiratory syncytial virus. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3208–3212. doi: 10.1073/pnas.80.11.3208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dickens L. E., Collins P. L., Wertz G. W. Transcriptional mapping of human respiratory syncytial virus. J Virol. 1984 Nov;52(2):364–369. doi: 10.1128/jvi.52.2.364-369.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Garcin D., Kolakofsky D. Tacaribe arenavirus RNA synthesis in vitro is primer dependent and suggests an unusual model for the initiation of genome replication. J Virol. 1992 Mar;66(3):1370–1376. doi: 10.1128/jvi.66.3.1370-1376.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grosfeld H., Hill M. G., Collins P. L. RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA. J Virol. 1995 Sep;69(9):5677–5686. doi: 10.1128/jvi.69.9.5677-5686.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iverson L. E., Rose J. K. Localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription. Cell. 1981 Feb;23(2):477–484. doi: 10.1016/0092-8674(81)90143-4. [DOI] [PubMed] [Google Scholar]
  15. Johnson P. R., Collins P. L. The A and B subgroups of human respiratory syncytial virus: comparison of intergenic and gene-overlap sequences. J Gen Virol. 1988 Nov;69(Pt 11):2901–2906. doi: 10.1099/0022-1317-69-11-2901. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  17. Kuo L., Fearns R., Collins P. L. The structurally diverse intergenic regions of respiratory syncytial virus do not modulate sequential transcription by a dicistronic minigenome. J Virol. 1996 Sep;70(9):6143–6150. doi: 10.1128/jvi.70.9.6143-6150.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kuo L., Grosfeld H., Cristina J., Hill M. G., Collins P. L. Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus. J Virol. 1996 Oct;70(10):6892–6901. doi: 10.1128/jvi.70.10.6892-6901.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Samal S. K., Collins P. L. RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end. J Virol. 1996 Aug;70(8):5075–5082. doi: 10.1128/jvi.70.8.5075-5082.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yu Q., Hardy R. W., Wertz G. W. Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication. J Virol. 1995 Apr;69(4):2412–2419. doi: 10.1128/jvi.69.4.2412-2419.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zamora M., Samal S. K. Gene junction sequences of bovine respiratory syncytial virus. Virus Res. 1992 Jun;24(1):115–121. doi: 10.1016/0168-1702(92)90035-8. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES