An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis

Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides.

[1]  D. Baltimore,et al.  Defective Viral Particles and Viral Disease Processes , 1970, Nature.

[2]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[3]  E. Wimmer,et al.  The 5' end of poliovirus mRNA is not capped with m7G(5')ppp(5')Np. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Baltimore,et al.  5'-terminal structure of poliovirus polyribosomal RNA is pUp. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[5]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[6]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[7]  B. Semler,et al.  Primary structure, gene organization and polypeptide expression of poliovirus RNA , 1981, Nature.

[8]  D. Baltimore,et al.  Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Baltimore,et al.  Cloned poliovirus complementary DNA is infectious in mammalian cells. , 1981, Science.

[10]  N. Sonenberg,et al.  Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex. , 1982, The Journal of biological chemistry.

[11]  E. Wimmer,et al.  Identification of the initiation site of poliovirus polyprotein synthesis , 1982, Journal of virology.

[12]  H. Luthman,et al.  High efficiency polyoma DNA transfection of chloroquine treated cells. , 1983, Nucleic acids research.

[13]  D. Melton,et al.  Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. , 1984, Nucleic acids research.

[14]  A. Nomoto,et al.  Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants. , 1984, Journal of molecular biology.

[15]  J. Maizel,et al.  Increased neurovirulence associated with a single nucleotide change in a noncoding region of the Sabin type 3 poliovaccine genome , 1985, Nature.

[16]  V. Agol,et al.  The genomes of attenuated and virulent poliovirus strains differ in their in vitro translation efficiencies. , 1985, Virology.

[17]  D. Baltimore,et al.  Poliovirus mutant that does not selectively inhibit host cell protein synthesis , 1985, Molecular and cellular biology.

[18]  D. Baltimore,et al.  A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[19]  V. Racaniello,et al.  Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA. , 1986, Virology.

[20]  D. Baltimore,et al.  Genetic complementation among poliovirus mutants derived from an infectious cDNA clone , 1986, Journal of virology.

[21]  B. Semler,et al.  A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[22]  S. Kuge,et al.  Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5' noncoding sequence in viral replication , 1987, Journal of virology.

[23]  N. Sonenberg,et al.  Proteolysis of the p220 component of the cap-binding protein complex is not sufficient for complete inhibition of host cell protein synthesis after poliovirus infection , 1987, Journal of virology.

[24]  N. Sonenberg.  Regulation of translation by poliovirus. , 1987, Advances in virus research.

[25]  N. Sonenberg,et al.  Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region , 1988, Molecular and cellular biology.