Sridhar Byrappa - Academia.edu (original) (raw)
Papers by Sridhar Byrappa
Journal of general virology, 1999
RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphopr... more RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66 % similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side.
RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphopr... more RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66 % similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side. 0001-6071 # 1999 SGM
Careful titration of Vent polymerase activity allows efficient amplification of full-length plasm... more Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotidedirected site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with >90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient. i Corresponding author. E-MAIL kgupta@rpslmc-edu; FAX (312) 226-6020. 404 ~ GENOME RESEARCH 5:404-407 ©1995 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/95 $5.00 Cold Spring Harbor Laboratory Press on March 22, 2015 -Published by genome.cshlp.org Downloaded from PLASMID MUTAGENESIS enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.
Previously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in viri... more Previously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in virions was primarily and constitutively phosphorylated on serine(s) in a single tryptic phosphopeptide TP1. By two-dimensional thin-layer electrophoresis and chromatography analysis of tryptic phosphopeptides of several deletion and point mutants of the P protein, we now show that the sole phosphorylation site in TP1 is serine249. Interestingly, when serine249 was deleted or mutagenized alternate potential serine sites were more heavily phosphorylated. A similar effect was observed when the deletion was very close to serine249 (D208-236). Mutagenesis of proline250 to alanine abrogated phosphorylation at serine249 suggesting that proline250 is essential for the primary phosphorylation of the P protein. Conceivably, serine249 phosphorylation is mediated by a proline-directed protein kinase. This finding is unusual because a majority of the P proteins from other negative-strand RNA viruses have been shown to be phosphorylated primarily by casein kinase II. Our results demonstrate that the P protein has a strong potency to remain phosphorylated. Based on our previous and present results, we suggest that the phosphorylation sites on P are dependent on the accessibility of phosphatases rather than kinases as all potential sites are about equally competent for phosphorylation. We propose that phosphorylation is important for maintaining the structural integrity of the Sendai virus P protein. ᭧
Journal of general virology, 1999
RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphopr... more RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66 % similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side.
RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphopr... more RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66 % similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side. 0001-6071 # 1999 SGM
Careful titration of Vent polymerase activity allows efficient amplification of full-length plasm... more Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotidedirected site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with >90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient. i Corresponding author. E-MAIL kgupta@rpslmc-edu; FAX (312) 226-6020. 404 ~ GENOME RESEARCH 5:404-407 ©1995 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/95 $5.00 Cold Spring Harbor Laboratory Press on March 22, 2015 -Published by genome.cshlp.org Downloaded from PLASMID MUTAGENESIS enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.
Previously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in viri... more Previously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in virions was primarily and constitutively phosphorylated on serine(s) in a single tryptic phosphopeptide TP1. By two-dimensional thin-layer electrophoresis and chromatography analysis of tryptic phosphopeptides of several deletion and point mutants of the P protein, we now show that the sole phosphorylation site in TP1 is serine249. Interestingly, when serine249 was deleted or mutagenized alternate potential serine sites were more heavily phosphorylated. A similar effect was observed when the deletion was very close to serine249 (D208-236). Mutagenesis of proline250 to alanine abrogated phosphorylation at serine249 suggesting that proline250 is essential for the primary phosphorylation of the P protein. Conceivably, serine249 phosphorylation is mediated by a proline-directed protein kinase. This finding is unusual because a majority of the P proteins from other negative-strand RNA viruses have been shown to be phosphorylated primarily by casein kinase II. Our results demonstrate that the P protein has a strong potency to remain phosphorylated. Based on our previous and present results, we suggest that the phosphorylation sites on P are dependent on the accessibility of phosphatases rather than kinases as all potential sites are about equally competent for phosphorylation. We propose that phosphorylation is important for maintaining the structural integrity of the Sendai virus P protein. ᭧