Site-specific phosphorylation regulates the transcriptive activity of vesicular stomatitis virus NS protein

Abstract

In vitro transcription by vesicular stomatitis virus nucleocapsids is inhibited by enzymatic dephosphorylation of the NS protein. We provide evidence that specific, partial dephosphorylation of NS molecules is the only detectable change in nucleocapsids treated with bacterial alkaline phosphatase under conditions that prevent the action of adventitious protease. Dephosphorylation appeared to affect only the rate of transcription; there were no changes in sedimentation rates of transcripts. To identify the sites of phosphorylation required for NS activity in transcription, we examined phosphopeptides produced by chymotrypsin digestion of the two electrophoretic classes of NS molecules found in virions and infected cells. The electrophoretically slower class, NS1, abundant in the intracellular soluble pool, has a lower activity in transcription; it contained six chymotryptic phosphopeptides. Five of these peptides contained both phosphoserine and phosphothreonine, indicating that this peptide cluster represents at least 11 separate sites of phosphorylation. In the electrophoretically faster nucleocapsid-associated NS2 class of molecules, which support a higher rate of transcription, another group of eight phosphopeptides was superimposed on this pattern. Two of these peptides contained both phosphoserine and phosphothreonine, so this cluster of peptides represents at least 10 additional phosphorylation sites. These sites were especially sensitive to dephosphorylation by bacterial alkaline phosphatase. One or more of them appears to be responsible for the higher transcription rates medicated by NS2 molecules.

Vesicular stomatitis virus NS proteins: structural similarity without extensive sequence homology

“…Except for a wide difference in the content of alanine, cysteine, and histidine residues, many of the remaining 20 amino acids are present in similar mole percent in both proteins. Also, the numbers of serine and threonine residues, the presumptive phosphorylation sites, in each polypeptide (12,17) as well as the deduced molecular weights are very similar. Interestingly, 7 out of a total of 30 serine residues of NS(NJ) are positioned in the same place as in NS(IND).…”

“…Dideoxy sequencing was carried out by the method of Sanger et al (22) with the 53-base-pair restriction fragment of cloned DNA as primer, total VSV mRNA as template, and reverse transcriptase. The ratio of ddNTP to dNTP was 15 to 60:40, 15 to 30:40, 12 to 48:40, and 0.9 to 3.6:10 for T, G, A, and C, respectively, and [co-32PJdCTP was the labeled precursor.…”

“…It has been well established that both L and NS proteins are required to transcribe the N-RNA complex in vitro into five monocistronic mRNAs and a leader RNA (47 bases) in a sequential manner, 3'-N-NS-M-G-L-5' (1). Although the precise functions of L and NS proteins in the transcription process have not been fully established, different phosphorylated states of the NS protein have been shown to play a role in the transcription process in vitro (12,13). Recently, it has been shown that L protein interacts with the N-RNA template to initiate RNA chains and that the NS protein plays a role in chain elongation (5).…”

Resolution of multiple complexes of phosphoprotein NS with nucleocapsid protein N of vesicular stomatitis virus

“…This result suggested that either N or NS protein underwent some irreversible change shortly after its translation, which then prevented it from associating with the other protein. Since NS protein is phosphorylated at multiple sites in a manner that may affect the conformation and function of this molecule (19,24), mixing experiments were also carried out in the presence of either ATP or calf intestinal alkaline phosphatase. However, neither of these conditions increased the amount of complex formation (data not shown).…”

Transcription and replication of rhabdoviruses.

“…Both phosphorylated species contain phosphothreonine and phosphoserine, with the latter being the principal phosphorylated amino acid. Hsu et al (105,107,108) have established that within these NS1 and NS2 species there are subsets of as many as 21 phosphorylated serine and threonine residues. In addition, the NS1 species is more resistant to bacterial alkaline phosphatase than NS2.…”