Mutation of Capsid Protein Phosphorylation Sites Abolishes Cauliflower Mosaic Virus Infectivity

Abstract

The cauliflower mosaic virus (CaMV) capsid protein is derived by bidirectional processing of the precapsid protein (CP56). We expressed several derivatives of CP56 in Escherichia coli and used them as substrates for virus-associated kinase and casein kinase II purified from plant cells. Three serine residues located at the N terminus of the mature viral protein CP44 were identified as phosphorylation targets. A mutation of one of them in the viral context had little or no effect on viral infectivity, but a mutation of all three serines abolished infectivity. The mapping of phosphorylation sites in CP44, but not CP39 or CP37, and immunodetection of the Zn finger motif in CP44 and CP39, but not CP37, support the model that CP39 is produced from CP44 by N-terminal processing and CP37 is produced from CP39 by C-terminal processing. We discuss the possible role of phosphorylation in the processing and assembly of CaMV capsid protein.The structural proteins of retroviruses and pararetroviruses are modified by proteolysis, phosphorylation (5, 6), ubiquitination (23, 18), glycosylation (3, 19), meristylation, and other reactions. In many cases, these modifications have been shown to control assembly, genome packaging and release, and intracellular transport and distribution (2). The best studied of the plant pararetroviruses is cauliflower mosaic virus (CaMV). Its precapsid protein (CP56) is 489 amino acids long (Fig. 1A). Several processing steps lead to three main capsid protein species, CP44, CP39, and CP37 (named after their respective mobilities, in kilodaltons, on sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis [PAGE]), that share the same central sequence but differ in their termini (1,4,16). This is in contrast to the structural proteins of retroviruses, which represent different, nonoverlapping domains (MA, CA, and NC) of the precursor Gag protein, or to the hepatitis B virus (HBV) C protein, which is not cleaved.CP44, but not CP39 or CP37, contains an acidic N-terminal sequence, which also includes serine, threonine, and proline residues, a combination that frequently renders proteins unstable (12). The acidic domain is followed by a short nuclear localization signal (13), an assembly domain (1), and a large cluster of basic amino acids involved in nucleic acid binding (1, 4), including a Zn finger motif specifying interaction with a packaging signal located on the CaMV RNA leader sequence (7). CP44 starts with amino acid 77 of the precursor protein (16); however, neither the N termini of CP39 and CP37 nor the C termini of any of these proteins are precisely known. Additional domains characterized by acidic residues, serine, and threonine and leading to protein instability make up the N and C termini of CP56 (12).A virion-associated enzyme (17) with casein kinase II (CK II) properties (16) phosphorylates CP44, but not CP39 or CP37. In the experiments leading to these results, substrates and enzymatic activity in the same preparation were evaluated. In the present study, CaMV CP derivatives were...

Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots

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Architecture and Assembly of Virus Particles

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