HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases

Abstract

Nonsegmented negative-stranded RNA viruses, or members of the order Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have coevolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes the L protein through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-dimethylaminoethylamino-17-demethoxygeldanamycin as specific inhibitors of cellular heat shock protein 90 (HSP90), we found that efficient chaperoning of L by HSP90 requires P in the measles, Nipah, and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L, as evidenced by a luciferase reporter insert fused within measles virus L. HSP90 acts as a true chaperon; its activity is transient and dispensable for the activity of measles and Nipah virus polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order. IMPORTANCEViruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus, successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, while P protein binds to the N terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded, and functional L proteins. Once formed, the mature P؉L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved in several members of the Mononegavirales order, including the Nipah and vesicular stomatitis viruses. Viruses with a nonsegmented negative-stranded RNA genome, or members of the order Mononegavirales, share a common and highly conserved genomic organization and replication machinery that are unique in the living world. Indeed, the L protein, or polymerase, which is endowed with all of the enzymatic activities required for the synthesis of RNA and the capping and polyadenylation of viral transcripts, cannot use naked viral genomic RNA in a processive way (1). Instead, L associates with the phosphoprotein (P) to dynamically anchor the polymerase complex to the nucleocapsid and/or to enable...

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“…We next analyzed whether the involvement of Hsp70 and CHIP in the maturation and degradation of the L protein was common among other paramyxoviruses. Consistent with a previous study (12), we found that the MeV L protein was also degraded by 17-AAG (Fig. 9A).…”

“…The data showed that the amount of the L protein in the soluble fraction was increased when the L and P proteins were expressed together ( Fig. 4C and D), suggesting that the MuV P protein also functions as a chaperone for the MuV L protein, as shown for other paramyxoviruses (10)(11)(12). Therefore, both Hsp90 and the P protein contributed to the stability of the L protein.…”

“…Previous studies of paramyxoviruses have shown that the P protein is required for proper folding of the L protein and prevention of L-protein aggregation (10)(11)(12). In the present study, therefore, we used expression plasmids to express the MuV L protein alone or together with the MuV P protein.…”

“…Hsp90 plays a role in the regulation of viral polymerase activity, rather than protein folding, during herpes simplex virus (27) and hepatitis B virus infections (28,29) and is important for proper cleavage of the newly synthesized hepatitis C virus NS2/3 protein (30) and for RNP assembly of influenza virus (31,32). In the cases of mononegavirus infections, the L protein has been identified as a client protein of Hsp90, and its stability and maturation are dependent on the Hsp90 chaperone machinery (12,(33)(34)(35)(36).…”

“…Recent studies have shown that the MuV P protein directly interacts with the N-terminal core region of the N protein so that the L protein locates in close proximity to the NC (8,9). In addition, the P protein of paramyxoviruses, such as Sendai virus, human parainfluenza virus (HPIV), measles virus (MeV), and Nipah virus (NiV), acts as a chaperone, which prevents the L protein from aggregation and ensures the proper RdRp formation (10)(11)(12). A similar chaperone-like function of the P protein has been reported for the N proteins of several mononegaviruses (13,14).…”