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Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase.

ABSTRACT: Mononegavirales is an order of viruses with a genome in the form of a non-segmented negative-strand RNA that encodes several proteins. The functional polymerase complex of these viruses is composed of two proteins: a large protein (L) and a phosphoprotein (P). The replication of viruses from this order depends on Hsp90 chaperone activity. Previous studies have demonstrated that Hsp90 inhibition results in the degradation of mononegaviruses L protein, with exception of the rabies virus, for which the degradation of P protein was observed. Here, we demonstrated that Hsp90 inhibition does not affect the expression of rabies L and P proteins, but it inhibits binding of the P protein and L protein into functional viral polymerase. Rabies and the vesicular stomatitis virus, but not the measles virus, L proteins can be expressed independently of the presence of a P protein and in the presence of an Hsp90 inhibitor. Our results suggest that the interaction of L proteins with P proteins and Hsp90 in the process of polymerase maturation may be a process specific to particular viruses.

SUBMITTER: Dalidowska I

PROVIDER: S-EPMC9266396 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase.

Dalidowska Iga I Orlowska Anna A Smreczak Marcin M Bieganowski Pawel P

International journal of molecular sciences 20220622 13

<i>Mononegavirales</i> is an order of viruses with a genome in the form of a non-segmented negative-strand RNA that encodes several proteins. The functional polymerase complex of these viruses is composed of two proteins: a large protein (L) and a phosphoprotein (P). The replication of viruses from this order depends on Hsp90 chaperone activity. Previous studies have demonstrated that Hsp90 inhibition results in the degradation of mononegaviruses L protein, with exception of the rabies virus, fo ...[more]

PMID: 35805948

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Project description:We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the full-length L protein of RABV and the characterization of its RdRP activity in vitro The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity.

Project description:While the RNA-dependent RNA polymerase L protein of rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, has potential to be a therapeutic target for rabies, the molecular functions of this protein have remained largely unknown. In this study, to obtain a novel experimental tool for molecular function analysis of the RABV L protein, we established by using a reverse genetics approach an L gene-deficient RABV (Nishi-ΔL/Nluc), which infects, propagates, and correspondingly produces NanoLuc luciferase in cultured neuroblastoma cells transfected to express the L protein. trans-Complementation with wild-type L protein, but not that with a functionally defective L protein mutant, efficiently supported luciferase production by Nishi-ΔL/Nluc, confirming its potential for function analysis of the L protein. Based on the findings obtained from comprehensive genetic analyses of L genes from various RABV and other lyssavirus species, we examined the functional importance of a highly conserved L protein region at positions 1914 to 1933 by a trans-complementation assay with Nishi-ΔL/Nluc and a series of L protein mutants. The results revealed that the amino acid sequence at positions 1929 to 1933 (NPYNE) is functionally important, and this was supported by other findings that this sequence is critical for binding of the L protein with its essential cofactor, P protein, and thus also for L protein's RNA polymerase activity. Our findings provide useful information for the development of an anti-RABV drug targeting the L-P protein interaction.IMPORTANCE To the best of our knowledge, this is the first report on the establishment of an L gene-deficient, reporter gene-expressing virus in all species of the order Mononegavirales, also highlighting its applicability to a trans-complementation assay, which is useful for molecular function analyses of their L proteins. Moreover, this study revealed for the first time that the NPYNE sequence at positions 1929 to 1933 in the RABV L protein is important for L protein's interaction with the P protein, consistent with and extending the results of a previous study showing that the P protein-binding domain in the L protein is located in its C-terminal region, at positions 1562 to 2127. This study indicates that the NPYNE sequence is a promising target for the development of an inhibitor of viral RNA synthesis, which has high potential as a therapeutic drug for rabies.

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Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase.

Publications

Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase.

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