Involvement of Hsp90 in Assembly and Nuclear Import of Influenza Virus RNA Polymerase Subunits

Abstract

Transcription and replication of the influenza virus RNA genome occur in the nuclei of infected cells through the viral RNA-dependent RNA polymerase consisting of PB1, PB2, and PA. We previously identified a host factor designated RAF-1 (RNA polymerase activating factor 1) that stimulates viral RNA synthesis. RAF-1 is found to be identical to Hsp90. Here, we examined the intracellular localization of Hsp90 and viral RNA polymerase subunits and their molecular interaction. Hsp90 was found to interact with PB2 and PB1, and it was relocalized to the nucleus upon viral infection. We found that the nuclear transport of Hsp90 occurs in cells expressing PB2 alone. The nuclear transport of Hsp90 was in parallel with that of the viral RNA polymerase binary complexes, either PB1 and PB2 or PB1 and PA, as well as with that of PB2 alone. Hsp90 also interacted with the binary RNA polymerase complex PB1-PB2, and it was dissociated from the PB1-PB2 complex upon its association with PA. Furthermore, Hsp90 could form a stable PB1-PB2-Hsp90 complex prior to the formation of a ternary polymerase complex by the assembly of PA in the infected cells. These results suggest that Hsp90 is involved in the assembly and nuclear transport of viral RNA polymerase subunits, possibly as a molecular chaperone for the polymerase subunits prior to the formation of a mature ternary polymerase complex.The influenza A virus contains eight segmented and negative-stranded RNAs as its genome. The viral RNAs (vRNA) are associated with the viral RNA-dependent RNA polymerase subunits (PB1, PB2, and PA) and nucleoprotein (NP), forming structurally distinct viral ribonucleoprotein (vRNP) complexes (36). The vRNP complex is a basic unit for active transcription and replication. Transcription and replication of vRNA occur in the nuclei of infected cells. The PB1 subunit plays a central role in the catalysis of the polymerization of the RNA chain. It contains amino acid motifs that are common to RNA-dependent RNA polymerases and RNA-dependent DNA polymerases (2). The PB2 subunit is required for the transcription of vRNA. It binds to the methylated cap-1 structure of host RNAs, and the capped oligonucleotide RNA is endonucleolytically cleaved by the PB1 subunits (8, 15). The resultant 10-to 13-nucleotide-long capped RNA fragment serves as a primer for viral mRNA synthesis. Genetic analyses suggest that the PA subunit is required for vRNA replication (14). The PA subunit induces a generalized proteolytic process (23, 34), and it is involved in the assembly of the polymerase subunits (13).In negative-strand RNA viruses, RNA-dependent RNA polymerases are present in the virion. Purified vRNP complexes or RNA polymerases catalyze transcription from vRNA in vitro; however, the vRNP complexes alone are not sufficient for genome replication or for the efficient transcription of viral RNAs. Some of the paramyxoviruses and rhabdoviruses have been shown to require host factors for efficient RNA synthesis in vitro. Tubulin is involved in the transcription of vesicular stom...

The influenza virus RNA synthesis machine

“…Thus, it has been suggested that PB1 and PA form a heterodimeric complex that is transported to the nucleus by association to RanBP5, 77,78 but other studies suggested a role for Hsp90 in the transport of PB1-PB2 and PB1-PA heterodimers. 79 Recently, studies of crosscorrelation fluorescence spectroscopy have supported the formation of PB1-PA heterodimers in the cytoplasm and their transport to the nucleus independently of the PB2 subunit. 80 In addition, mutation analyses of the PB2 NLS indicated that the transport of PB2 to the nucleus and the formation of a functional polymerase complex are closely correlated events 14 and represent host-regulated steps in the virus multiplication cycle.…”

Oligomerization of the influenza virus polymerase complex in vivo

“…On the contrary, replication of viral RNPs does not require capped primers and occurs by the synthesis of a complementary RNA (cRNA) intermediate, which is a complete copy of the virion RNA (vRNA) (Hay et al, 1982), and serves as template for the generation of progeny vRNAs. Both vRNAs and cRNAs are encapsidated into RNP structures during replication (reviewed by Elton et al, 2005).In addition to the RNP-associated polymerase, soluble polymerase complexes have been detected in infected cells (Detjen et al, 1987) and the interaction of cellular proteins with the polymerase complex has been described (Deng et al, 2006;Mayer et al, 2007;Momose et al, 2002;Naito et al, 2007) (N. Jorba and others, unpublished data). To characterize these soluble intracellular polymerase complexes we have addressed their expression and purification from human cells.…”

“…In addition to the RNP-associated polymerase, soluble polymerase complexes have been detected in infected cells (Detjen et al, 1987) and the interaction of cellular proteins with the polymerase complex has been described (Deng et al, 2006;Mayer et al, 2007;Momose et al, 2002;Naito et al, 2007) (N. Jorba and others, unpublished data). To characterize these soluble intracellular polymerase complexes we have addressed their expression and purification from human cells.…”

Antiviral evaluation of an Hsp90 inhibitor, gedunin, against dengue virus

“…Hsp90 has been believed to facilitate viral protein folding and activity of non-structural proteins such as polymerase, protease and helicase as well as the structural proteins [3]. The polymerase of several viruses require Hsp90 for genome replication which include influenza virus A [12], herpes simplex virus [26], flock house virus [27] and vesicular stomatitis virus and treatment with Hsp90 inhibitors such as geldanamycin and 17-AAG lead to degradation of polymerase complexes [28,22].…”

“…Other studies have suggested that Hsp90 also binds to the viral components mainly to the RNA polymerase affecting the assembly and nuclear transport of polymerase [12]. Researchers have found a tetranortriterpenoid compound called gedunin, obtained from the Indian neem tree (Azadirachta indica), to be active against the 90 kDa Hsp90 by binding directly to its helper protein p23 leading to its inactivation [13,14].…”