Cellular Splicing Factor RAF-2p48/NPI-5/BAT1/UAP56 Interacts with the Influenza Virus Nucleoprotein and Enhances Viral RNA Synthesis

Abstract

Previous biochemical data identified a host cell fraction, designated RAF-2, which stimulated influenza virus RNA synthesis. A 48-kDa polypeptide (RAF-2p48), a cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases previously designated BAT1 (also UAP56), has now been identified as essential for RAF-2 stimulatory activity. Additionally, RAF-2p48 was independently identified as an influenza virus nucleoprotein (NP)-interacting protein, NPI-5, in a yeast two-hybrid screen of a mammalian cDNA library. In vitro, RAF-2p48 interacted with free NP but not with NP bound to RNA, and the RAF-2p48-NP complex was dissociated following addition of free RNA. Furthermore, RAF-2p48 facilitated formation of the NP-RNA complexes that likely serve as templates for the viral RNA polymerase. RAF-2p48 was shown, in both in vitro binding assays and the yeast two-hybrid system, to bind to the amino-terminal region of NP, a domain essential for RNA binding. Together, these observations suggest that RAF-2p48 facilitates NP-RNA interaction, thus leading to enhanced influenza virus RNA synthesis.The genome of influenza A virus consists of eight singlestranded RNA segments of negative polarity. These viral RNA (vRNA) segments exist as ribonucleoprotein (vRNP) complexes with nucleocapsid proteins (NP) and viral RNA polymerases as components. Each RNA segment contains highly conserved 3Ј-and 5Ј-terminal untranslated regions which function as regulatory signals for transcription and replication of the genome. The partially hybridized terminal regions have been referred to as panhandle (9), fork, hook, and corkscrew (14,15,25,44) forms. In vRNP complexes prepared from purified virions, viral RNA polymerase is found bound to the panhandle region (33), and NP is bound to vRNA such that each NP monomer occupies approximately 20 nucleotides (6, 56).Studies using the vRNP isolated from virions have revealed that viral RNA polymerase and NP are essential for transcription (3,21,24). The viral RNA polymerase consists of PB2, PB1, and PA subunits and is capable of initiating primerdependent RNA synthesis (20, 27). However, for synthesis of full-length RNA, NP is required (21,22). Transcription is initiated by recognition by PB2 of the cap structure of nuclear pre-mRNA. PB2 truncates the capped RNA at 10 to 13 bases downstream from the 5Ј end (3, 42). After the capped oligonucleotide is cleaved, it serves as a primer for viral mRNA synthesis catalyzed by PB1 (17). Elongation of the RNA chain proceeds until the polymerase reaches a polyadenylation signal, consisting of five to seven U residues located near the 5Ј-terminal region of the vRNA (29). The viral RNA polymerase polyadenylates the nascent RNA chain, possibly by a slippage mechanism at the U stretch (43). Replication of vRNA is a primer-independent two-step reaction: first, cRNAs are synthesized from vRNA templates; and second, the progeny vRNAs are amplified from cRNA templates. Genetic analyses suggest that PA participates in the replication process. However, vRNP co...

The influenza virus RNA synthesis machine

“…61,117 The incorporation of NP into new vRNPs is facilitated by the activity of cellular factors UAP56 and Tat-SF1, that may act as chaperones. 118,119 Viral polymerase should also be required stoichiometrically, as the end product is not vRNA but a vRNP. The question remains whether the polymerase complex associated to the progeny vRNP is identical or distinct to the polymerase that actually performs the replication reaction.…”

Oligomerization and GTP-binding Requirements of MxA for Viral Target Recognition and Antiviral Activity against Influenza A Virus

“…We have shown recently that MxA binds to UAP56, a DEAD-box helicase. UAP56 is also an interaction partner of NP and is required for efficient replication of IAV and evasion of the IFN response (22)(23)(24). Therefore, UAP56 may recruit MxA and NP to form a complex involving all three proteins.…”

Influenza A Virus Multiplication and the Cellular SUMOylation System

“…Although UAP56 is clearly a factor in cellular RNA splicing, in vitro studies indicate that UAP56 forms heterodimers with NP in the absence of vRNA. Upon addition of vRNA the heterodimer dissociates and, through an unknown mechanism, facilitates vRNA synthesis [117] suggesting higher affinity interaction of one of these proteins for vRNA. Altogether, this not only suggests a pro-viral functionality of UAP56 in enhancing vRNA production, but that efficient viral replication exploits multiple functionalities of cellular host factors in a well orchestrated manner.…”