ABSTRACT
Influenza A virus controls replication and transcription of its genome through the tight regulation of interaction between the ribonucleoprotein (RNP) complex subunits. The helical scaffold of RNP is maintained by nucleoprotein (NP). Previous studies have revealed that NP interacts with both PB2 N-terminal and C-terminal regions, with both regions sharing similar affinity to NP as revealed in co-immunoprecipitation assay. Our work here suggests that the interaction between NP and PB2 N-terminal region lies in the cap-binding domain (residue 320-483). By co-immunoprecipitation assay, the interaction was found to involve RNA. On the other hand, the cap-binding activity was not essential in the interaction. As shown by the NHS pull-down assay, a specific RNA sequence was not required. Among the cap-binding domain, residues K331 and R332 of PB2 play a role in RNP function so that polymerase activity was reduced when these residues were mutated, while K331 was found to be more crucial in the NP interaction. Collectively, our findings suggest a new binding mode between NP and PB2 which was mediated by RNA, and such interaction may provide a novel interacting site for influenza drug development.
Subject(s)
Host Microbial Interactions/genetics , Protein Domains , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , RNA-Dependent RNA Polymerase/metabolism , Viral Core Proteins/metabolism , Viral Proteins/metabolism , Binding Sites , HEK293 Cells , Humans , Immunoprecipitation , Nucleocapsid Proteins , Plasmids/genetics , Protein Binding/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/genetics , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Transfection , Viral Core Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Replication/geneticsABSTRACT
The influenza virus RNA genome is transcribed and replicated in the context of the viral ribonucleoprotein (vRNP) complex by the viral RNA polymerase. The nucleoprotein (NP) is the structural component of the vRNP providing a scaffold for the viral RNA. In the vRNP as well as during transcription and replication the viral polymerase interacts with NP but it is unclear which parts of the polymerase and NP mediate these interactions. Previously the C-terminal '627' domain (amino acids 538-693) of PB2 was shown to interact with NP. Here we report that a fragment encompassing amino acids 146-185 of NP is sufficient to mediate this interaction. Using NMR chemical shift perturbation assays we show that amino acid region 601 to 607 of the PB2 '627' domain interacts with this fragment of NP. Substitutions of these PB2 amino acids resulted in diminished RNP activity and surface plasmon resonance assays showed that amino acids D605 was essential for the interaction with NP and V606 may also play a partial role in the interaction. Collectively these results reveal a possible interaction surface between NP and the PB2 subunit of the RNA polymerase complex.
Subject(s)
Influenza A Virus, H5N1 Subtype/chemistry , Influenza A Virus, H5N1 Subtype/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Viral Core Proteins/chemistry , Viral Core Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Amino Acid Substitution , Aspartic Acid/chemistry , Genome, Viral , HEK293 Cells , Humans , Influenza A Virus, H5N1 Subtype/physiology , Influenza, Human/virology , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nucleocapsid Proteins , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Surface Plasmon Resonance , Valine/chemistry , Virus ReplicationABSTRACT
SRPKs (serine/arginine protein kinases) are highly specific kinases that recognize and phosphorylate RS (Arg-Ser) dipeptide repeats. It has been shown previously that SRPK1 phosphorylates the RS domain of SRSF1 (serine/arginine splicing factor 1) at multiple sites using a directional and processive mechanism. Such ability to processively phosphorylate substrates is proposed to be an inherent characteristic of SRPKs. SRPK2 is highly related to SRPK1 in sequence and in vitro properties, yet it has been shown to have distinct substrate specificity and physiological function in vivo. To study the molecular basis for substrate specificity of SRPK2, we investigated the roles of the non-kinase regions and a conserved docking groove of SRPK2 in the recognition and phosphorylation of different substrates: SRSF1 and acinusS. Our results reveal that a conserved electronegative docking groove in SRPK2, but not its non-kinase regions, is responsible for substrate binding regardless of their identities. Although SRPK2 phosphorylates SRSF1 in a processive manner as predicted, an electronegative region on acinusS restricts SRPK2 phosphorylation to a single specific site despite the presence of multiple RS dipeptides. These results suggest that primary structural elements on the substrates serve as key regulatory roles in determining the phosphorylation mechanism of SRPK2.
