Modulation of IAV polymerase activity is a contested issue, with many seemingly disparate mechanisms having been proposed as to how the virus transitions from primary transcription to genome replication. Here, we demonstrate that IAV-derived svRNAs function to promote replicase activity and template elongation in a segment-specific manner by interacting with the RNA binding cleft within the PA subunit, allowing the polymerase to maintain the stoichiometric balance of all eight genomic segments. This function, as an allosteric small RNA enhancer, sets svRNAs apart from other classes of small regulatory RNAs. While its activity is somewhat reminiscent of that of microRNA (miRNA) miR-122 for hepatitis C virus (HCV), whereby miR-122 directly interacts with the 5′ untranslated region (UTR) of HCV to promote translation and viral replication, svRNAs are produced from the viral genome and directly interact with the viral polymerase (20
). Apart from miRNAs, svRNAs could be, and indeed have been, compared to the leader RNAs of paramyxoviruses and rhabdoviruses (49
). However, this also seems inappropriate as leader RNAs have not been found to directly associate with RdRps and generally exist for both the 5′ and 3′ ends of the viral RNA genome (3
). As svRNAs have been found to directly interact with the IAV RdRp and do not require incorporation into nascent vRNAs for activity, they are unlikely to function as leader RNAs and most likely behave as segment-specific enhancers.
Given that the cRNA intermediate could serve as a template for svRNA synthesis, it is likely that svRNA production relies solely on viral proteins. While the roles of PB2, PB1, and PA could be extrapolated from their known viral functions, the need for NP and NEP was less clear. The dependence on NP may be a result of needing a functional polymerase with the ability to elongate products (18
); likewise, as svRNA is approximately the same length as an RNA molecule wrapped over an NP monomer, it is also possible that NP serves as a size marker for generation of svRNA of the appropriate length (26
). In addition, NEP has been ascribed a number of distinct functions, the primary activities being nuclear export of progeny vRNPs, the enhancement of cRNA synthesis, and, most recently, determining host tropism (27
). As dose dependency and truncation analysis demonstrated a correlation between svRNA production and nuclear export-independent cRNA synthesis, we conclude that the NEP requirement is the result of inefficient cRNA concentrations to template svRNA production.
Following cRNA-dependent svRNA synthesis, this small regulatory RNA loads into the RdRp to promote replicase activity. Truncation analysis and available structural information for the PA/PB1 heterodimer revealed a putative RNA binding cleft, which could accommodate svRNA. Subsequent mutation of these key basic residues abrogated binding to synthetic svRNA, demonstrating the crucial role this RNA binding cleft plays in mediating polymerase-svRNA interactions. As neither the 5′ triphosphate nor nucleotides beyond position 13 were required for enhancing polymerase activity, it is likely that the sequence and structure of the 5′ end of svRNA are sufficient to mediate its function. While sequence-specific interactions between proteins and dsRNAs are rare, given the uniform face of the minor groove and the depth of the major groove, the binding of the corkscrew structure by the RdRp has been proposed to be sequence specific, with interactions occurring at the ACAA at positions 8 to 11 (44
). This suggests that the interactions between the PA RNA binding cleft and svRNA involve both sequence and structural elements, which may alter the overall conformation of the polymerase. Furthermore, as the PA RNA binding cleft mutants were incapable of both transcription and replication, it is possible that substrate-specific occupancy may determine polymerase activity. Interactions between the host 5′ cap and the cleft may promote viral transcription, whereas interactions with svRNA promote replication. This is in concordance with work demonstrating that changes in the concentrations of both the 5′ host cap and 5′ vRNA end have the ability to bias polymerase activity to transcription and replication, respectively (28
Loading of svRNA into the PA RNA binding cleft likely modifies the polymerase to become replication competent, promoting full-length vRNA synthesis from the cRNA intermediate. While only trace amounts of vRNA synthesis can be detected in the absence of svRNA, administration of various synthetic svRNAs demonstrates enhancement of full-length vRNA synthesis. As these in vitro
reactions lack recombinant NP, it is not surprising that de novo
svRNA synthesis is inhibited, which relies on NP for proper production. Therefore, synthetic svRNAs must be supplied in order to detect vRNA synthesis from the cRNA template. This is in stark contrast to the RdRp reconstitution assays, in which basal levels of svRNA production may be sufficient to mediate vRNA synthesis in the absence of NEP. In an NP-free system, svRNA is capable of promoting full-length vRNA synthesis, suggesting that it may function to enhance the processivity of the RdRp. While NP has been characterized to promote polymerase elongation (18
), it does so in a non-segment-specific manner, whereas svRNA can promote processivity specifically for its cognate cRNP, ensuring proper vRNA synthesis for a given RNP. Although the short synthetic svRNA is capable of promoting full-length vRNA synthesis in vitro
, it may not be beneficial to the virus to utilize such a promiscuous regulatory system in vivo
As in vitro analysis demonstrated that svRNAs were capable of promoting full-length vRNA synthesis by the RdRp, we next wanted to investigate the contribution of svRNA to viral infection. To ascertain this, we engineered an IAV strain incapable of generating svRNA for a single genomic segment. Loss of the ability to produce svRNA from the viral promoter impaired vRNA synthesis in a segment-specific manner, such that the lack of NA svRNA resulted in a loss of NA vRNA without impacting the other viral segments. These data suggest that in addition to regulating vRNA synthesis by the polymerase, svRNA also functions to maintain the stoichiometric balance of each of the eight segments. In this way the IAV polymerase can produce equivalent amounts of each genomic segment to ensure that all eight vRNAs can be packaged into progeny virions.
Here, we propose a model in which PA is loaded with svRNA to convert the RdRp from a transcriptase into a replicase. Upon infection, the incoming polymerase, unoccupied by svRNA but associated with the vRNA promoter, acts predominantly as a transcriptase, utilizing host mRNAs as primers for synthesis of capped viral transcripts. As viral transcripts of NEP accumulate, cRNA is synthesized in what has been proposed to be a stochastic manner (27
), with newly synthesized RdRp serving to stabilize the genomic intermediate (). Nascent cRNA provides a template for svRNA synthesis which, once synthesized, associates with the basic cleft of PA (). svRNA-loaded PA then allows the RdRp to interact with the cognate cRNA template and synthesize full-length vRNA (). In this model, svRNA loading would generate segment-specific replication-competent RdRp populations as the 3′ complementarity of each svRNA would be distinct for a cRNA template. This would ensure the maintenance of the stoichiometric balance between each genomic segment as one RdRp complex would be devoted to the replication of one genomic segment. Taking these observations together, we have demonstrated that svRNA interacts with the RNA binding cleft within PA to promote full-length synthesis of vRNA in an allosteric manner, switching the polymerase from a transcriptase to a replicase. This finding is the first example of a small RNA capable of controlling RNA-dependent RNA polymerase activity and suggests that svRNA is a master regulator of IAV replication.
Fig 7 Schematic representation of IAV polymerase activity and svRNA-induced vRNA synthesis. The IAV polymerase enters the host nucleus tethered to the double-stranded ends of each negative-sense viral RNA (vRNA) segment. cRNA, the full-length copy of the vRNA, (more ...)