Stabilization of Influenza Virus Replication Intermediates Is Dependent on the RNA-Binding but Not the Homo-Oligomerization Activity of the Viral Nucleoprotein

Abstract

The influenza virus nucleoprotein (NP) is believed to play a central role in directing a switch from RNA genome transcription to replication by the viral RNA polymerase. However, this role has recently been disputed with the proposal of alternative regulatory mechanisms. It has been suggested that the expression of viral polymerase and NP allows genome replication by stabilization of cRNA replication intermediates and complementary ribonucleoprotein (cRNP) assembly. Here, we demonstrate that the RNA-binding activity of NP is necessary for stabilization of cRNA, whereas, surprisingly, homo-oligomerization of NP is not essential. However, both RNA binding and homo-oligomerization activities are essential for genome replication.Influenza virus, a member of the Orthomyxoviridae family, contains a segmented, negative-sense, single-stranded RNA (ssRNA) genome. Each viral RNA (vRNA) segment is bound by the viral RNA-dependent RNA polymerase (RdRp) and nucleoprotein (NP) to form viral ribonucleoprotein (vRNP) complexes. Within the vRNP, the RdRp binds to the corkscrew structure formed by the partially complementary conserved 5Ј and 3Ј ends of the vRNA representing the vRNA promoter. The rest of the vRNA interacts with multiple copies of NP, each molecule covering approximately 24 nucleotides (reviewed in reference 17). NP binds ssRNA with high affinity but little or no sequence specificity (2, 9, 22) and has been shown to homo-oligomerize and interact with the PB1 and PB2 subunits of the viral RdRp (4,8,14,15,23). Although cryoelectron microscopy reconstitution images of a mini-RNP containing nine NP molecules have been obtained (6), a detailed high-resolution structure of the vRNP is still lacking.During the viral life cycle, the vRNA is transcribed into mRNA and replicated through a cRNA intermediate into more copies of vRNA by the viral RdRp (reviewed in references 10, 17, and 18). Viral mRNAs associate with cellular factors normally associating with host mRNAs, e.g., nuclear and cytoplasmic cap-binding proteins, leading to the stabilization of viral mRNAs and their processing, nuclear export, and translation (3,16,21). In contrast, cRNAs are stabilized by the association of viral factors, i.e., the viral RdRp and NP, which together form a cRNP structure similar to that of vRNPs. During infection, viral mRNAs can be detected initially, while cRNAs become detectable only after the onset of viral protein synthesis (12). This has been interpreted to mean that a switch of polymerase function from a transcriptase to a replicase is required for cRNA synthesis, and various models have been proposed that implicate viral and host factors, as well as small viral RNAs (svRNAs), in the switching (reviewed in reference 17). However, an alternative interpretation is that both mRNA and cRNA are synthesized from early on in a stochastic manner but cRNA is degraded by host nucleases until sufficient amounts of viral RdRp and NP accumulate to stabilize it (20). In agreement with this interpretation, the overexpression of catalytically...

Modeling the Intracellular Dynamics of Influenza Virus Replication To Understand the Control of Viral RNA Synthesis

“…Consequently, viral genome replication starts 1.5 to 2 hpi and persists throughout infection, but it levels off from 6 hpi onwards. These dynamics are in general agreement with other experiments using different influenza virus strains and cell lines (15,21,53,62).…”

“…Hence, NP would not induce a switch but merely act as a cofactor during cRNA encap-sidation. This hypothesis is consistent with other observations showing that the relative rate of genome replication is independent of the abundance of NP (40), and that the RNA-binding activity of NP is necessary for cRNA accumulation (62). However, definite proof is still lacking.…”

“…In contrast, the accumulation of vRNAs continues unhindered. These dynamics were also observed in several independent experiments (21,31,54,62). Hence, we proposed that M1 proteins regulate viral RNAs during the late phase of infection by interacting with vRNPs and inhibiting positive-strand RNA synthesis.…”

Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus

“…These results suggest that FMRP interacts with NP in an RNA-dependent manner. In support of these observations, the association between FMRP and an RNA-binding-deficient NP mutant 28,29 was substantially disrupted (Fig. 4d), confirming the role of RNAs in mediating the FMRP-NP association.…”

“…corresponding to natural viral RNA promoter region at the 3 0 end. The TAP-tagged or non-tagged pcDNA plasmids encoding PB1, PB2, PA, NP, NP-G1(4) and two viral RNA expressing plasmids pPOLI-NA-RT, pPOLI-HA-RT derived from influenza A/WSN/33 have been described previously 16,29 . Rabbit anti-Lamin A (1:2,000, L1293), rabbit anti-GAPDH (1:4,000, G9545), mouse monoclonal anti-bactin (1:4,000, A5441) and mouse monoclonal anti-Flag M2 (1:4,000, F3165) were purchased from Sigma-Aldrich.…”