Model Suggesting that Replication of Influenza Virus Is Regulated by Stabilization of Replicative Intermediates

Abstract

The RNA-dependent RNA polymerase of influenza A virus is responsible for both transcription and replication of negative-sense viral RNA. It is thought that a "switching" mechanism regulates the transition between these activities. We demonstrate that, in the presence of preexisting viral RNA polymerase and nucleoprotein (NP), influenza A virus synthesizes both mRNA (transcription) and cRNA (replication) early in infection. We suggest that there may be no switch regulating the initiation of RNA synthesis and present a model suggesting that nascent cRNA is degraded by host cell nucleases unless it is stabilized by newly synthesized viral RNA polymerase and NP.

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

“…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 inactive RdRp and NP prior to viral infection allows the accumulation of cRNA in the presence of cycloheximide, an inhibitor of protein translation (19,20).…”

“…Only vRNAs introduced into the cell by the infecting virions could be detected. However, preexpression of NP, together with a catalytically inactive viral RdRp (containing D445A/D446A mutant PB1) (20), resulted in the linear accumulation of cRNA (Fig. 1B).…”

“…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 inactive RdRp and NP prior to viral infection allows the accumulation of cRNA in the presence of cycloheximide, an inhibitor of protein translation (19,20). The expression of RdRp is absolutely essential for the stabilization of cRNA, while NP, although not essential, greatly increases the amounts of accumulated cRNA.…”

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

“…Because this could be caused by impaired cRNA synthesis and/or rapid degradation of synthesized cRNA, we performed a cRNA stabilization assay 19 . As described by others, cRNA synthesis can be observed by pre-expression of NP and a catalytically inactive polymerase, containing an inactive PB1 subunit 19 before viral infection in the presence of cycloheximide 19 . As shown in Fig.…”

NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome

“…Over the last decade, reconstitution of recombinant vRNP complexes in transfected cells from co-expressed PB1, PB2 and PA polymerase subunits, NP and vRNA, followed by the analysis of reporter gene expression/activity or a direct analysis of accumulating viral RNAs by a primer extension assay, has become the method of choice for studying influenza virus polymerase activity in vivo (Deng et al, 2006;Fodor et al, 2002;Gabriel et al, 2005;Labadie et al, 2007;Mullin et al, 2004;Pleschka et al, 1996;Salomon et al, 2006;Vreede et al, 2004). However, as the RNP reconstitution assay involves only the minimal components required for viral transcription and replication, it might not reflect all of the regulatory events that occur during transcription and replication in virus-infected cells.…”