Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition

Abstract

Negative-sense RNA viruses (NSVs) rely on prepackaged viral RNA-dependent RNA polymerases (RdRp) to replicate and transcribe their viral genomes. Their replication machinery consists of an RdRp bound to viral RNA which is wound around a nucleoprotein (NP) scaffold, forming a viral ribonucleoprotein complex. NSV NP is known to regulate transcription and replication of genomic RNA, however its role in maintaining and protecting the viral genetic material is unknown. Here, we exploited host microRNA expression to target NP of influenza A virus and Sendai virus to ascertain how this would impact genomic levels and the host response to infection. We find that in addition to inducing a drastic decrease in genome replication, the antiviral host response in the absence of NP is dramatically enhanced. Additionally, our data shows that insufficient levels of NP prevent the replication machinery of these NSVs to process full-length genomes, resulting in aberrant replication products which form pathogen-associated molecular patterns in the process. These dynamics facilitate immune recognition by cellular pattern recognition receptors leading to a strong host antiviral response. Moreover, we observe that the consequences of limiting NP levels are universal amongst NSVs including Ebola virus, Lassa virus and Measles virus. Overall, these results provide new insights into viral genome replication of negative-sense RNA viruses and highlight novel avenues towards developing effective antiviral strategies, adjuvants, and/or live-attenuated vaccines. IMPORTANCE Negative-sense RNA viruses comprise some of the most important known human pathogens, including influenza A virus, measles virus and Ebola virus. These viruses possess RNA genomes that are unreadable to the host as they require specific viral RNA dependent RNA polymerases in conjunction with other viral proteins such as nucleoprotein to be replicated and transcribed. As this process generates a significant amount of pathogen-associated molecular patterns, this phylum of viruses can result in a robust induction of the intrinsic host cellular response. To circumvent these defenses, these viruses form tightly regulated ribonucleoprotein replication complexes in order to protect their genomes from detection and to prevent excessive aberrant replication. Here we demonstrate the balance that negative-sense RNA viruses must achieve to both replicate efficiently and to avoid induction of the host defenses.

Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

“…Thus, length would be observed to correlate well with interferon induction in natural populations, even if length was not the underlying causal factor. This does not preclude the role of mvRNAs <125nt in interferon induction, and these incredibly small RNA species likely contribute alongside defective genomes to the observed interferon response ( Velthuis et al, 2018b ; Nilsson-Payant et al, 2021 ).…”

The influenza virus RNA polymerase as an innate immune agonist and antagonist

“…What might be the mechanism behind DVG formation in this case? As mentioned above, elongation defects can be induced experimentally by limiting the availability of NP during viral replication [24,110,111]. Although not experimentally confirmed, the transcriptional defects induced by reduced Pol II binding likely result in lower NP levels, which subsequently promote aberrant polymerase activity [24,110,111].…”

“…Several viral factors have been associated with the formation of aberrant RNAs. Recent studies have shown that elongation defects can be induced experimentally by limiting the availability of NP, an important elongation factor and key component of RNPs, suggesting that impaired elongation or aberrant encapsidation play a role in DVG or mvRNA formation [24,110,111]. In addition, mutations in several viral proteins, such as nuclear export protein, matrix protein 1 and 2, and the RNA polymerase subunits, also promote the formation of DVGs [34,[112][113][114].…”

Influenza Virus RNA Synthesis and the Innate Immune Response

“…Another trigger for aberrant RNA synthesis and, in particular, mvRNA synthesis appears to be limiting NP levels, as shown by experiments that either overexpressed the viral RNA polymerase subunits or knocked down NP expression [ 21 , 72 , 73 ]. This suggests that an incompletely encapsidated RNA template (aberrant RNP) or an interrupted encapsidation process (limiting free NP) may lead to impaired RNA polymerase processivity and/or a higher propensity to realign product and template, and the generation of an aberrant RNA.…”

“…Theoretically, this could happen when cells are infected with a high MOI, when RNA polymerase mutations reduce Pol II binding or cap-snatching, when mutations increase viral replication, or when viral RNA polymerase or NP levels are not sufficient to complete encapsidation of nascent vRNA or cRNAs. Interestingly, recent studies have indeed shown that limiting NP levels leads to the formation of aberrant viral RNAs and RIG-I activation during IAV infection as well as other negative-sense RNA virus infections [ 21 , 72 , 73 ]. Moreover, in vivo studies by Tapia et al confirmed that DI-rich influenza viruses are associated with stronger IFN expression in lungs of infected mice [ 114 ].…”