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Lassa virus NP DEDDh 3'-5' exoribonuclease activity is required for optimal viral RNA replication.

ABSTRACT: Mammarenavirus include several pathogens that can cause severe and fatal zoonotic diseases in humans. Lassa virus (LASV) is the causative agent for Lassa fever (LF) currently endemic in West Africa. There is no approved vaccines and antivirals against LASV infection. Despite the substantial threat of LASV to public health, important questions in the basic biology of LASV still exist. LASV nucleoprotein has a DEDDH 3'- to-5' exoribonuclease motif (ExoN), which is known to degrade virus-derived double-stranded RNA and thereby is key to LASV evasion of interferon response. Intriguingly, the NP ExoN motif is highly conserved in mammarenaviruses, regardless of pathogenicity, suggesting that NP ExoN has important function in virus life cycle in addition to immune evasion. By using reverse genetics system, we rescued an ExoN-deficient LASV mutant (ExoN- rLASV). Compared to wild-type rLASV, the ExoN- rLASV demonstrated impaired virus growth and decreased viral RNA production in interferon-deficient cells. For the first time, we found that abrogation of LASV ExoN activity led to increased aberrant viral RNA production in infection. Furthermore, NP ExoN deficiency increased LASV sensitivity to mutagenic nucleoside analogue. Our study provided evidence for an additional role of LASV NP ExoN in viral RNA replication besides its recognized function in immune evasion. LASV ExoN activity may be required for optimal viral RNA synthesis and control of aberrant viral RNA and viral RNA mutation.

SUBMITTER: Huang C

PROVIDER: S-EPMC10120729 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Lassa virus NP DEDDh 3'-5' exoribonuclease activity is required for optimal viral RNA replication and mutation control.

Huang Cheng C Mantlo Emily E Paessler Slobodan S

bioRxiv : the preprint server for biology 20231230

Lassa virus (LASV), a mammarenavirus from <i>Arenaviridae</i>, is the causative agent of Lassa fever (LF) endemic in West Africa. Currently, there are no vaccines or antivirals approved for LF. The RNA-dependent RNA polymerases (RdRp) of RNA viruses are error-prone. As a negative-sense RNA virus, how LASV copes with errors in RNA synthesis and ensures optimal RNA replication are not well elucidated. LASV nucleoprotein (NP) contains a DEDDH 3'-to-5' exoribonuclease motif (ExoN), which is known to ...[more]

PMID: 37090668

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Project description:BackgroundCoronavirus (CoV) nonstructural protein 14 (nsp14) has exoribonuclease (ExoN) activity, responsible for proofreading and contributing to replication fidelity. It has been reported that CoVs exhibit variable sensitivity to nsp14-ExoN deficiency. Betacoronavirus murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV were viable upon nsp14-ExoN deficiency. While betacoronavirus Middle East respiratory syndrome (MERS)-CoV and SARS-CoV-2 were non-viable with disabled nsp14-ExoN. In this study, we investigated the nsp14-ExoN deficiency of alphacoronavirus porcine epidemic diarrhea virus (PEDV) in viral pathogenesis using reverse genetics.ResultsEight nsp14-ExoN deficient mutants, targeting the predicted active sites and the Zinc finger or mental-coordinating sites, of PEDV were designed. Only one mutant E191A with a mutation in the Mg2+-binding site was rescued using the infectious clone of PEDV PC22A strain (icPC22A). The passage no.1-3 (P1-3) of E191A grew to very low titers in Vero cells. To evaluate the pathogenesis of the E191A, 4 or 5-day-old gnotobiotic pigs were inoculated orally with 100 TCID50/pig of the E191A-P1, icPC22A, or mock. All mock pigs did not shed virus in feces or show clinical signs. All pigs inoculated with icPC22A shed high viral RNA levels, had severe diarrhea, and died by 6 days post-inoculation (dpi). In contrast, only 3 pigs (3/4, 75%) in the E191A-P1 group shed low levels of viral RNA and 2 pigs had moderate diarrhea at acute infection phase. At 22 dpi, each pig was challenged orally with 106 plaque forming unit of virulent icPC22A. All pigs in the mock group developed severe diarrhea and 2 of the 5 pigs died. Pigs in the E191A-P1 group had less severe diarrhea and no pigs died. Sanger sequencing analysis revealed that the viral genome in the fecal sample of one E191A-P1-inoculated pig and the P4 virus passaged in vitro lost the E191A mutation, suggesting the genetic instability of the E191A mutant.ConclusionThe recombinant PEDV variants carrying mutations at the essential functional sites within nsp14-ExoN were either lethal or genetically unstable. Our finding further confirmed the critical role of nsp14-ExoN in CoV life cycle, suggesting that it may be a target for the design of universal anti-CoV drugs.

Project description:Among RNA viruses, the order Nidovirales stands out for including viruses with the largest RNA genomes currently known. Nidoviruses employ a complex RNA-synthesizing machinery comprising a variety of non-structural proteins (nsps). One of the postulated drivers of the expansion of nidovirus genomes is the presence of a proofreading 3'-to-5' exoribonuclease (ExoN) belonging to the DEDDh family. ExoN may enhance the fidelity of RNA synthesis by correcting nucleotide incorporation errors made by the RNA-dependent RNA polymerase. Here, we review our current understanding of ExoN evolution, structure, and function. Most experimental data are derived from studies of the ExoN domain of coronaviruses (CoVs), which were triggered by the bioinformatics-based identification of ExoN in the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) and its relatives in 2003. Although convincing data supporting the proofreading hypothesis have been obtained, from biochemical assays and studies with CoV mutants lacking ExoN functionality, the features of ExoN from most other nidovirus families remain to be characterized. Remarkably, viable ExoN knockout mutants were obtained only for two CoVs, mouse hepatitis virus (MHV) and SARS-CoV, whose RNA synthesis and replication kinetics were mildly affected by the lack of ExoN function. In several other CoV species, ExoN inactivation was not tolerated, and knockout mutants could not be rescued when launched using a reverse genetics system. This suggests that ExoN is also critical for primary viral RNA synthesis, a property that poorly matches the profile of an enzyme that would merely boost long-term replication fidelity. In CoVs, ExoN resides in a bifunctional replicase subunit (nsp14) whose C-terminal part has (N7-guanine)-methyltransferase activity. The crystal structure of SARS-CoV nsp14 has shed light on the interplay between these two domains, and on nsp14's interactions with nsp10, a co-factor that strongly enhances ExoN activity in vitro assays. Further elucidation of the structure-function relationships of ExoN and its interactions with other (viral and/or host) members of the CoV replication machinery will be key to understanding the enzyme's role in viral RNA synthesis and pathogenesis, and may contribute to the design of new approaches to combat emerging nidoviruses.

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Lassa virus NP DEDDh 3'-5' exoribonuclease activity is required for optimal viral RNA replication.

Publications

Lassa virus NP DEDDh 3'-5' exoribonuclease activity is required for optimal viral RNA replication and mutation control.

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