The Nucleocapsid of Paramyxoviruses: Structure and Function of an Encapsidated Template

Viruses of the Paramyxoviridae family share a common and complex molecular machinery for transcribing and replicating their genomes. Their non-segmented, negative-strand RNA genome is encased in a tight homopolymer of viral nucleoproteins (N). This ribonucleoprotein complex, termed a nucleocapsid, is the template of the viral polymerase complex made of the large protein (L) and its co-factor, the phosphoprotein (P). This review summarizes the current knowledge on several aspects of paramyxovirus transcription and replication, including structural and functional data on (1) the architecture of the nucleocapsid (structure of the nucleoprotein, interprotomer contacts, interaction with RNA, and organization of the disordered C-terminal tail of N), (2) the encapsidation of the genomic RNAs (structure of the nucleoprotein in complex with its chaperon P and kinetics of RNA encapsidation in vitro), and (3) the use of the nucleocapsid as a template for the polymerase complex (release of the encased RNA and interaction network allowing the progress of the polymerase complex). Finally, this review presents models of paramyxovirus transcription and replication.

Comparison of Zaire and Bundibugyo ebolavirus polymerase complexes and susceptibility to antivirals through a newly developed Bundibugyo minigenome system

Members of the genus Ebolavirus cause lethal disease in humans with Zaire ebolavirus (EBOV) being the most pathogenic (up to 90% morality) and Bundibugyo ebolavirus (BDBV) the least pathogenic (∼37% mortality). Historically, there has been a lack of research on BDBV and there is no means to study BDBV outside of a high-containment laboratory. Here, we describe a minigenome replication system to study BDBV transcription and compare the efficacy of small molecule inhibtors between EBOV and BDBV. Using this system, we examined the ability of the polymerase complex proteins from EBOV and BDBV to interact and form a functional unit as well as the impact of the genomic untranslated ends, known to contain important signals for transcription (3’-untranslated region) and replication (5’-untranslated region). Varying levels of compatibility were observed between proteins of the polymerase complex from each ebolavirus resulting in differences in genome transcription efficiency. Most pronounced was the effect of the nucleoprotein and the 3’-untranslated region. These data suggest that there are intrinsic specificities in the polymerase complex and untranslated signaling regions that could offer insight regarding observed pathogenic differences. Further adding to the differences in the polymerase complexes, post-transfection/infection treatment with the compound remdesivir (GS-5734) showed a greater inhibitory effect against BDBV compared to EBOV. The delayed growth kinetics of BDBV and the greater susceptibility to polymerase inhibitors indicate that disruption of the polymerase complex may be a viable target for therapeutics. Importance Ebolavirus disease is a viral infection and is fatal in 25-90% of cases, depending on the viral species and the amount of supportive care available. Two species have caused outbreaks in the Democratic Republic of the Congo, Zaire ebolavirus (EBOV) and Bundibugyo ebolavirus (BDBV). Pathogenesis and clinical outcome differ between these two species but there is still limited information regarding the viral mechanism for these differences. Previous studies suggest that BDBV replicates slower than EBOV but it is unknown if this is due to differences in the polymerase complex and its role in transcription and replication. This study details the construction of a minigenome replication system which can be used in a biosafety level (BSL) 2 laboartory. This system will be important for studying the polymerase complex of BDBV and comparing it with other filoviruses and can be used as a tool for screening inhibitors of viral growth.

Meta-Analysis and Structural Dynamics of the Emergence of Genetic Variants of SARS-CoV-2

Graphical AbstractErrors are regularly made when SARS-CoV-2 replicates its RNA genome. The viral polymerase complex is error-prone with imperfect proofreading abilities. These errors or mutations often lead to deleterious or neutral effects on the virus. However, sometimes these mutations have a positive effect and create genetic variants of the virus with different features including increased transmissibility, pathogenicity, and immune escape capabilities. When mutations work collaboratively to create a new virus feature, this is called epistasis.

Surveillance of Influenza A resistance to polymerase complex inhibitors by whole genome analysis, 2016-17, 2017-18 and 2018-19 Seasons, Eastern Spain

Molecular surveillance by whole genome sequencing was used to monitor the susceptibility of circulating Influenza A viruses to three polymerase complex inhibitors. A total of 12 resistance substitutions were found among 285 genomes analysed, but none associated with high levels of resistance. Natural resistance to these influenza A antivirals is currently uncommon.

Therapeutic targeting of measles virus polymerase with ERDRP-0519 suppresses all RNA synthesis activity

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.

Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

Regulation of VP30-Dependent Transcription by RNA Sequence and Structure in the Genomic Ebola Virus Promoter

Viral transcription and replication of Ebola virus (EBOV) is balanced by transcription factor VP30, an RNA binding protein. An RNA hairpin at the transcription start site (TSS) of the first gene (NP hairpin) in the 3′-leader promoter is thought to mediate the VP30 dependency of transcription. Here, we investigated the constraints of VP30 dependency using a series of monocistronic minigenomes with sequence, structure and length deviations from the native NP hairpin. Hairpin stabilizations decreased while destabilizations increased transcription in the absence of VP30, but in all cases, transcription activity was higher in the presence versus absence of VP30. This also pertains to a mutant that is unable to form any RNA secondary structure at the TSS, demonstrating that the activity of VP30 is not simply determined by the capacity to form a hairpin structure at the TSS. Introduction of continuous 3′-UN5 hexamer phasing between promoter elements PE1 and PE2 by a single point mutation in the NP hairpin boosted VP30-independent transcription. Moreover, this point mutation, but also hairpin stabilizations, impaired the relative increase of replication in the absence of VP30. Our results suggest that the native NP hairpin is optimized for tight regulation by VP30 while avoiding an extent of hairpin stability that impairs viral transcription, as well as for enabling the switch from transcription to replication when VP30 is not part of the polymerase complex. IMPORTANCE A detailed understanding is lacking how the Ebola virus (EBOV) protein VP30 regulates activity of the viral polymerase complex. Here, we studied how RNA sequence, length and structure at the transcription start site (TSS) in the 3′-leader promoter influence the impact of VP30 on viral polymerase activity. We found that hairpin stabilizations tighten the VP30 dependency of transcription but reduce transcription efficiency and attenuate the switch to replication in the absence of VP30. Upon hairpin destabilization, VP30-independent transcription - already weakly detectable at the native promoter - increases, but never reaches the same extent as in the presence of VP30. We conclude that the native hairpin structure involving the TSS (i) establishes an optimal balance between efficient transcription and tight regulation by VP30, (ii) is linked to hexamer phasing in the promoter, and (iii) favors the switch to replication when VP30 is absent.