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Hepatitis C virus (HCV) genotype 3 presents a high level of both baseline and acquired resistance to direct-acting antivirals (DAAs), particularly those targeting the NS5A protein. To understand this resistance we studied a cohort of Brazilian patients treated with the NS5A DAA, daclatasvir and the nucleoside analogue, sofosbuvir. We observed a novel substitution at NS5A amino acid residue 98 [serine to glycine (S98G)] in patients who relapsed post-treatment. The effect of this substitution on both replication fitness and resistance to DAAs was evaluated using two genotype 3 subgenomic replicons. S98G had a modest effect on replication, but in combination with the previously characterized resistance-associated substitution (RAS), Y93H, resulted in a significant increase in daclatasvir resistance. This result suggests that combinations of substitutions may drive a high level of DAA resistance and provide some clues to the mechanism of action of the NS5A-targeting DAAs.

Non-coding regions of viral RNA (vRNA) genomes are critically important in the regulation of gene expression. In particular, pseudoknot (PK) structures, which are present in a wide range of RNA molecules, have a variety of roles. The 5′ untranslated region (5′ UTR) of foot-and-mouth disease virus (FMDV) vRNA is considerably longer than in other viruses from the picornavirus family and consists of a number of distinctive structural motifs that includes multiple (2, 3 or 4 depending on the virus strain) putative PKs linked in tandem. The role(s) of the PKs in the FMDV infection are not fully understood. Here, using bioinformatics, sub-genomic replicons and recombinant viruses we have investigated the structural conservation and importance of the PKs in the FMDV lifecycle. Our results show that despite the conservation of two or more PKs across all FMDVs, a replicon lacking PKs was replication competent, albeit at reduced levels. Furthermore, in competition experiments, GFP FMDV replicons with less than two (0 or 1) PK structures were outcompeted by a mCherry FMDV wt replicon that had 4 PKs, whereas GFP replicons with 2 or 4 PKs were not. This apparent replicative advantage offered by the additional PKs correlates with the maintenance of at least two PKs in the genomes of FMDV field isolates. Despite a replicon lacking any PKs retaining the ability to replicate, viruses completely lacking PK were not viable and at least one PK was essential for recovery of infections virus, suggesting a role for the PKs in virion assembly. Thus, our study points to roles for the PKs in both vRNA replication and virion assembly, thereby improving understanding the molecular biology of FMDV replication and the wider roles of PK in RNA functions.

Made available in DSpace on 2021-06-26T02:36:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 MRC China Scholarship Council Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Hepatitis C virus (HCV) genotype 3 presents a high level of both baseline and acquired resistance to direct-acting antivirals (DAAs), particularly those targeting the NS5A protein. To understand this resistance we studied a cohort of Brazilian patients treated with the NS5A DAA, daclatasvir and the nucleoside analogue, sofosbuvir. We observed a novel substitution at NS5A amino acid residue 98 [serine to glycine (S98G)] in patients who relapsed post-treatment. The effect of this substitution on both replication fitness and resistance to DAAs was evaluated using two genotype 3 subgenomic replicons. S98G had a modest effect on replication, but in combination with the previously characterized resistance-associated substitution (RAS), Y93H, resulted in a significant increase in daclatasvir resistance. This result suggests that combinations of substitutions may drive a high level of DAA resistance and provide some clues to the mechanism of action of the NS5A-targeting DAAs. Sao Paulo State Univ, Inst Biosci Languages & Exact Sci, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil Univ Leeds, Fac Biol Sci, Sch Mol & Cellular Biol, Leeds LS2 9JT, W Yorkshire, England Univ Leeds, Astbury Ctr Struct Mol Biol, Leeds LS2 9JT, W Yorkshire, England Univ Sao Paulo, Ribeirao Preto Sch Med, BR-14049900 Ribeirao Preto, SP, Brazil Univ Sao Paulo, Ribeirao Preto Fac Pharmaceut Sci, BR-14040903 Ribeirao Preto, SP, Brazil Sao Paulo State Univ, Inst Biosci Languages & Exact Sci, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil MRC: MR/S001026/1 FAPESP: 2016/03807-0 FAPESP: 2018/04678-5

This research was partially supported by a Cheney Fellowship to S. J. P. from the University of Leeds. Support from BBSRC grant BB/P001459/1 to N. J. S. and BBSRC Studentship BB/F01614X/1 to J. W. is also gratefully acknowledged. Arbidol (ARB, also known as umifenovir) is used clinically in several countries as an anti-influenza virus drug. ARB inhibits multiple enveloped viruses in vitro and the primary mode of action is inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. ARB is also an effective inhibitor of non-enveloped poliovirus types 1 and 3. In the current report, we evaluate the antiviral potential of ARB against another picornavirus, foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus and an important veterinary pathogen. ARB inhibits the replication of FMDV RNA sub-genomic replicons. ARB inhibition of FMDV RNA replication is not a result of generalized inhibition of cellular uptake of cargo, such as transfected DNA, and ARB can be added to cells up to 3 h post-transfection of FMDV RNA replicons and still inhibit FMDV replication. ARB prevents the recovery of FMDV replication upon withdrawal of the replication inhibitor guanidine hydrochloride (GuHCl). Although restoration of FMDV replication is known to require de novo protein synthesis upon GuHCl removal, ARB does not suppress cellular translation or FMDV internal ribosome entry site (IRES)-driven translation. ARB also inhibits infection with the related Aphthovirus, equine rhinitis A virus (ERAV). Collectively, the data demonstrate that ARB can inhibit some non-enveloped picornaviruses. The data are consistent with inhibition of picornavirus genome replication, possibly via the disruption of intracellular membranes on which replication complexes are located. Publisher PDF

RNA aptamers are synthetic single stranded RNA oligonucleotides that function analogously to antibodies. Recently, they have shown promise for use in treating inflammatory skin disease as, unlike antibody-based biologics, they are able to enter the skin following topical administration. However, it is important to understand the inflammatory milieu into which aptamers are delivered, as numerous immune-modulating mediators will be present at abnormal levels. LL-37 is an important immune-modifying protein upregulated in several inflammatory skin conditions, including psoriasis, rosacea and eczema. This inflammatory antimicrobial peptide is known to complex nucleic acids and induce both inflammatory and interferon responses from keratinocytes. Given the attractive notion of using RNA aptamers in topical medication and the prevalence of LL-37 in these inflammatory skin conditions, we examined the effect of LL-37 on the efficacy and safety of the anti-IL-17A RNA aptamer, Apt 21-2. LL-37 was demonstrated to complex with the RNA aptamer by electrophoretic mobility shift and filter binding assays. In contrast to free Apt 21-2, LL-37-complexed Apt 21-2 was observed to efficiently enter both keratinocytes and fibroblasts by confocal microscopy. Despite internalization of LL-37-complexed aptamers, measurement of inflammatory mediators and interferon stimulated genes showed LL-37-complexed Apt 21-2 remained immunologically inert in keratinocytes, fibroblasts, and peripheral blood mononuclear cells including infiltrating dendritic cells and monocytes. The findings of this study suggest RNA aptamers delivered into an inflammatory milieu rich in LL-37 may become complexed and subsequently internalized by surrounding cells in the skin. Whilst the results of this study indicate delivery of RNA aptamers into tissue rich in LL-37 should not cause an unwarranted inflammatory of interferon response, these results have significant implications for the efficacy of aptamers with regards to extracellular vs. intracellular targets that should be taken into consideration when developing treatment strategies utilizing RNA aptamers in inflamed tissue.

RNA viruses, including poliovirus, evolve rapidly due to the error-prone nature of the polymerase enzymes involved in genome replication. Fixation of advantageous mutations may require the acquisition of complementary mutations which can act in concert to achieve a favorable phenotype. This study highlights a compensatory role of a nonstructural regulatory protein, 2Apro, for an otherwise lethal mutation of the structural VP1 protein to facilitate increased thermal resistance. Studying how viruses respond to selection pressures is important for understanding mechanisms which underpin emergence of resistance and could be applied to the future development of antiviral agents and vaccines., Virus capsid proteins must perform a number of roles. These include self-assembly and maintaining stability under challenging environmental conditions, while retaining the conformational flexibility necessary to uncoat and deliver the viral genome into a host cell. Fulfilling these roles could place conflicting constraints on the innate abilities encoded within the protein sequences. In a previous study, we identified a number of mutations within the capsid-coding sequence of poliovirus (PV) that were established in the population during selection for greater thermostability by sequential treatment at progressively higher temperatures. Two mutations in the VP1 protein acquired at an early stage were maintained throughout this selection procedure. One of these mutations prevented virion assembly when introduced into a wild-type (wt) infectious clone. Here we show, by sequencing beyond the capsid-coding region of the heat-selected virions, that two mutations had arisen within the coding region of the 2A protease. Both mutations were maintained throughout the selection process. Introduction of these mutations into a wt infectious clone by site-directed mutagenesis considerably reduced replication. However, they permitted a low level of assembly of infectious virions containing the otherwise lethal mutation in VP1. The 2Apro mutations were further shown to slow the kinetics of viral polyprotein processing, and we suggest that this delay improves the correct folding of the mutant capsid precursor protein to permit virion assembly. IMPORTANCE RNA viruses, including poliovirus, evolve rapidly due to the error-prone nature of the polymerase enzymes involved in genome replication. Fixation of advantageous mutations may require the acquisition of complementary mutations which can act in concert to achieve a favorable phenotype. This study highlights a compensatory role of a nonstructural regulatory protein, 2Apro, for an otherwise lethal mutation of the structural VP1 protein to facilitate increased thermal resistance. Studying how viruses respond to selection pressures is important for understanding mechanisms which underpin emergence of resistance and could be applied to the future development of antiviral agents and vaccines.