Biology subjects

Goldhill, D.

Publications and source records attributed to Goldhill, D..

5 recordsLinked to original sources

bioRxiv2021

TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

Ebola virus (EBOV) causes highly pathogenic disease in primates. Through screening a library of human interferon-stimulated genes (ISGs), we identified TRIM25 as a potent inhibitor of EBOV transcription-and-replication-competent virus-like particle (trVLP) propagation. TRIM25 overexpression inhibited the accumulation of viral genomic and messenger RNAs independently of the RNA sensor RIG-I or secondary proinflammatory gene expression. Deletion of TRIM25 strongly attenuated the sensitivity of trVLPs to inhibition by type-I interferon. The antiviral activity of TRIM25 required ZAP and the effect of type-I interferon was modulated by the CpG dinucleotide content of the viral genome. We find that TRIM25 interacts with the EBOV vRNP, resulting in its autoubiquitination and ubiquitination of the viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry, and leads to dissociation of NP from the vRNA. We propose that TRIM25 targets the EBOV vRNP, exposing CpG-rich viral RNA species to restriction by ZAP. HighlightsO_LITRIM25 and ZAP play a major role on type I IFN-mediated inhibition of EBOV trVLP replication C_LIO_LITRIM25 interacts with the EBOV NP and is recruited to vRNPs in the cytoplasm after viral entry C_LIO_LITRIM25 ubiquitinates NP and displaces it from the viral genome, facilitating ZAP interaction C_LIO_LIZAP targets CpGs in the EBOV genome to inhibit EBOV trVLP replication C_LI

microbiology↗

bioRxiv2020

Mammalian ANP32A and ANP32B proteins drive alternative avian influenza virus polymerase adaptations

ANP32 proteins, which act as influenza polymerase co-factors, vary between birds and mammals. The well-known mammalian adaptation, PB2-E627K, enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbour this adaptation. Here, we show that alternative PB2 adaptations, Q591R and D701N also allow influenza polymerase to use mammalian ANP32 proteins. PB2-E627K strongly favours use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs and horses where ANP32A proteins are more strongly pro-viral. In an experimental evolution approach, passage of avian viruses in human cells drives acquisition of PB2-E627K, but not when ANP32B is ablated. The strong pro-viral support of ANP32B for PB2-E627K maps to the LCAR region of ANP32B.

microbiology↗

bioRxiv2020

Favipiravir-resistant influenza A virus shows potential for transmission

Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain. Author SummaryIn the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipiravir is a novel drug which might be used against influenza virus in the event of a new pandemic. Is resistance likely to be a problem for the use of favipiravir? Our previous work has shown that resistance to favipiravir can be generated in cell culture but we dont know whether there will be a cost preventing the spread of resistance in whole organisms. Here, we used a mix of wild-type and resistant influenza viruses from early in the 2009 pandemic to test whether viruses resistant to favipiravir could transmit between ferrets. We found that the resistant viruses could transmit but that the resistance mutation was selected against within some ferrets. Using modelling and in vitro experiments, we found that the resistant mutation was selected against in the influenza strain from our experiment but not in more recently evolved strains. Our results show that favipiravir resistant viruses could spread if resistance is generated but the probability will depend on the genetic background of the virus.

microbiology↗

bioRxiv2019

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.\n\nRational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic \"swine\" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

microbiology↗

bioRxiv2018

Determining the Mutation Bias of Favipiravir in Influenza Using Next-generation Sequencing

AbstractFavipiravir is a broad spectrum antiviral drug that may be used to treat influenza. Previous research has identified that favipiravir likely acts as a mutagen but the precise mutation bias that favipiravir induces in influenza virus RNAs has not been described. Here, we use next-generation sequencing (NGS) with barcoding of individual RNA molecules to accurately and quantitatively detect favipiravir-induced mutations and to sample orders of magnitude more mutations than would be possible through Sanger sequencing. We demonstrate that favipiravir causes mutations and show that favipiravir primarily acts as a guanine analogue and secondarily as an adenine analogue resulting in the accumulation of transition mutations. We also use a standard NGS pipeline to show that the mutagenic effect of favipiravir can be measured by whole genome sequencing of virus.\n\nImportanceNew antiviral drugs are needed as a first line of defence in the event of a novel influenza pandemic. Favipiravir is a broad-spectrum antiviral which is effective against influenza. The exact mechanism of how favipiravir works to inhibit influenza is still unclear. We used next-generation sequencing (NGS) to demonstrate that favipiravir causes mutations in influenza RNA. The greater depth of NGS sequence information over traditional sequencing methods allowed us to precisely determine the bias of particular mutations caused by favipiravir. NGS can also be used in a standard diagnostic pipeline to show that favipiravir is acting on the virus by revealing the mutation bias pattern typical to the drug. Our work will aid in testing whether viruses are resistant to favipiravir and may help demonstrate the effect of favipiravir on viruses in a clinical setting. This will be important if favipiravir is used during a future influenza pandemic.

microbiology↗