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1.
Curr Opin Virol ; 52: 71-77, 2022 02.
Article in English | MEDLINE | ID: covidwho-1936243

ABSTRACT

Flaviviruses are zoonotic pathogens transmitted by the bite of infected mosquitos and ticks and represent a constant burden to human health. Here we review recent literature aimed at uncovering how flaviviruses interact with the cells that they infect. A better understanding of these interactions may ultimately lead to novel therapeutic targets. We highlight several studies that employed low-biased methods to discover new protein-protein, protein-RNA, and genetic interactions, and spotlight recent work characterizing the host protein, TMEM41B, which has been shown to be critical for infection by diverse flaviviruses and coronaviruses.


Subject(s)
Flavivirus Infections , Flavivirus , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Flavivirus/genetics , Flavivirus/metabolism , Host-Pathogen Interactions/genetics , Humans , Proviruses , Virus Replication
2.
Adv Virus Res ; 112: 1-29, 2022.
Article in English | MEDLINE | ID: covidwho-1763516

ABSTRACT

Reverse genetics is the prospective analysis of how genotype determines phenotype. In a typical experiment, a researcher alters a viral genome, then observes the phenotypic outcome. Among RNA viruses, this approach was first applied to positive-strand RNA viruses in the mid-1970s and over nearly 50 years has become a powerful and widely used approach for dissecting the mechanisms of viral replication and pathogenesis. During this time the global health importance of two virus groups, flaviviruses (genus Flavivirus, family Flaviviridae) and betacoronaviruses (genus Betacoronavirus, subfamily Orthocoronavirinae, family Coronaviridae), have dramatically increased, yet these viruses have genomes that are technically challenging to manipulate. As a result, several new techniques have been developed to overcome these challenges. Here I briefly review key historical aspects of positive-strand RNA virus reverse genetics, describe some recent reverse genetic innovations, particularly as applied to flaviviruses and coronaviruses, and discuss their benefits and limitations within the larger context of rigorous genetic analysis.


Subject(s)
Flavivirus , RNA Viruses , Flavivirus/genetics , Genome, Viral , Positive-Strand RNA Viruses , RNA Viruses/genetics , Reverse Genetics/methods , Virus Replication/genetics
3.
Microbiol Spectr ; 10(1): e0165521, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1673364

ABSTRACT

Although lessons have been learned from previous severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) outbreaks, the rapid evolution of the viruses means that future outbreaks of a much larger scale are possible, as shown by the current coronavirus disease 2019 (COVID-19) outbreak. Therefore, it is necessary to better understand the evolution of coronaviruses as well as viruses in general. This study reports a comparative analysis of the amino acid usage within several key viral families and genera that are prone to triggering outbreaks, including coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2], SARS-CoV, MERS-CoV, human coronavirus-HKU1 [HCoV-HKU1], HCoV-OC43, HCoV-NL63, and HCoV-229E), influenza A (H1N1 and H3N2), flavivirus (dengue virus serotypes 1 to 4 and Zika) and ebolavirus (Zaire, Sudan, and Bundibugyo ebolavirus). Our analysis reveals that the distribution of amino acid usage in the viral genome is constrained to follow a linear order, and the distribution remains closely related to the viral species within the family or genus. This constraint can be adapted to predict viral mutations and future variants of concern. By studying previous SARS and MERS outbreaks, we have adapted this naturally occurring pattern to determine that although pangolin plays a role in the outbreak of COVID-19, it may not be the sole agent as an intermediate animal. In addition to this study, our findings contribute to the understanding of viral mutations for subsequent development of vaccines and toward developing a model to determine the source of the outbreak. IMPORTANCE This study reports a comparative analysis of amino acid usage within several key viral genera that are prone to triggering outbreaks. Interestingly, there is evidence that the amino acid usage within the viral genomes is not random but in a linear order.


Subject(s)
Coronavirus/genetics , Ebolavirus/genetics , Evolution, Molecular , Flavivirus/genetics , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H3N2 Subtype/genetics , Codon , Coronavirus/classification , Genome, Viral , Humans , Linear Models , Mutation , SARS-CoV-2/genetics , Virus Diseases/virology
4.
J Virol ; 95(24): e0059621, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1443352

ABSTRACT

Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.


Subject(s)
CRISPR-Cas Systems , Flavivirus/genetics , Neoplasm Proteins/genetics , Receptors for Activated C Kinase/genetics , Virus Replication , A549 Cells , Aedes , Animals , COVID-19 , Chlorocebus aethiops , Culicidae , Dengue Virus/genetics , Genome-Wide Association Study , HEK293 Cells , Host-Pathogen Interactions/genetics , Humans , RNA, Small Interfering/metabolism , RNA, Viral/metabolism , SARS-CoV-2 , Vero Cells , West Nile virus/genetics , Zika Virus/genetics , Zika Virus Infection/virology
5.
J Mol Biol ; 434(6): 167277, 2022 03 30.
Article in English | MEDLINE | ID: covidwho-1440208

ABSTRACT

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.


Subject(s)
COVID-19 , Flavivirus , Zika Virus Infection , Zika Virus , Antiviral Agents/pharmacology , Flavivirus/genetics , Histone Deacetylases , Humans , Interferons/genetics , Membrane Proteins , RNA-Binding Proteins , Repressor Proteins , SARS-CoV-2 , Virus Replication/genetics , Zika Virus/genetics , Zika Virus Infection/genetics
6.
Molecules ; 25(23)2020 Dec 03.
Article in English | MEDLINE | ID: covidwho-963646

ABSTRACT

The RNA-dependent RNA polymerase (RdRp) is an essential enzyme for the viral replication process, catalyzing the viral RNA synthesis using a metal ion-dependent mechanism. In recent years, RdRp has emerged as an optimal target for the development of antiviral drugs, as demonstrated by recent approvals of sofosbuvir and remdesivir against Hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively. In this work, we overview the main sequence and structural features of the RdRp of emerging RNA viruses such as Coronaviruses, Flaviviruses, and HCV, as well as inhibition strategies implemented so far. While analyzing the structural information available on the RdRp of emerging RNA viruses, we provide examples of success stories such as for HCV and SARS-CoV-2. In contrast, Flaviviruses' story has raised attention about how the lack of structural details on catalytically-competent or ligand-bound RdRp strongly hampers the application of structure-based drug design, either in repurposing and conventional approaches.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , RNA Viruses/enzymology , RNA-Dependent RNA Polymerase/chemistry , Amides/chemistry , Amides/pharmacology , Coronavirus/drug effects , Coronavirus/enzymology , Coronavirus/genetics , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Flavivirus/drug effects , Flavivirus/enzymology , Flavivirus/genetics , Hepacivirus/drug effects , Hepacivirus/enzymology , Hepacivirus/genetics , Humans , Pyrazines/chemistry , Pyrazines/pharmacology , RNA Virus Infections/epidemiology , RNA Viruses/drug effects , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology
7.
Virology ; 547: 35-46, 2020 08.
Article in English | MEDLINE | ID: covidwho-343623

ABSTRACT

Spondweni virus (SPONV) is the most closely related known flavivirus to Zika virus (ZIKV). Its pathogenic potential and vector specificity have not been well defined. SPONV has been found predominantly in Africa, but was recently detected in a pool of Culex quinquefasciatus mosquitoes in Haiti. Here we show that SPONV can cause significant fetal harm, including demise, comparable to ZIKV, in a mouse model of vertical transmission. Following maternal inoculation, we detected infectious SPONV in placentas and fetuses, along with significant fetal and placental histopathology, together suggesting vertical transmission. To test vector competence, we exposed Aedes aegypti and Culex quinquefasciatus mosquitoes to SPONV-infected bloodmeals. Aedes aegypti could efficiently transmit SPONV, whereas Culex quinquefasciatus could not. Our results suggest that SPONV has the same features that made ZIKV a public health risk.


Subject(s)
Aedes/virology , Flavivirus Infections/virology , Flavivirus/physiology , Mosquito Vectors/virology , Receptor, Interferon alpha-beta/genetics , Aedes/physiology , Animals , Disease Models, Animal , Female , Flavivirus/genetics , Flavivirus Infections/genetics , Flavivirus Infections/metabolism , Flavivirus Infections/mortality , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mosquito Vectors/physiology , Receptor, Interferon alpha-beta/deficiency
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