Pharmacological perturbation of intracellular dynamics as a SARS-CoV-2 antiviral strategy

SARS-CoV-2 (CoV2) is the viral agent responsible for the pandemic of the coronavirus disease 2019 (COVID-19). Vaccines are being deployed all over the world with good efficacy, but there is no approved antiviral treatment to date. This is particularly needed since the emergence of variants and the potential immune escape may prolong pandemic spreading of the infection for much longer than anticipated. Here, we developed a series of small molecules and identified RG10 as a potent antiviral compound against SARS-CoV-2 in cell lines and human airway epithelia (HAE). RG10 localizes to endoplasmic reticulum (ER) membranes, perturbing ER morphology and inducing ER stress. Yet, RG10 does not associate with SARS-CoV-2 replication sites although preventing virus replication. To further investigate the antiviral properties of our compound, we developed fluorescent SARS-CoV-2 viral particles allowing us to track virus arrival to ER membranes. Live cell imaging of replication-competent virus infection revealed that RG10 stalls the intracellular virus-ER dynamics. Finally, we synthesized RG10b, a stable version of RG10, that showed increased potency in vitro and in HAE with a pharmacokinetic half-life greater than 2 h. Together, our work reports on a novel fluorescent virus model and innovative antiviral strategy consisting of the perturbation of ER/virus dynamics, highlighting the promising antiviral properties of RG10 and RG10b.

SARS COV-2: Exploring the Virus of the Century

Coronaviruses comprise a large family of viruses that cause respiratory and intestinal infections in animals and humans. This recent outbreak of unusual respiratory disease plaguing the entire world has been named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the basis of phylogenetic analysis of related coronaviruses. Its transmission occurs mainly through airborn, fomite and other modes. Structurally, it is similar to other coronaviruses and has four major structural proteins; the spike surface glycoprotein (S), small envelope protein (E), matrix protein (M) and nucleocapsid protein (N). The M protein is most abundant and is responsible for intracellular formation of virus particles. S protein induces antibody generation and is involved in intracellular virus entry. Drug combinations are being tried on the basis of structural and genomic knowledge of the virus. Various researchers have found that the SARS CoV2 has many strains among which L type is most pathogenic and D614 type is most infective. All this information has been collected in this review to understand the virus behind this calamity in depth and to make it handy for the researchers to search literature related to SARS COV2. Keywords: SARS COV2, spike glycoprotein, L type strain, D614 strain.

Adaptive evolution of nontransitive fitness in yeast

A common misconception is that evolution is a linear 'march of progress', where each organism along a line of descent is more fit than all those that came before it. Rejecting this misconception implies that evolution is nontransitive: a series of adaptive events will, on occasion, produce organisms that are less fit compared to a distant ancestor. Here we identify a nontransitive evolutionary sequence in a 1,000-generation yeast evolution experiment. We show that nontransitivity arises due to adaptation in the yeast nuclear genome combined with the stepwise deterioration of an intracellular virus, which provides an advantage over viral competitors within host cells. Extending our analysis, we find that nearly half of our ~140 populations experience multilevel selection, fixing adaptive mutations in both the nuclear and viral genomes. Our results provide a mechanistic case-study for the adaptive evolution of nontransitivity due to multilevel selection in a 1,000-generation host/virus evolution experiment.

A single-cell polony method reveals low levels of infected Prochlorococcus in oligotrophic waters despite high cyanophage abundances

Long-term stability of picocyanobacteria in the open oceans is maintained by a balance between synchronous division and death on daily timescales. Viruses are considered a major source of microbial mortality, however, current methods to measure infection have significant methodological limitations. Here we describe a method that pairs flow-cytometric sorting with a PCR-based polony technique to simultaneously screen thousands of taxonomically resolved individual cells for intracellular virus DNA, enabling sensitive, high-throughput, and direct quantification of infection by different virus lineages. Under controlled conditions with picocyanobacteria-cyanophage models, the method detected infection throughout the lytic cycle and discriminated between varying infection levels. In North Pacific subtropical surface waters, the method revealed that only a small percentage of Prochlorococcus (0.35–1.6%) were infected, predominantly by T4-like cyanophages, and that infection oscillated 2-fold in phase with the diel cycle. This corresponds to 0.35–4.8% of Prochlorococcus mortality daily. Cyanophages were 2–4-fold more abundant than Prochlorococcus, indicating that most encounters did not result in infection and suggesting infection is mitigated via host resistance, reduced phage infectivity and inefficient adsorption. This method will enable quantification of infection for key microbial taxa across oceanic regimes and will help determine the extent that viruses shape microbial communities and ecosystem level processes.

Differential Alphavirus Defective RNA Diversity between Intracellular and Extracellular Compartments Is Driven by Subgenomic Recombination Events

ABSTRACT Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective viral RNAs featuring large deletions during replication. These defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity-of-infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early-passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated next-generation sequencing libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than P2 virions and that specific populations of D-RNAs are differentially expressed between intracellular and extracellular compartments. Importantly, these trends were likewise observed in a murine model of CHIKV AF15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that is conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which were confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template. IMPORTANCE Our understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between the capsid-E3 region and the E1-3′ untranslated region (UTR) and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally, they may exist as subgenome-derived transcripts.

Differential alphavirus defective RNA diversity between intracellular and encapsidated compartments is driven by subgenomic recombination events

ABSTRACTAlphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective genomes featuring large deletions during replication. Defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity of infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated NGS libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and encapsidated P2 virus and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than encapsidated and specific populations of D-RNAs are differentially expressed between intracellular and encapsidated compartments. Importantly, these trends were likewise observed in a murine model of CHIKV 15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that are conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which was confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template.IMPORTANCEOur understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between capsid/E3 and E1/3’UTR regions and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally may exist as subgenome-derived transcripts.

Adaptive evolution of nontransitive fitness in yeast

Nontransitivity – commonly illustrated by the rock-paper-scissors game – is well documented among extant species as a contributor to biodiversity. However, it is unclear if nontransitive interactions also arise by way of genealogical succession, and if so, through what mechanisms. Here we identify a nontransitive evolutionary sequence in the context of yeast experimental evolution in which a 1,000-generation evolved clone outcompetes a recent ancestor but loses in direct competition with a distant ancestor. We show that nontransitivity arises due to the combined forces of adaptation in the yeast nuclear genome and the stepwise deterioration of an intracellular virus. We show that, given the initial conditions of the experiment, this outcome likely to arise: nearly half of all populations experience multilevel selection, fixing adaptive mutations in both the nuclear and viral genomes. In contrast to conventional views of virus-host coevolution, we find no evidence that viral mutations (including loss of the virus) increase the fitness of the host. Instead, the evolutionary success of evolved viral variants results from their selective advantage over viral competitors within the context of individual cells. Our results provide the first mechanistic case-study of the adaptive evolution of nontransitivity, in which a series of adaptive replacements produce organisms that are less fit when compared to a distant genealogical ancestor.

Mechanisms of action and efficacy of HCV direct-acting antiviral agents and the host-targeting agent Cyclosporin A

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Hepatitis C virus (HCV) remains a significant public health burden worldwide, with an approximated 170 million people infected globally. Chronic HCV infection is associated with increased risk of liver fibrosis, cirrhosis and hepatocellular carcinoma. In developed countries, it is the leading cause of liver transplantation. The previous standard of care (SOC) for HCV was composed of pegylated interferon alpha (pegIFN-[alpha]) and ribavirin (RBV) for 48 weeks. This SOC is associated with about 50% virologic response rate and with severe side effects leading to treatment discontinuation. Since 2011, several direct-acting antiviral agents (DAAs) have been approved, aiming for interferon (IFN)-sparing regimens with shorter duration and fewer side effects. However, the mechanism of action (MOA) and the kinetics of DAAs are still not known. In this Ph.D. thesis work, we first established a multiplex assay approach to compare the relative efficacy of various DAAs and the kinetics of their antiviral activities, as a potential pre-clinical measure of their clinical utility. This approach employs flow cytometry, a Gaussia luciferase reporter system, western blot analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR), limited dilution assay [tissue culture infectious dose 50 (TCID50)], and an image profiling assay that quantifies the NS5A redistribution in response to drug treatment. Using this multiplex assay approach, we evaluated three classes of DAAs, including NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). Our results suggest that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, our data demonstrate that LDV has a more pronounced effect than DCV on viral replication, assembly, and infectivity of released virus. In contrast to DAAs, host-targeting agents (HTAs) affect host factors that are involved in the viral life cycle, conferring a higher barrier to viral resistance. Using the previously established multiplex assay approach plus a strand-specific nucleic acid visualization method, we compared the inhibition kinetics, MOA, and relative potency of cyclosporin A (CsA), an HTA that targets host factor cyclophilin A (CypA), to DAAs, including LDV, DCV, DNV and SOF. Our data show that CsA suppresses viral protein production more rapidly than other DAAs and causes lipid droplets (LDs) enlarged in size. However, LDV has the fastest effect on viral RNA and NS5A redistribution and disrupts the association of double-stranded RNA (dsRNA) with NS5A. Furthermore, inhibitors of NS5A, NS3/4A, but not of NS5B, suppressed the infectivity of released virus. NS5A inhibitors LDV and DCV rapidly suppressed the infectivity of intracellular virus,suggesting that they exert effects on HCV assembly. CypA inhibitor CsA rapidly suppressed the infectivity of extracellular virus, but significantly less efficient in inhibiting the infectivity of intracellular virus, indicating that it blocks a step of the viral life cycle after viral assembly, but before, or at the step of virus release. Taken together, my Ph.D. thesis work has led to the establishment of a multiplex assay approach and shed light on the MOA and efficacy of HCV antivirals. We used this approach to compare the kinetics and the MOA of DAAs and HTA. In addition, this approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations for therapeutic interventions.

Hepatitis C virus promotes virion secretion through cleavage of the Rab7 adaptor protein RILP

Hepatitis C virus (HCV) is an enveloped RNA virus that modifies intracellular trafficking processes. The mechanisms that HCV and other viruses use to modify these events are poorly understood. In this study, we observed that two different RNA viruses, HCV and Sendai, cause inhibition of ras-related protein Rab-7 (Rab7)-dependent endosome–lysosome fusion. In both cases, viral infection causes cleavage of the Rab7 adaptor protein RILP (Rab interacting lysosomal protein), which is responsible for linking Rab7 vesicles to dynein motor complexes. RILP cleavage results in the generation of a cleaved RILP fragment (cRILP) missing the N terminus of the molecule. Although RILP localizes in a perinuclear fashion, cRILP moves to the cell periphery. Both knockdown of RILP and expression of cRILP reproduced the HCV-induced trafficking defect, and restoring full-length RILP reversed the trafficking effects of virus. For the first 3 d after electroporation of HCV RNA, intracellular virus predominates over secreted virus, but the quantity of intracellular virus then rapidly declines as secreted virus dominates. The transition from the intracellular-predominant to the secretion-predominant phenotype corresponds to the time course of cRILP generation. Expressing cRILP directly prevents intracellular virus accumulation at early times without affecting net virus production. The ability of cRILP to promote virus secretion could be prevented by a kinesin inhibitor. HCV thus modifies cellular trafficking by cleaving RILP, which serves to redirect Rab7-containing vesicles to a kinesin-dependent trafficking mode promoting virion secretion. Cleavage of a Rab adaptor protein is thus a mechanism by which viruses modify trafficking patterns of infected cells.