Viral Replication Complex Research Articles

A pan-serotype dengue virusinhibitor targeting the NS3-NS4Binteraction.

Dengue virus causes approximately 96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue1,2. There are no antiviralagents available to prevent or treat dengue. Here, we describe a highly potent dengue virus inhibitor (JNJ-A07) that exerts nanomolar to picomolar activity against a panel of 21 clinical isolates that represent the natural genetic diversity of known genotypes and serotypes. The molecule has a high barrier to resistance and prevents the formation of the viral replication complex by blocking the interaction between two viral proteins (NS3 and NS4B), thus revealing a previously undescribed mechanism of antiviral action. JNJ-A07 has a favourable pharmacokinetic profile that results in outstanding efficacy against dengue virus infection in mouse infection models. Delaying start of treatment until peak viraemia results in a rapid and significant reduction in viral load. An analogue is currently in further development.

Cellular tropism of SARS-CoV-2 in the respiratory tract of Syrian hamsters and B6.Cg-Tg(K18-ACE2)2Prlmn/J transgenic mice.

Several animal models have been developed to study the pathophysiology of SARS-CoV-2 infection and to evaluate vaccines and therapeutic agents for this emerging disease. Similar to infection with SARS-CoV-1, infection of Syrian hamsters with SARS-CoV-2 results in moderate respiratory disease involving the airways and lung parenchyma but does not lead to increased mortality. Using a combination of immunohistochemistry and transmission electron microscopy, we showed that the epithelium of the conducting airways of hamsters was the primary target for viral infection within the first 5 days of infection, with little evidence of productive infection of pneumocytes. At 6 days postinfection, antigen was cleared but parenchymal damage persisted, and the major pathological changes resolved by day 14. These findings are similar to those previously reported for hamsters with SARS-CoV-1 infection. In contrast, infection of K18-hACE2 transgenic mice resulted in pneumocyte damage, with viral particles and replication complexes in both type I and type II pneumocytes together with the presence of convoluted or cubic membranes; however, there was no evidence of virus replication in the conducting airways. The Syrian hamster is a useful model for the study of SARS-CoV-2 transmission and vaccination strategies, whereas infection of the K18-hCE2 transgenic mouse results in lethal disease with fatal neuroinvasion but with sparing of conducting airways.

Role of Heat Shock Proteins (HSP70 and HSP90) in Viral Infection.

Heat shock proteins (HSPs) are a large group of chaperones found in most eukaryotes and bacteria. They are responsible for the correct protein folding, protection of the cell against stressors, presenting immune and inflammatory cytokines; furthermore, they are important factors in regulating cell differentiation, survival and death. Although the biological function of HSPs is to maintain cell homeostasis, some of them can be used by viruses both to fold their proteins and increase the chances of survival in unfavorable host conditions. Folding viral proteins as well as replicating many different viruses are carried out by, among others, proteins from the HSP70 and HSP90 families. In some cases, the HSP70 family proteins directly interact with viral polymerase to enhance viral replication or they can facilitate the formation of a viral replication complex and/or maintain the stability of complex proteins. It is known that HSP90 is important for the expression of viral genes at both the transcriptional and the translational levels. Both of these HSPs can form a complex with HSP90 and, consequently, facilitate the entry of the virus into the cell. Current studies have shown the biological significance of HSPs in the course of infection SARS-CoV-2. A comprehensive understanding of chaperone use during viral infection will provide new insight into viral replication mechanisms and therapeutic potential. The aim of this study is to describe the molecular basis of HSP70 and HSP90 participation in some viral infections and the potential use of these proteins in antiviral therapy.

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector.

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.

Nuclear exportin 1 facilitates turnip mosaic virus infection by exporting the sumoylated viral replicase and by repressing plant immunity.

Exportin 1/XPO1 is an important nuclear export receptor that binds directly to cargo proteins and translocates the cargo proteins to the cytoplasm. To understand XPO1 protein functions during potyvirus infections, we investigated the nuclear export of the NIb protein encoding the RNA-dependent RNA polymerase (RdRp) of turnip mosaic virus (TuMV). Previously, we found that NIb is transported to the nucleus after translation and sumoylated by the sumoylation (small ubiquitin-like modifier) pathway to support viral infection. Here, we report that XPO1 interacts with NIb to facilitate translocation from the nucleus to the viral replication complexes (VRCs) that accumulate in the perinuclear regions of TuMV-infected cells. XPO1 contains two NIb-binding domains that recognize and interact with NIb in the nucleus and in the perinuclear regions, respectively, which facilitates TuMV replication. Moreover, XPO1 is involved in nuclear export of the sumoylated NIb and host factors tagged with SUMO3 that is essential for suppression of plant immunity in the nucleus. Deficiencies of XPO1 in Arabidopsis and Nicotiana benthamiana plants inhibit TuMV replication and infection. These data demonstrate that XPO1 functions as a host factor in TuMV infection by regulating NIb nucleocytoplasmic transport and plant immunity.

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism.

Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.

Molecular dynamics simulations of allosteric motions and competitive inhibition of the Zika virus helicase

The 2015 Zika outbreak sparked major global concern and emphasized the reality and dangers still posed by mosquito borne pathogens. While efforts have been made to develop a vaccine and other therapeutics, there is still a great demand for antiviral drugs targeting Zika and other flaviviruses. The non-structural protein 3 (NS3) helicase is a vital component of the viral replication complex, tasked with unwinding the viral dsRNA molecule into single strands. Given this critical function, the Zika virus helicase is a potential therapeutic target and the focus of many ongoing research efforts. Using a combination of drug docking and molecular dynamics simulations, we have identified a list of competitive helicase inhibitors targeting the ATP hydrolysis site and have discovered a potential allosteric site capable of distorting both of the protein's active sites.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

ABSTRACTCharacterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation.IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Role of Lipid Transfer Proteins (LTPs) in the Viral Life Cycle.

Viruses are obligate parasites that depend on the host cell machinery for their replication and dissemination. Cellular lipids play a central role in multiple stages of the viral life cycle such as entry, replication, morphogenesis, and egress. Most viruses reorganize the host cell membranes for the establishment of viral replication complex. These specialized structures allow the segregation of replicating viral RNA from ribosomes and protect it from host nucleases. They also facilitate localized enrichment of cellular components required for viral replication and assembly. The specific composition of the lipid membrane governs its ability to form negative or positive curvature and possess a rigid or flexible form, which is crucial for membrane rearrangement and establishment of viral replication complexes. In this review, we highlight how different viruses manipulate host lipid transfer proteins and harness their functions to enrich different membrane compartments with specific lipids in order to facilitate multiple aspects of the viral life cycle.

Membrane-Associated Flavivirus Replication Complex-Its Organization and Regulation.

Flavivirus consists of a large number of arthropod-borne viruses, many of which cause life-threatening diseases in humans. A characteristic feature of flavivirus infection is to induce the rearrangement of intracellular membrane structure in the cytoplasm. This unique membranous structure called replication organelle is considered as a microenvironment that provides factors required for the activity of the flaviviral replication complex. The replication organelle serves as a place to coordinate viral RNA amplification, protein translation, and virion assembly and also to protect the viral replication complex from the cellular immune defense system. In this review, we summarize the current understanding of how the formation and function of membrane-associated flaviviral replication organelle are regulated by cellular factors.

NAP1L1 and NAP1L4 Binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein Is Bivalent and Requires Phosphorylation.

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that (i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication, (ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD, (iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD, (iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD, and HVD phosphorylation is mediated by CK2 kinase, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCE Cellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We show that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection.

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.

TMEM41B is a host factor required for the replication of diverse coronaviruses including SARS-CoV-2.

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics.

A Novel Series of Indole Alkaloid Derivatives Inhibit Dengue and Zika Virus Infection by Interference with the Viral Replication Complex.

Here, we identified a novel class of compounds which demonstrated good antiviral activity against dengue and Zika virus infection. These derivatives constitute intermediates in the synthesis of indole (ervatamine-silicine) alkaloids and share a tetracyclic structure, with an indole and a piperidine fused to a seven-membered carbocyclic ring. Structure-activity relationship studies indicated the importance of substituent at position C-6 and especially the presence of a benzyl ester for the activity and cytotoxicity of the molecules. In addition, the stereochemistry at C-7 and C-8, as well as the presence of an oxazolidine ring, influenced the potency of the compounds. Mechanism of action studies with two analogues of this family (compounds 22 and trans-14) showed that this class of molecules can suppress viral infection during the later stages of the replication cycle (RNA replication/assembly). Moreover, a cell-dependent antiviral profile of the compounds against several Zika strains was observed, possibly implying the involvement of a cellular factor(s) in the activity of the molecules. Sequencing of compound-resistant Zika mutants revealed a single nonsynonymous amino acid mutation (aspartic acid to histidine) at the beginning of the predicted transmembrane domain 1 of NS4B protein, which plays a vital role in the formation of the viral replication complex. To conclude, our study provides detailed information on a new class of NS4B-associated inhibitors and strengthens the importance of identifying host-virus interactions in order to tackle flavivirus infections.

Therapeutic Potential of Exploiting Autophagy Cascade Against Coronavirus Infection.

Since its emergence in December 2019 in Wuhan, China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created a worldwide pandemic of coronavirus disease (COVID-19) with nearly 136 million cases and approximately 3 million deaths. Recent studies indicate that like other coronaviruses, SARS-CoV-2 also hijacks or usurps various host cell machineries including autophagy for its replication and disease pathogenesis. Double membrane vesicles generated during initiation of autophagy cascade act as a scaffold for the assembly of viral replication complexes and facilitate RNA synthesis. The use of autophagy inhibitors - chloroquine and hydroxychloroquine initially appeared to be as a potential treatment strategy of COVID-19 patients but later remained at the center of debate due to high cytotoxic effects. In the absence of a specific drug or vaccine, there is an urgent need for a safe, potent as well as affordable drug to control the disease spread. Given the intricate connection between autophagy machinery and viral pathogenesis, the question arises whether targeting autophagy pathway might show a path to fight against SARS-CoV-2 infection. In this review we will discuss about our current knowledge linking autophagy to coronaviruses and how that is being utilized to repurpose autophagy modulators as potential COVID-19 treatment.

Light and electron microscopy imaging unveils new aspects of the antiviral capacity of silver nanoparticles in bunyavirus-infected cells

Drug repurposing is an important source of new antivirals because many compounds used to treat a variety of pathologies also hamper viral infections. Habitually, silver nanoparticles (AgNPs) have been used to treat bacterial and fungal infections and their antiviral properties have been also reported. In this work, we have studied the antiviral capacity of AgNPs in cells infected with Bunyamwera virus (BUNV), the prototype of the Bunyavirales order. This group of viruses contains important pathogens for humans, animals and plants. Incubation of BUNV-infected Vero cells with non-toxic concentrations of AgNPs, reduced the production of extracellular infectious viruses in up to three orders of magnitude. With a combination of imaging techniques, we have visualized the intracellular distribution of AgNPs in mock- and BUNV-infected cells and studied their effects on intracellular organelles. In mock-infected cells and at short times post-incubation, AgNPs were detected inside nuclei and mitochondria by transmission electron microscopy (TEM). At long times post-treatment, they accumulated inside lysosome-like organelles. Cell compartments did not exhibit any appreciable ultrastructural alterations after incubation with AgNPs. In BUNV-infected cells, AgNPs attached to extracellular virions, that showed a disrupted morphology. Inside cells, they were detected inside the nucleus, in mitochondria and around characteristic Golgi-associated, single-membrane spherules. These membranous structures are the replication organelles (ROs) of bunyaviruses and contain active viral replication complexes (VRCs). Compared to normal spherules that are round, compact and have an electron-dense core, spherules in AgNPs-treated cells were deformed and their core was electron-lucent. Interestingly, in BUNV-infected cells treated with the typical antiviral ribavirin (RBV), spherules with VRCs exhibit also an anomalous morphology and an electron-lucent core. Both AgNPs and RBV might interfere with BUNV-induced dismantling of cell nucleoli and with the intercellular propagation of large groups of virions, a mechanism of BUNV transmission observed for the first time in cultured cells. Our results point to silver nanoparticles as good candidates for antiviral therapy, either alone or in combination with other antiviral drugs, such as RBV-related compounds.

Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods.

Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.

Differences in Interactions Within Viral Replication Complexes of SARS-CoV-2 (COVID-19) and SARS-CoV Coronaviruses Control RNA Replication Ability.

COVID-19 has become a global pandemic caused by the SARS-CoV-2 coronavirus. SARS-CoV-2 shares many similarities with SARS coronavirus (SARS-CoV). A viral replication complex containing non-structural proteins (nsps) is the toolbox for RNA replication and transcription of both coronaviruses. In both cases, the RNA-dependent RNA polymerase (RdRp) domain of the coronaviral replication complex dictates the primary polymerase activity by cooperating with cofactors. The higher transmissibility and mortality due to SARS-CoV-2 are related to its higher RNA replication activity compared to SARS-CoV. The discrepancy between the RNA replication efficiency of SARS-CoV and SARS-CoV-2 can be understood by exploring interactions within their viral replication complexes. Our modeling of molecular interactions within the viral replication complexes of SARS-CoV and SARS-CoV-2 using molecular dynamics simulations suggests that in contrast to SARS-CoVnsp12, SARS-CoV2nsp12 prefers helices as the dominant interacting secondary motifs. The relative differences in nonbonded interactions between nsps could suggest viral RNA replication ability in coronaviruses.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11837-021-04662-6.

Novel Analogues of the Chikungunya Virus Protease Inhibitor: Molecular Design, Synthesis, and Biological Evaluation.

The Chikungunya virus (CHIKV) is an arbovirus belonging to the genus Alphavirus of the Togaviridae family. CHIKV is transmitted by the mosquitoes and causes Chikungunya fever. CHIKV outbreaks have occurred in Africa, Asia, Europe, and the countries of Indian and Pacific Oceans. In 2013, CHIKV cases were registered for the first time in the Americas on the Caribbean islands. There is currently no vaccine to prevent or medicines to treat CHIKV infection. The CHIKV nonstructural protease (nsP2) is a promising potential target for the development of drugs against CHIKV infection because this protein is one of the key components of the viral replication complex and is involved in multiple steps of virus infection. In this work, novel analogues of the potential CHIKV nsP2 protease inhibitor, first reported by Das et al. in 2016, were identified using molecular modeling methods, synthesized, and evaluated in vitro. The optimization of the structure of the inhibitor allowed to increase the antiviral activity of the compound 2–10 times. The possible mechanism of action of the identified potential inhibitors of the CHIKV nsP2 protease was studied in detail using molecular dynamics (MD) simulations. According to the MD results, the most probable mechanism of action is the blocking of conformational changes in the nsP2 protease required for substrate recognition and binding.

HSP70 plays an ambiguous role during viral infections in plants

Heat shock and almost all types of stresses associated with oxidative stress are accompanied by heat shock protein (HSP) expression. HSPs are involved in refolding denatured proteins and directing unrepairable proteins for degradation. Thus, under stress conditions, HSPs help to restore cellular balance. However, in virus-infected plants, HSP70 can have both positive and negative effects because viruses usually recruit HSP70. HSP70 can promote the replication and translation of the viral genome, the formation of viral replication complexes, and the propagation of viral particles from cell to cell and throughout the plant. HSP gene silencing in various virus-host plants systems and the comparison of susceptible and resistant species have shown that HSPs70 accelerate the development of infection. Conversely, during the process known as thermotherapy, the temperature increase inhibits viral replication in some host and virus systems. The success of thermotherapy depends not only on the temperature and treatment period or duration but also on the plant species and viral strain. In this review, we discuss the ambiguous role that HSPs70 play during viral infections in plants towards weighing the balance between their positive and negative functions.