Early Stages Of Replication Research Articles

Antiviral effect in association with anti-apoptosis and anti-autophagy of repurposing formoterol fumarate dihydrate on enterovirus A71-infected neuronal cells

ObjectiveTo investigate antiviral activity, anti-apoptosis and anti-autophagy associated with antiviral effect of repurposing formoterol fumarate dihydrate (FFD) against enterovirus A71 (EV-A71) infection in human neuroblastoma cells. MethodsIn vitro antiviral effects of FFD against EV-A71 infection were examined in human neuroblastoma SK-N-SH cells. The impacts on EV-A71 replication were evaluated by progeny virus production, viral RNA synthesis, and viral protein expression. The target of action of FFD against EV-A71 was determined from the effective stage by time-of-addition assay. Moreover, the anti-apoptosis and anti-autophagy activities associated with antiviral effect were observed by detection of apoptosis- and autophagy-related proteins. ResultsFFD significantly inhibited EV-A71 replication in neuronal cells through interfering the early stages of replication cycle which might be the steps during uncoating to viral protein synthesis. Additionally, FFD culminated in reducing of EV-A71-induced apoptosis and autophagy with caspase-3-cleaved form and LC3-II expression levels showed markedly decreased while increasing of Bcl-2 and mTOR expression levels. These might indicate the neuroprotective effect of FFD on EV-A71-induced apoptosis and autophagy. ConclusionsPreliminary mode of action studies showed that repurposing FFD significantly inhibited EV-A71 replication at early stage of viral replication and exhibited anti-apoptosis and anti-autophagy activities in neuronal cells. These findings may provide an opportunity, via drug repurposing of FFD, for a candidate antiviral drug against EV-A71 infection.

Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences.

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.

Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

Glycomic Analysis Reveals That Sialyltransferase Inhibition Is Involved in the Antiviral Effects of Arbidol

ABSTRACTDue to the high mutation rate of influenza virus and the rapid increase of drug resistance, it is imperative to discover host-targeting antiviral agents with broad-spectrum antiviral activity. Considering the discrepancy between the urgent demand of antiviral drugs during an influenza pandemic and the long-term process of drug discovery and development, it is feasible to explore host-based antiviral agents and strategies from antiviral drugs on the market. In the current study, the antiviral mechanism of arbidol (ARB), a broad-spectrum antiviral drug with potent activity at early stages of viral replication, was investigated from the aspect of hemagglutinin (HA) receptors of host cells. N-glycans that act as the potential binding receptors of HA on 16-human bronchial epithelial (16-HBE) cells were comprehensively profiled for the first time by using an in-depth glycomic approach based on TiO2-PGC chip-Q-TOF MS. Their relative levels upon the treatment of ARB and virus were carefully examined by employing an ultra-high sensitive qualitative method based on Chip LC-QQQ MS, showing that ARB treatment led to significant and extensive decrease of sialic acid (SA)-linked N-glycans (SA receptors), and thereby impaired the virus utilization on SA receptors for rolling and entry. The SA-decreasing effect of ARB was demonstrated to result from its inhibitory effect on sialyltransferases (ST), ST3GAL4 and ST6GAL1 of 16-HBE cells. Silence of STs, natural ST inhibitors, as well as sialidase treatment of 16-HBE cells, resulted in similar potent antiviral activity, whereas ST-inducing agent led to the diminished antiviral effect of ARB. These observations collectively suggesting the involvement of ST inhibition in the antiviral effect of ARB.IMPORTANCE This study revealed, for the first time, that ST inhibition and the resulted destruction of SA receptors of host cells may be an underlying mechanism for the antiviral activity of ARB. ST inhibition has been proposed as a novel host-targeting antiviral approach recently and several compounds are currently under exploration. ARB is the first antiviral drug on the market that was found to possess ST inhibiting function. This will provide crucial evidence for the clinical usages of ARB, such as in combination with neuraminidase (NA) inhibitors to exert optimized antiviral effect, etc. More importantly, as an agent that can inhibit the expression of STs, ARB can serve as a novel lead compound for the discovery and development of host-targeting antiviral drugs.

The possible role of ursolic acid in Covid-19: A real game changer.

Ursolic acid (UA) is a pentacyclic terpenoid is usually found in the fruit peels and stem bark as secondary metabolites. UA has antiviral, antibacterial, and antiparasitic properties. UA has a wide spectrum of pharmacological activities against different infections. Because of the greatest antiviral and anti-inflammatory properties of UA, so it could be a plausible therapeutic herbal medicine in Covid-19 treatment. Covid-19 is a recent worldwide virulent disease pandemic due to severe acute respiratory coronavirus disease 2 (SARS-CoV-2). The pathogenesis of SARS-CoV-2 infection is related to the direct cytopathic effect and exaggerated immune response by which acute lung injury (ALI) and/or acute respiratory distress syndrome might be developed in critical cases. UA may inhibit main protease of SARS-CoV-2, and inhibits the interface flanked by SARS-CoV-2 viral proteins and its entry point commonly recognized as angiotensin converting enzyme 2 (ACE2). In addition, UA attenuates SARS-CoV-2-induced inflammatory reactions and oxidative stress.Therefore, UA could avert SARS-CoV-2 infection from causing ALI. This opinion proposed that UA might be a potential candidate therapy against Covid-19 and can mitigate post-Covid-19 complications such as lung fibrosis. In this regards, forthcoming studies are reasonable to substantiate the therapeutic role of UA in Covid-19. However, taken into account that Covid-19 is yet to be investigating for further evaluations, therefore, clinical trials are recommended regarding use and dose of UA in Covid-19 treatment, as well as secondary effects.

Severity of COVID-19 patients with coexistence of asthma and vitamin D deficiency

Coronavirus disease 2019 (COVID-19)-driven global pandemic triggered innumerable health complications, imposing great challenges in managing other respiratory diseases like asthma. Furthermore, increases in the underlying inflammation involved in the fatality of COVID-19 have been linked with lack of vitamin D. In this research work, we intend to investigate the possible genetic linkage of asthma and vitamin D deficiency with the severity and fatality of COVID-19 using a network-based approach. We identified and analysed 41 and 14 differentially expressed genes (DEGs) of COVID-19 being common with asthma and vitamin D deficiency, respectively, through the comparative differential gene expression analysis and their footprints on signalling pathways. Gene set enrichment analysis for GO terms and signalling pathways reveals key biological activities, including inflammatory response-related pathways (e.g., cytokine- and chemokine-mediated signalling pathways, IL-17, and TNF signalling pathways). Besides, the Protein–Protein Interaction network analysis of those DEGs reveals hub proteins, some of which are reported as inflammatory antiviral interferon-stimulated biomarkers that potentially drive the cytokine storm leading to COVID-19 severity and fatality, and contributes in the early stage of viral replication, respectively. Moreover, the regulatory network analysis found these DEGs associated with antiviral and tumour inhibitory transcription factors and micro-RNAs. Finally, drug–target enrichment analysis yields tetradioxin, estradiol, arsenenous acid, and zinc, which have been reported to be effective in suppressing the pro-inflammatory cytokines production, and other respiratory tract infections. Our results yield shared biomarker-driven key hypotheses followed by network-based analytics, demystifying the mechanistic details of COVID-19 comorbidity of asthma and vitamin D deficiency with their potential therapeutic implications.

HSP90 inhibitors 17-AAG and VER-82576 inhibit porcine deltacoronavirus replication in vitro

Porcine deltacoronavirus (PDCoV) is highly pathogenic to piglets, and no specific drugs or vaccines are available for the prevention and treatment of PDCoV infection, the need for antiviral therapies is pressing. HSP90 inhibitors have potent inhibitory effects against the replication of numerous viruses, hence we evaluated three HSP90 inhibitors, 17-AAG, VER-82576, and KW-2478, for their effects on PDCoV infection in vitro. We evaluated their effectivenesses at suppressing PDCoV by qRT-PCR, western blot, and TCID50 assay, and found that 17-AAG and VER-82576 inhibited PDCoV at the early stage of replication, while KW-2478 showed no significant antiviral activity at any stage of infection. These results indicated that the PDCoV-inhibitory effects of 17-AAG and VER-82576 might be exerted by targeting host cell factor HSP90AB1 but not HSP90AA1. Further study showed that HSP90AB1 mRNA and protein levels were not significantly different in 17-AAG and VER-82576-treated cells versus control cells. 17-AAG and VER-82576 were also evaluated for their effects on the expressions of TNF-α, IL-6, and IL-12, which are PDCoV-induced proinflammatory cytokines. We found that both 17-AAG and VER-82576 inhibited the expressions of TNF-α, IL-6, and IL-12 to varying degrees, but in a dose dependent manner. From our data we can conclude that the HSP90 inhibitors 17-AAG and VER-82576 are promising candidates for the treatment of PDCoV infection.

Dengue virus replication enhances labile zinc pools by modulation of ZIP8

Zinc-dependent viral proteins rely on intracellular zinc homeostasis for successful completion of infectious life-cycle. Here, we report that the intracellular labile zinc levels were elevated at early stages of dengue virus (DENV) infection in hepatic cells and this increase in free zinc was abolished in cells infected with UV-inactivated virus or with a DENV replication inhibitor implicating a role for zinc homeostasis in viral RNA replication. This change in free zinc was mediated by zinc transporter, ZIP8, as siRNA-mediated knockdown of ZIP8 resulted in abrogation of increase in free zinc levels leading to significant reduction in DENV titers suggesting a crucial role for ZIP8 in early stages of DENV replication. Furthermore, elevated free zinc levels correlated with high copy numbers of dengue genome in peripheral blood leukocytes obtained from dengue patients compared to healthy controls suggesting a critical role for zinc homeostasis in dengue infection.Take AwaysDengue virus utilises cellular zinc homeostasis during replication of its RNA.ZIP8 upregulates free zinc levels during dengue virus replication.Enhanced viremia associates with elevated intracellular free zinc in dengue.

Design, synthesis, and mechanistic investigations of phenylalanine derivatives containing a benzothiazole moiety as HIV-1 capsid inhibitors with improved metabolic stability

Further clinical development of PF74, a lead compound targeting HIV-1 capsid, is impeded by low antiviral activity and inferior metabolic stability. By modifying the benzene (region I) and indole of PF74, we identified two potent compounds (7m and 7u) with significantly improved metabolic stability. Compared to PF74, 7u displayed greater metabolic stability in human liver microsomes (HLMs) with half-life (t1/2) 109-fold that of PF74. Moreover, mechanism of action (MOA) studies demonstrated that 7m and 7u effectively mirrored the MOA of compounds that interact within the PF74 interprotomer pocket, showing direct and robust interactions with recombinant CA, and 7u displaying antiviral effects in both the early and late stages of HIV-1 replication. Furthermore, MD simulation corroborated that 7u was bound to the PF74 binding site, and the results of the online molinspiration software predicted that 7m and 7u had desirable physicochemical properties. Unexpectedly, this series of compounds exhibited better antiviral activity than PF74 against HIV-2, represented by compound 7m whose anti-HIV-2 activity was almost 5 times increased potency over PF74. Therefore, we have rationally redesigned the PF74 chemotype to inhibitors with novel structures and enhanced metabolic stability in this study. We hope that these new compounds can serve as a blueprint for developing a new generation of HIV treatment regimens.

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture

We have previously reported that bovine papillomavirus type 1 (BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae (budding yeast) cultures (Zhao and Frazer 2002, Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication (day 3–16), middle weak replication (day 23–34/45) and late stable replication (day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs (cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.

The Role of Capsid in HIV-1 Nuclear Entry.

HIV-1 can infect non-dividing cells. The nuclear envelope therefore represents a barrier that HIV-1 must traverse in order to gain access to the host cell chromatin for integration. Hence, nuclear entry is a critical step in the early stages of HIV-1 replication. Following membrane fusion, the viral capsid (CA) lattice, which forms the outer face of the retroviral core, makes numerous interactions with cellular proteins that orchestrate the progress of HIV-1 through the replication cycle. The ability of CA to interact with nuclear pore proteins and other host factors around the nuclear pore determines whether nuclear entry occurs. Uncoating, the process by which the CA lattice opens and/or disassembles, is another critical step that must occur prior to integration. Both early and delayed uncoating have detrimental effects on viral infectivity. How uncoating relates to nuclear entry is currently hotly debated. Recent technological advances have led to intense discussions about the timing, location, and requirements for uncoating and have prompted the field to consider alternative uncoating scenarios that presently focus on uncoating at the nuclear pore and within the nuclear compartment. This review describes recent advances in the study of HIV-1 nuclear entry, outlines the interactions of the retroviral CA protein, and discusses the challenges of investigating HIV-1 uncoating.

The Putative Roles and Functions of Indel, Repetition and Duplication Events in Alphavirus Non-Structural Protein 3 Hypervariable Domain (nsP3 HVD) in Evolution, Viability and Re-Emergence.

Alphavirus non-structural proteins 1–4 (nsP1, nsP2, nsP3, and nsP4) are known to be crucial for alphavirus RNA replication and translation. To date, nsP3 has been demonstrated to mediate many virus–host protein–protein interactions in several fundamental alphavirus mechanisms, particularly during the early stages of replication. However, the molecular pathways and proteins networks underlying these mechanisms remain poorly described. This is due to the low genetic sequence homology of the nsP3 protein among the alphavirus species, especially at its 3′ C-terminal domain, the hypervariable domain (HVD). Moreover, the nsP3 HVD is almost or completely intrinsically disordered and has a poor ability to form secondary structures. Evolution in the nsP3 HVD region allows the alphavirus to adapt to vertebrate and insect hosts. This review focuses on the putative roles and functions of indel, repetition, and duplication events that have occurred in the alphavirus nsP3 HVD, including characterization of the differences and their implications for specificity in the context of virus–host interactions in fundamental alphavirus mechanisms, which have thus directly facilitated the evolution, adaptation, viability, and re-emergence of these viruses.

Increased CDC6 Expression Associates With Poor Prognosis in Patients With Clear Cell Renal Cell Carcinoma.

BackgroundCDC6 (Cell division control protein 6), located at chromosome 17q21.3, plays an important role in the early stage of DNA replication and has unique functions in various malignant tumors. Here, we evaluate the relationship between CDC6 expression and oncology outcomes in patients with clear cell renal cell carcinoma (ccRCC).MethodsA retrospective analysis of 118 ccRCC patients in Affiliated Hospital of Nantong University from 2015 to 2017 was performed. Triplicate tissue microarrays (TMA) were prepared from formalin-fixed and paraffin-embedded specimens. Immunohistochemistry (IHC) was conducted to evaluate the relationship between CDC6 expression and standard pathological features and prognosis. The RNA sequencing data and corresponding clinical information were acquired from the TCGA database. GSEA was used to identify signal pathways related to CDC6. Cox regression analysis was used to assess independent prognostic factors. In addition, the relationship between CDC6 and immunity was also investigated.ResultsThe results of Kaplan–Meier curve indicated that the OS of the patients with high expression of CDC6 was shorter than that of the patients with low CDC6 expression. Integrating the TCGA database and IHC staining, the results showed that CDC6 in ccRCC tissue was obviously up-regulated compared with adjacent normal kidney tissue. The results of Logistic regression analysis demonstrated that ccRCC patients with high expression of CDC6 are more likely to develop advanced disease than ccRCC patients with low CDC6 expression. The results of GSEA showed that the high expression of CDC6 was related to multiple signaling pathways. As for immunity, it was also related to TMB, immune checkpoint molecules, tumor microenvironment and immune infiltration. There were significantly correlations with CDC6 and immune cell infiltration levels and tumor microenvironment. The results of further results of the TCGA database showed that CDC6 was obviously related to immune checkpoint molecules and immune cells.ConclusionsIncreased expression of CDC6 is a potentially prognostic factor of poor prognosis in ccRCC patients.

GS-441524 inhibits African swine fever virus infection in vitro

African swine fever virus (ASFV) is a highly infectious and lethal swine pathogen that causes serious socio-economic consequences in endemic countries for which no safe and effective vaccine is currently available. GS-441524, a 1-cyano-substituted adenine C-nucleoside ribose analogue, inhibits viral RNA transcription by competing with natural nucleosides (ATP, TTP, CTP, and GTP) and effectively inhibits viral RNA-dependent RNA polymerase activity. However, whether GS-441524 can inhibit the replication of DNA viruses is unknown. In this study, we confirmed that GS-441524 inhibits ASFV infection in porcine alveolar macrophages (PAMs) in a dose-dependent manner; GS-441524 significantly inhibited ASFV replication at different time points after ASFV infection, particularly at the early stages of viral replication. Notably, GS-441524 did not increase the levels of antiviral cytokines or ATP in PAMs. However, an increase in the concentration of natural ATP in PAMs promoted the replication of ASFV and attenuated the inhibitory effect of GS-441524 in a dose-dependent manner. Our results suggest that GS-441524 is an effective antiviral against ASFV.

Oxidative stress specifically inhibits replication of dengue virus.

Reactive oxygen species (ROS) are chemically active species which are involved in maintaining cellular and signalling processes at physiological concentrations. Therefore, cellular components that regulate redox balance are likely to play a crucial role in viral life-cycle either as promoters of viral replication or with antiviral functions. Zinc is an essential micronutrient associated with anti-oxidative systems and helps in maintaining a balanced cellular redox state. Here, we show that zinc chelation leads to induction of reactive oxygen species (ROS) in epithelial cells and addition of zinc restores ROS levels to basal state. Addition of ROS (H2O2) inhibited dengue virus (DENV) infection in a dose-dependent manner indicating that oxidative stress has adverse effects on DENV infection. ROS affects early stages of DENV replication as observed by quantitation of positive and negative strand viral RNA. We observed that addition of ROS specifically affected viral titres of positive strand RNA viruses. We further demonstrate that ROS specifically altered SEC31A expression at the ER suggesting a role for SEC31A-mediated pathways in the life-cycle of positive strand RNA viruses and provides an opportunity to identify drug targets regulating oxidative stress responses for antiviral development.

Alpha-mangostin inhibits dengue virus production and pro-inflammatory cytokine/chemokine expression in dendritic cells.

Dengue virus (DENV) is transmitted to humans via the bite of an Aedes mosquito, causing dengue fever, dengue hemorrhagic fever, or dengue shock syndrome. In the human skin, DENV first infects keratinocytes, dendritic cells, and macrophages. Monocytes that are recruited to the site of infection and differentiate into monocyte-derived dendritic cells (moDCs) are also infected by DENV. DENV-infected DCs secrete pro-inflammatory cytokines and chemokines to modulate the immune response. The viral load and massive pro-inflammatory cytokine/chemokine production, referred to as a 'cytokine storm', are associated with disease severity. We propose that an ideal drug for treatment of DENV infection should inhibit both virus production and the cytokine storm, and previously, we reported that alpha-mangostin (α-MG) inhibits both DENV replication and cytokine production in hepatocytes. However, the effect of α-MG on DENV-infected moDCs remains unknown. In this study, we investigated the effects of α-MG on DENV infection and pro-inflammatory cytokine/chemokine production in primary moDCs generated ex vivo from monocytes of healthy individuals. α-MG at the non-toxic concentrations of 20 and 25 μM reduced DENV production by more than 10-fold and 1,000-fold, respectively. Treatment with α-MG efficiently inhibited the infection of immature moDCs by all four serotypes of DENV. Time-of-addition studies suggested that α-MG (25 μM) inhibits DENV at the early stage of replication. In addition, α-MG markedly reduced cytokine/chemokine (TNF-α, CCL4, CCL5, CXCL10, IL6, IL1β, IL10, and IFN-α) transcription in DENV-infected immature moDCs. These findings suggest the potential of α-MG to be developed as a novel anti-DENV drug.

Clofazimine: A Promising Inhibitor of Rabies Virus.

With an almost 100% mortality rate, rabies virus (RABV) infection is a global concern. Limited post-exposure prophylaxis and lack of an effective treatment necessitate novel antiviral therapies against RABV. Here, using a high-throughput screening (HTS) method developed in our lab, 11 candidates with anti-RABV activity were identified from a library of 767 clinical drugs. Clofazimine (CFZ), an anti-leprosy drug, displayed an EC50 of 2.28 μM, and SI over 967 against RABV. Investigations into the underlying mechanisms revealed that CFZ targeted viral membrane fusion at the early stages of virus replication. Moreover, CFZ and Clofazimine salicylates (CFZS) exhibited elevated survival rates in vivo, compared with the positive control T-705. Thus, this study revealed CFZ as a promising drug against RABV infection.

Interactions of HIV-1 Capsid with Host Factors and Their Implications for Developing Novel Therapeutics.

The Human Immunodeficiency Virus type 1 (HIV-1) virion contains a conical shell, termed capsid, encasing the viral RNA genome. After cellular entry of the virion, the capsid is released and ensures the protection and delivery of the HIV-1 genome to the host nucleus for integration. The capsid relies on many virus–host factor interactions which are regulated spatiotemporally throughout the course of infection. In this paper, we will review the current understanding of the highly dynamic HIV-1 capsid–host interplay during the early stages of viral replication, namely intracellular capsid trafficking after viral fusion, nuclear import, uncoating, and integration of the viral genome into host chromatin. Conventional anti-retroviral therapies primarily target HIV-1 enzymes. Insights of capsid structure have resulted in a first-in-class, long-acting capsid-targeting inhibitor, GS-6207 (Lenacapavir). This inhibitor binds at the interface between capsid protein subunits, a site known to bind host factors, interferes with capsid nuclear import, HIV particle assembly, and ordered assembly. Our review will highlight capsid structure, the host factors that interact with capsid, and high-throughput screening techniques, specifically genomic and proteomic approaches, that have been and can be used to identify host factors that interact with capsid. Better structural and mechanistic insights into the capsid–host factor interactions will significantly inform the understanding of HIV-1 pathogenesis and the development of capsid-centric antiretroviral therapeutics.

Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry

Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmi...

In Silico, In Vitro and In Cellulo Models for Monitoring SARS-CoV-2 Spike/Human ACE2 Complex, Viral Entry and Cell Fusion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.