Production Of Infectious Virus Particles Research Articles

Neural Infection by Oropouche Virus in Adult Human Brain Slices Induces an Inflammatory and Toxic Response.

Oropouche virus (OROV) is an emerging arbovirus in South and Central Americas with high spreading potential. OROV infection has been associated with neurological complications and OROV genomic RNA has been detected in cerebrospinal fluid from patients, suggesting its neuroinvasive potential. Motivated by these findings, neurotropism and neuropathogenesis of OROV have been investigated in vivo in murine models, which do not fully recapitulate the complexity of the human brain. Here we have used slice cultures from adult human brains to investigate whether OROV is capable of infecting mature human neural cells in a context of preserved neural connections and brain cytoarchitecture. Our results demonstrate that human neural cells can be infected ex vivo by OROV and support the production of infectious viral particles. Moreover, OROV infection led to the release of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and diminished cell viability 48 h post-infection, indicating that OROV triggers an inflammatory response and tissue damage. Although OROV-positive neurons were observed, microglia were the most abundant central nervous system (CNS) cell type infected by OROV, suggesting that they play an important role in the response to CNS infection by OROV in the adult human brain. Importantly, we found no OROV-infected astrocytes. To the best of our knowledge, this is the first direct demonstration of OROV infection in human brain cells. Combined with previous data from murine models and case reports of OROV genome detection in cerebrospinal fluid from patients, our data shed light on OROV neuropathogenesis and help raising awareness about acute and possibly chronic consequences of OROV infection in the human brain.

Characterisation of Antiviral Activity of Cathelicidins from Naked Mole Rat and Python bivittatus on Human Herpes Simplex Virus 1.

Hg-CATH and Pb-CATH4 are cathelicidins from Heterocephalus glaber and Python bivittatus that have been previously identified as potent antibacterial peptides. However, their antiviral properties were not previously investigated. In this study, their activity against the herpes simplex virus (HSV)-1 was evaluated during primary human keratinocyte infection. Both of them significantly reduced HSV-1 DNA replication and production of infectious viral particles in keratinocytes at noncytotoxic concentrations, with the stronger activity of Pb-CATH4. These peptides did not show direct virucidal activity and did not exhibit significant immunomodulatory properties, except for Pb-CATH4, which exerted a moderate proinflammatory action. All in all, our results suggest that Hg-CATH and Pb-CATH4 could be potent candidates for the development of new therapies against HSV-1.

In Vivo Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

ABSTRACTDefective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.IMPORTANCE Defective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens; no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes, with the majority affecting genes required for viral replication. Longitudinal analysis showed that these DVGs can persist during an infection but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests that these parasitic sequences exist across the many classes of viruses capable of causing human disease.

A Thioester-Containing Protein Controls Dengue Virus Infection in Aedes aegypti Through Modulating Immune Response.

Complement-like proteins in arthropods defend against invading pathogens in the early phases of infection. Thioester-containing proteins (TEPs), which exhibit high similarity to mammalian complement C3, are thought to play a key role in the innate immunity of arthropods. We identified and characterized anti-dengue virus (DENV) host factors, in particular complement-like proteins, in the mosquito Aedes aegypti. Our results indicate that TEP1 limits DENV infection in Ae. aegypti. We showed that TEP1 transcription is highly induced in mosquitoes following DENV infection. Silencing TEP1 resulted in the up-regulation of viral RNA and proteins. In addition, the production of infectious virus particles increased in the absence of TEP1. We generated a transgenic mosquito line with a TEP1 loss-of-function phenotype under a blood meal-inducible promoter. We showed that viral protein and titers increased in transgenic mosquitoes after an infectious blood meal. Interestingly, expression of transcription factor Rel2 and certain anti-microbial peptides (AMPs) were inhibited in transgenic mosquitoes. Overall, our results suggest that TEP1 regulates the immune response and consequently controls the replication of dengue virus in mosquitoes. This finding provides new insight into the molecular mechanisms of mosquito host factors in the regulation of DENV replication.

Characterizing infection of B lymphocytes with vaccine and wild-type strains of measles virus

Abstract Wild-type (w.t.) measles virus (MeV) infection can result in immunosuppression that does not occur following vaccination. A rhesus macaque model indicated that B cells are a target of infection. The role of B cell infection in MeV-induced immune suppression is not fully understood. We characterize the differences in viral replication in human B cells infected with vaccine (Edmonston Zagreb [EZ]) or w.t. virus. B cells from healthy donor blood were infected (MOI 1.0) with a GFP-expressing EZ virus or a w.t. virus to evaluate viral replication after 24 and 48 hours. The number of GFP+ cells (EZ infected) or hemagglutinin (H)+ cells (w.t. infected) was measured via flow cytometry. Transcription of the viral nucleoprotein (N) gene was measured by mRNA-specific cDNA generation and N gene specific qPCR. Production of infectious viral particles was measured via endpoint dilution. In B cells infected with EZ, 30% of cells expressed GFP at 24 hours and 45% of cells expressed GFP at 48 hours. There was a 4.4-fold increase in N gene transcription from 0 to 48 hours. There was no detectable increase in viral titer over input after 48 hours. In B cells infected with w.t. virus, 10% of cells expressed viral H at 24 hours with no increase in H expression at 48 hours. The w.t. virus had a 2.4-fold increase in viral transcription from 0 to 48 hours. There was no detectable increase in viral titer over input after 48 hours. MeV-infected B cells show viral protein production and N gene transcription at 24 and 48 hours. However, w.t. virus replication may be less efficient as shown by lower percentages of H+ cells at 24 and 48 hours and lower levels of N gene transcription at 48 hours. There is no evidence of infectious virus production by either strain of MeV despite evidence of replication.

Mayaro Virus Infects Human Brain Cells and Induces a Potent Antiviral Response in Human Astrocytes.

Mayaro virus (MAYV) and chikungunya virus (CHIKV) are known for their arthrotropism, but accumulating evidence shows that CHIKV infections are occasionally associated with serious neurological complications. However, little is known about the capacity of MAYV to invade the central nervous system (CNS). We show that human neural progenitors (hNPCs), pericytes and astrocytes are susceptible to MAYV infection, resulting in the production of infectious viral particles. In primary astrocytes, MAYV, and to a lesser extent CHIKV, elicited a strong antiviral response, as demonstrated by an increased expression of several interferon-stimulated genes, including ISG15, MX1 and OAS2. Infection with either virus led to an enhanced expression of inflammatory chemokines, such as CCL5, CXCL10 and CXCL11, whereas MAYV induced higher levels of IL-6, IL-12 and IL-15 in these cells. Moreover, MAYV was more susceptible than CHIKV to the antiviral effects of both type I and type II interferons. Taken together, this study shows that although MAYV and CHIKV are phylogenetically related, they induce different types of antiviral responses in astrocytes. This work is the first to evaluate the potential neurotropism of MAYV and shows that brain cells and particularly astrocytes and hNPCs are permissive to MAYV, which, consequently, could lead to MAYV-induced neuropathology.

Human NLRP1 Is a Sensor of 3CL Proteases from Pathogenic Coronaviruses in Lung Epithelial Cells

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly, cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also cleave and inactivate the pyroptosis executioner Gasdermin (GSDM)-D. Consequently, Caspase-3 and GSDM-E promote alternative cell pyroptosis, a process exacerbated in cells exhibiting imparied type I interferon production. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to Interferon alterations identify GSDM-E/Caspase-3 as biological markers of disease severity. Overall, our findings identify NLRP1 as a key sensor of SARS-CoV-2 infection in lung epithelia.Funding Information: This project has been funded on lab own funds from unrelated grants from the Fondation pour la Recherche Médicale (FRM) and ERC StG (INFLAME) to EM, from ERC StG (ANTIViR) to CG, by the French Ministry of Health with the participation of the Groupement Interrégional de Recherche Clinique et d’Innovation Sud-Ouest Outre-Mer (PHRCI 2020 IMMUNOMARK-COV) to G-M.B. The ASB3 structure is supported by LABEX, Investissement d’Avenir and foundation Bettencourt grants to ON. MP and RP were respectively funded by a CIFRE PhD fellowship and a research grant from Invivogen. SB is supported by a PhD fellowship from Mali ministry of education and from the FRM (FDT 12794). SALC is supported by a Vaincre La Mucoviscidose (VLM) PhD fellowship.Declaration of Interests: Authors declare no conflict of interest. Ethics Approval Statement: All donors had given written informed consent and the study was approved by the ethical review board “Comité de Protection des Personnes Est-III” (ID-RCB 2020-A01292-37).

Role of the lysosomal‐associated membrane protein 2 in the AD‐like neurodegeneration induced by HSV‐1

AbstractBackgroundA growing number of studies have shown that herpes simplex virus type 1 (HSV‐1) infection could be involved in the pathogenesis of Alzheimer´s disease. Our group have developed a neuronal cell model of HSV‐1 infection showing the characteristic markers of Alzheimer‐type neurodegeneration. Gene expression studies of this cell model identified a set of oxidative stress‐regulated genes in infected cells. Analysis of these genes revealed that the main pathway altered was the lysosomal system (Neurobiol Aging. 2018 Aug;68:5‐17. doi:0.1016/j.neurobiolaging.2018.03.025). The lysosomal‐associated membrane protein 2 (LAMP2) gene was one of the most strongly modulated ones, pointing to LAMP2 as a strong candidate to mediate the lysosomal alterations induced by the virus. The objective of the present work is to study the involvement of LAMP2 in the neurodegeneration events induced by HSV‐1 infection.MethodsIn this work, we used murine neuroblastoma cell lines (N2a) with a stable knockdown for LAMP2. LAMP2 deficient and wild type N2a cells were infected by HSV‐1 for different times. Virus replication was assessed by quantitative PCR of viral genes. Levels of viral proteins were determined by western blot and infectious virus production was evaluated by titer plaque assays. Immunocytochemistry, western blot and ELISA assays were used to monitor AD neurodegeneration markers—tau hyperphosphorylation and Aβ accumulation.ResultsIn this project, we have focused on studying the role of LAMP2 in the neurodegeneration induced by HSV‐1 using a cellular model of neuroblastoma cells that expresses very low LAMP2 levels. The results show that LAMP2 deficiency deeply affects the efficiency of HSV‐1 infection reducing: i) the levels of viral DNA, ii) the amount of viral proteins and iii) the production of infectious viral particles. In addition, LAMP2 deficiency reduces tau hyperphosphorylation and increases Aβ secretion in HSV‐1‐infected cells. These findings indicate that LAMP2 deficiency partially blocks the neurodegeneration markers induced by the virus.ConclusionAll these findings suggest that the lysosomal gene LAMP2 could be a suitable candidate to mediate the AD‐like phenotype induced by HSV‐1.

Antiviral Effects of Oleandrin.

Over the past 15 years, investigators have reported on the utility and safety of cardiac glycosides for numerous health benefits including those as treatments for malignant disease, stroke-mediated ischemic injury and certain neurodegenerative diseases. In addition to those, there is a growing body of evidence for novel antiviral effects of selected cardiac glycoside molecules. One unique cardiac glycoside, oleandrin derived from Nerium oleander, has been reported to have antiviral activity specifically against ‘enveloped’ viruses including HIV and HTLV-1. Importantly, a recent publication has presented in vitro evidence for oleandrin’s ability to inhibit production of infectious virus particles when used for treatment prior to, as well as after infection by SARS-CoV-2/COVID-19. This review will highlight the known in vitro antiviral effects of oleandrin as well as present previously unpublished effects of this novel cardiac glycoside against Ebola virus, Cytomegalovirus, and Herpes simplex viruses.

Ubiquitination of Zika virus precursor membrane protein promotes the release of viral proteins

Zika virus (ZIKV) is an important human pathogen associated with severe neurological disorders. Ubiquitination of viral proteins has diverse roles in viral life cycle and pathogenesis. Here, we found that perturbation of ubiquitin-proteasome system significantly suppressed production of infectious viral particles. Moreover, we demonstrated that ZIKV precursor membrane (prM) protein underwent ubiquitination and proteasomal degradation. Furthermore, we showed that co-expression of E protein with ubiquitination-deficient prM-6 K/6R mutant protein did not affect translocation of viral proteins into endoplasmic reticulum and trans-Golgi networks. Intriguingly, the co-expression of E and prM-6 K/6R mutant proteins led to formation of relatively aggregated viral protein complexes and resulted in diminishing secretion of viral proteins as compared to wild-type prM. Collectively, these results suggest that ubiquitinated ZIKV prM protein contributes to the release of viral proteins and provide a new insight into the mechanism involved in ZIKV replication biology.

Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication.

There are approximately 20 million events of hepatitis E virus (HEV) infection worldwide annually. The genome of HEV is a single-strand, positive-sense RNA containing 5’ and 3’ untranslated regions and three open reading frames (ORF). HEV genome has 5’ cap and 3’ poly(A) tail to mimic host mRNA to escape the host innate immune surveillance and utilize host translational machineries for viral protein translation. The replication mechanism of HEV is poorly understood, especially how the viral polymerase distinguishes viral RNA from host mRNA to synthesize new viral genomes. We hypothesize that the HEV genome contains cis-acting elements that can be recognized by the virally encoded polymerase as “self” for replication. To identify functional cis-acting elements systematically across the HEV genome, we utilized an ORF1 transcomplementation system. Ultimately, we found two highly conserved cis-acting RNA elements within the ORF1 and ORF2 coding regions that are required for viral genome replication in a diverse panel of HEV genotypes. Synonymous mutations in the cis-acting RNA elements, not altering the ORF1 and ORF2 protein sequences, significantly impaired production of infectious viral particles. Mechanistic studies revealed that the cis-acting elements form secondary structures needed to interact with the HEV ORF1 protein to promote HEV replication. Thus, these cis-acting elements function as a scaffold, providing a specific “signal” that recruits viral and host factors to assemble the viral replication complex. Altogether, this work not only facilitates our understanding of the HEV life cycle and provides novel, RNA-directed targets for potential HEV treatments, but also sheds light on the development of HEV as a therapeutic delivery vector.

Selective and ATP-competitive kinesin KIF18A inhibitor suppresses the replication of influenza A virus.

The influenza virus is one of the major public health threats. However, the development of efficient vaccines and therapeutic drugs to combat this virus is greatly limited by its frequent genetic mutations. Because of this, targeting the host factors required for influenza virus replication may be a more effective strategy for inhibiting a broader spectrum of variants. Here, we demonstrated that inhibition of a motor protein kinesin family member 18A (KIF18A) suppresses the replication of the influenza A virus (IAV). The expression of KIF18A in host cells was increased following IAV infection. Intriguingly, treatment with the selective and ATP‐competitive mitotic kinesin KIF18A inhibitor BTB‐1 substantially decreased the expression of viral RNAs and proteins, and the production of infectious viral particles, while overexpression of KIF18A enhanced the replication of IAV. Importantly, BTB‐1 treatment attenuated the activation of AKT, p38 MAPK, SAPK and Ran‐binding protein 3 (RanBP3), which led to the prevention of the nuclear export of viral ribonucleoprotein complexes. Notably, administration of BTB‐1 greatly improved the viability of IAV‐infected mice. Collectively, our results unveiled a beneficial role of KIF18A in IAV replication, and thus, KIF18A could be a potential therapeutic target for the control of IAV infection.

Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly.

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.

Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein-Barr Virus Particles.

BGLF2 is a tegument protein of the Epstein–Barr virus (EBV). This study finds that BGLF2 is expressed in the late stage of the EBV lytic cycle. Microscopic investigations reveal that BGLF2 is present in both the nucleus and the cytoplasm and colocalized with BBLF1 and gp350 at juxtanuclear regions in the cytoplasm. This study also finds that the basic KKK69 motif of BGLF2 and acidic DYEE31 motif of BBLF1 are crucial for the interaction between BGLF2 and BBLF1, which is required for the recruitment of BGLF2 to the BBLF1 that is anchored on the trans-Golgi-network (TGN). In addition, BGLF2 in a density gradient is co-sedimented with un-enveloped capsids, revealing that BGLF2 associates with the EBV capsid before the final envelopment. The knockout of BGLF2 expression is demonstrated to reduce the numbers of infectious virions that are released into the culture medium, but they do not affect the expression of lytic proteins and viral DNA replication. The production of infectious viral particles by a BGLF2-knockout mutant can be rescued by exogenously expressed BGLF2 but only partially rescued by BGLF2-3KA, which is a mutant with reduced ability to interact with BBLF1 but does not affect its ability to activate the MAPK pathway and the expression of the EBV lytic proteins, suggesting that the interaction of BGLF2 with BBLF1 is important to the efficient production of infectious viral particles during the maturation. The results of this study improve our understanding of how BGLF2 promotes EBV viral production.

In vitro and in silico anti-dengue activity of compounds obtained from Psidium guajava through bioprospecting

BackgroundFor decades, bioprospecting has proven to be useful for the identification of compounds with pharmacological potential. Considering the great diversity of Colombian plants and the serious worldwide public health problem of dengue—a disease caused by the dengue virus (DENV)—in the present study, we evaluated the anti-DENV effects of 12 ethanolic extracts derived from plants collected in the Colombian Caribbean coast, and 5 fractions and 5 compounds derived from Psidium guajava.MethodsThe cytotoxicity and antiviral effect of 12 ethanolic extracts derived from plants collected in the Colombian Caribbean coast was evaluated in epithelial VERO cells. Five fractions were obtained by open column chromatography from the ethanolic extract with the highest selectivity index (SI) (derived from P. guajava, SI: 128.2). From the fraction with the highest selectivity (Pg-YP-I-22C, SI: 35.5), five compounds were identified by one- and two-dimensional nuclear magnetic resonance spectroscopy. The antiviral effect in vitro of the fractions and compounds was evaluated by different experimental strategies (Pre- and post-treatment) using non-toxic concentrations calculated by MTT method. The DENV inhibition was evaluated by plate focus assay. The results were analyzed by means of statistical analysis using Student’s t-test. Finally the antiviral effect in Silico was evaluated by molecular docking.ResultsIn vitro evaluation of these compounds showed that three of them (gallic acid, quercetin, and catechin) were promising antivirals as they inhibit the production of infectious viral particles via different experimental strategies, with the best antiviral being catechin (100% inhibition with a pre-treatment strategy and 91.8% with a post-treatment strategy). When testing the interactions of these compounds with the viral envelope protein in silico by docking, only naringin and hesperidin had better scores than the theoretical threshold of − 7.0 kcal/mol (− 8.0 kcal/mol and − 8.2 kcal/mol, respectively). All ligands tested except gallic acid showed higher affinity to the NS5 protein than the theoretical threshold.ConclusionEven though bioprospecting has recently been replaced by more targeted tools for identifying compounds with pharmacological potential, our results show it is still useful for this purpose. Additionally, combining in vitro and in silico evaluations allowed us to identify promising antivirals as well as their possible mechanisms of action.

Calcium Influx Caused by ER Stress Inducers Enhances Oncolytic Adenovirus Enadenotucirev Replication and Killing through PKCα Activation

Oncolytic viruses represent an emerging approach to cancer therapy. However, better understanding of their interaction with the host cancer cell and approaches to enhance their efficacy are needed. Here, we investigate the effect of chemically induced endoplasmic reticulum (ER) stress on the activity of the chimeric group B adenovirus Enadenotucirev, its closely related parental virus Ad11p, and the archetypal group C oncolytic adenovirus Ad5. We show that treatment of colorectal and ovarian cancer cell lines with thapsigargin or ionomycin caused an influx of Ca2+, leading to an upregulation in E1A transcript and protein levels. Increased E1A protein levels, in turn, increased levels of expression of the E2B viral DNA polymerase, genome replication, late viral protein expression, infectious virus particle production, and cell killing during Enadenotucirev and Ad11p, but not Ad5, infection. This effect was not due to the induction of ER stress, but rather the influx of extracellular Ca2+ and consequent increase in protein kinase C activity. These results underscore the importance of Ca2+ homeostasis during adenoviral infection, indicate a signaling pathway between protein kinase C and E1A, and raise the possibility of using Ca2+ flux-modulating agents in the manufacture and potentiation of oncolytic virotherapies.

Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. We previously identified SRC proto-oncogene, nonreceptor tyrosine kinase (c-Src), as an essential host component of the HCV replication complex consisting of NS5A, the RNA-dependent RNA polymerase NS5B, and c-Src. Pulldown assays revealed an interaction between NS5A and the Src homology 2 (SH2) domain of c-Src; however, the precise binding mode remains undefined. In this study, using a variety of biochemical and biophysical techniques, along with molecular dynamics simulations, we demonstrate that the interaction between NS5A and the c-Src SH2 domain strictly depends on an intact phosphotyrosine-binding competent SH2 domain and on tyrosine phosphorylation within NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Tyr-93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation. In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Tyr-93 in NS5A plays an important role during viral replication by facilitating NS5A's interaction with the SH2 domain of c-Src.

Reciprocal Effects of Fibroblast Growth Factor Receptor Signaling on Dengue Virus Replication and Virion Production

Dengue virus (DENV) is a human arboviral pathogen accounting for 390 million infections every year. The available vaccine has limited efficacy, and DENV-specific drugs have not been generated. To better understand DENV-host cell interaction, we employed RNA interference-based screening of the human kinome and identified fibroblast growth factor receptor 4 (FGFR4) to control the DENV replication cycle. Pharmacological inhibition of FGFR exerts a reciprocal effect by reducing DENV RNA replication and promoting the production of infectious virus particles. Addressing the latter effect, we found that the FGFR signaling pathway modulates intracellular distribution of DENV particles in a PI3K-dependent manner. Upon FGFR inhibition, virions accumulate in the trans-Golgi network compartment, where they undergo enhanced maturation cleavage of theenvelope protein precursor membrane (prM), rendering virus particles more infectious. This study reveals an unexpected reciprocal role of a cellular receptor tyrosine kinase regulating DENV RNA replication and the production of infectious virions.

Effect of full-length and truncated variants of LL-37 on dengue virus infection and immunomodulatory effects of LL-37 in dengue virus infected U937-DC-SIGN cells

LL-37 is an antiviral and an immunomodulatory antimicrobial peptide present in humans. The present study investigated the inhibitory effect of LL-37 and its truncated variants on the infection of all the four serotypes of dengue virus (DENV) by immunofluorescence assays and the immunomodulatory activity of LL-37 on the expression of genes coding for pattern recognition receptors and components involved in type I interferon (IFN) signalling in DENV-2 infected U937-DC-SIGN cells using Taqman based assays. The results revealed the requirement of full length LL-37 to inhibit the infection of all the four serotypes of DENV when the viruses were pretreated with the peptide. However, the inhibitory effect of LL-37 on DENV-4 was not significant with regard to production of infectious virus particles. Truncated variants of LL-37 including LL-13, LL-23 and LL-18-37 had no effect on the viral infection. LL-37 significantly down regulated the expression of IFIH1, OAS1 and ISG15 genes in DENV-2 infected cells compared to DENV-2 infected cells without LL-37 treatment (P < 0.0001 for OAS1 and P < 0.05 for IFIH1 and ISG15). The results suggest the prophylactic utility of the full length LL-37 against all the four serotypes of DENV. Apart from its antiviral activity, LL-37 might also play an immunoregulatory role by modulating type I IFN response.

Autophagy Promotes Replication of Influenza A Virus In Vitro

Influenza A virus (IAV) infection could induce autophagosome accumulation. However, the impact of the autophagy machinery on IAV infection remains controversial. Here, we showed that induction of cellular autophagy by starvation or rapamycin treatment increases progeny virus production, while disruption of autophagy using a small interfering RNA (siRNA) and pharmacological inhibitor reduces progeny virus production. Further studies revealed that alteration of autophagy significantly affects the early stages of the virus life cycle or viral RNA synthesis. Importantly, we demonstrated that overexpression of both the IAV M2 and NP proteins alone leads to the lipidation of LC3 to LC3-II and a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Intriguingly, both M2 and NP colocalize and interact with LC3 puncta during M2 or NP transfection alone and IAV infection, leading to an increase in viral ribonucleoprotein (vRNP) export and infectious viral particle formation, which indicates that the IAV-host autophagy interaction plays a critical role in regulating IAV replication. We showed that NP and M2 induce the AKT-mTOR-dependent autophagy pathway and an increase in HSP90AA1 expression. Finally, our studies provided evidence that IAV replication needs an autophagy pathway to enhance viral RNA synthesis via the interaction of PB2 and HSP90AA1 by modulating HSP90AA1 expression and the AKT-mTOR signaling pathway in host cells. Collectively, our studies uncover a new mechanism that NP- and M2-mediated autophagy functions in different stages of virus replication in the pathogenicity of influenza A virus.IMPORTANCE Autophagy impacts the replication cycle of many viruses. However, the role of the autophagy machinery in IAV replication remains unclear. Therefore, we explored the detailed mechanisms utilized by IAV to promote its replication. We demonstrated that IAV NP- and M2-mediated autophagy promotes IAV replication by regulating the AKT-mTOR signaling pathway and HSP90AA1 expression. The interaction of PB2 and HSP90AA1 results in the increase of viral RNA synthesis first; subsequently the binding of NP to LC3 favors vRNP export, and later the interaction of M2 and LC3 leads to an increase in the production of infectious viral particles, thus accelerating viral progeny production. These findings improve our understanding of IAV pathogenicity in host cells.