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BackgroundHuman cytomegalovirus (HCMV) resides latently in cells of the myeloid compartment, including CD34+hematopoietic progenitor cells and circulating monocytes. Healthy hosts maintain the virus latently, and this infection is, for the most part, asymptomatic. However, given the proper external cues, HCMV reactivates from latency, at which point the virus disseminates, causing disease. The viral and cellular factors dictating the balance between these phases of infection are incompletely understood, though a large body of literature support a role for viral-mediated manipulation of host cell signaling.Main bodyTo establish and maintain latency, HCMV has evolved various means by which it usurps host cell factors to alter the cellular environment to its own advantage, including altering host cell signaling cascades. As early as virus entry into myeloid cells, HCMV usurps cellular signaling to change the cellular milieu, and this regulation includes upregulation, as well as downregulation, of different signaling cascades. Indeed, given proper reactivation cues, this signaling is again altered to allow for transactivation of viral lytic genes.ConclusionsHCMV modulation of host cell signaling is not binary, and many of the cellular pathways altered are finely regulated, wherein the slightest modification imparts profound changes to the cellular milieu. It is also evident that viral-mediated cell signaling differs not only between these phases of infection, but also is myeloid cell type specific. Nonetheless, understanding the exact pathways and the means by which HCMV mediates them will undoubtedly provide novel targets for therapeutic intervention.

Flaviviruses are enveloped viruses that infect multiple hosts. Envelope proteins are the outermost proteins in the structure of flaviviruses and mediate viral infection. Studies indicate that flaviviruses mainly use envelope proteins to bind to cell attachment receptors and endocytic receptors for the entry step. Here, we present current findings regarding key envelope protein amino acids that participate in the flavivirus early infection process. Among these sites, most are located in special positions of the protein structure, such as the α-helix in the stem region and the hinge region between domains I and II, motifs that potentially affect the interaction between different domains. Some of these sites are located in positions involved in conformational changes in envelope proteins. In summary, we summarize and discuss the key envelope protein residues that affect the entry process of flaviviruses, including the process of their discovery and the mechanisms that affect early infection.

The COVID-19 pandemic has put healthcare infrastructures and our social and economic lives under unprecedented strain. Effective solutions are needed to end the pandemic while significantly lessening its further impact on mortality and social and economic life. Effective and widely-available vaccines have appropriately long been seen as the best way to end the pandemic. Indeed, the current availability of several effective vaccines are already making a significant progress towards achieving that goal. Nevertheless, concerns have risen due to new SARS-CoV-2 variants that harbor mutations against which current vaccines are less effective. Furthermore, some individuals are unwilling or unable to take the vaccine. As health officials across the globe scramble to vaccinate their populations to reach herd immunity, the challenges noted above indicate that COVID-19 therapeutics are still needed to work alongside the vaccines. Here we describe the impact that neutralizing antibodies have had on those with early or mild COVID-19, and what their approval for early management of COVID-19 means for other viral entry inhibitors that have a similar mechanism of action. Importantly, we also highlight studies that show that therapeutic strategies involving various viral entry inhibitors such as multivalent antibodies, recombinant ACE2 and miniproteins can be effective not only for pre-exposure prophylaxis, but also in protecting against SARS-CoV-2 antigenic drift and future zoonotic sarbecoviruses.

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

Background Both vector borne and sexual transmission of Zika virus (ZIKV) involve infection of epithelial cells in the initial stages of infection. Epithelial cells are unique in their ability to form polarized monolayers and their barrier function. Cell polarity induces an asymmetry in the epithelial monolayer, which is maintained by tight junctions and specialized sorting machinery. This differential localization can have a potential impact of virus infection. Asymmetrical distribution of a viral receptor can restrict virus entry to a particular membrane while polarized sorting can lead to a directional release of virions. The present study examined the impact of cell polarity on ZIKV infection and release. Methods A polarized Caco-2 cell model we described previously was used to assess ZIKV infection. Transepithelial resistance (TEER) was used to assess epithelial cell polarity, and virus infection was measured by immunofluorescence microscopy and qRT-PCR. Cell permeability was measured using a fluorescein leakage assay. Statistical significance was calculated using one-way ANOVA and significance was set at p

Background Currently, no FDA-approved vaccines or treatments are available for Ebola virus disease (EVD), and therapy remains largely supportive. Ebola virus (EBOV) has broad tissue tropism and can infect a variety of cells including epithelial cells. Epithelial cells differ from most other cell types by their polarized phenotype and barrier function. In polarized cells, the apical and basolateral membrane domains are demarcated by tight junctions, and specialized sorting machinery, which results in a difference in composition between the two membrane domains. These specialized sorting functions can have important consequences for viral infections. Differential localization of a viral receptor can restrict virus entry to a particular membrane while polarized sorting can lead to a vectorial virus release. The present study investigated the impact of cell polarity on EBOV infection. Methods Characteristics of EBOV infection in polarized cells were evaluated in the polarized Caco-2 model grown on semipermeable transwells. Transepithelial resistance (TEER), which is a function of tight junctions, was used to assess epithelial cell polarization. EBOV infection was assessed with immunofluorescence microscopy and qPCR. Statistical significance was calculated using one-way ANOVA and significance was set at p

Background Duck tembusu virus (DTMUV, genus Flaviviruses, family Flaviviridae) is an emerging flavivirus that can infect a wide range of cells and cell lines in vitro, though the initial step of virus invasion remains obscure. Methods In this study, drug treatments that including heparin, chondroitin sulfate, heparinase I, chondroitinase ABC and trypsin were applied to detect the influence of DTMUV absorption, subsequently, the copy number of viral genome RNA was analyzed by quantitative real-time PCR. The inhibition process of viral absorption or entry by heparin was determined by western blotting, and the cytotoxicity of drug treated cells was detected by cell counting kit-8. Results We found that the desulfation of glycosaminoglycans (GAGs) with sodium chlorate had a significant effect on the adsorption of DTMUV in both BHK21 and DEF cells. Based on this result, we incubated cells with a mixture of DTMUV and GAGs competition inhibitors or pre-treated cells with inhibitors, after incubation with the virus, the NS5 expression of DTMUV and viral titers were detected. The data suggested that heparin can significantly inhibit the absorption of DTMUV in a dose dependent manner but not at the step of viral entry in BHK21 and DEF cells. Meanwhile, heparinase I can significantly inhibit DTMUV attachment step. Conclusions Our results clearly proved that heparin sulfate plays an important role in the first step of DTMUV entry, viral attachment, in both BHK21 and DEF cells, which sheds light on the entry mechanism of DTMUV.

Background Much evidence has demonstrated the influence of Hepatitis B virus (HBV) mutations on the clinical course of HBV infection. As large (L) protein plays a crucial role for viral entry, we hypothesized that mutations in the pre-S1 promoter region might affect the expression of L protein and subsequently change the biological characters of virus. Methods Patients infected with genotype C HBV were enrolled for analysis. HBV DNA sequences were inserted into a TA cloning vector and analyzed. To evaluate the effects of mutations in the pre-S1 promoter region, promoter activity and the expression of mRNA and L protein were analyzed using HepG2 cells. Results In total, 35 patients were enrolled and 13 patients (37.1%) had a single base substitution in the pre-S1 promoter region; the most frequent substitution was a G-to-A substitution at the 2765th base (G2765A) in the Sp1 region. The HBV viral load showed a negative correlation with the substitution ratio of the Sp1 region or G2765A (r = − 0.493 and − 0.473, respectively). Among those with a viral load ≤5.0 log IU/ml, patients with the G2765A substitution showed a significantly lower HBV viral load than those with the wild-type sequence. HepG2 cells transfected with the G2765A substitution vector showed reduced luciferase activity of the pre-S1 promoter, as well as reduced expression of pre-S1 mRNA and L protein. Furthermore, the G2765A substitution greatly reduced the L protein expression level of vector-produced virus particles. Conclusion G2765A substitution in the pre-S1 promoter reduced the expression of L protein and resulted in a low viral load and less severe disease in chronic HBV infections.

Background Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized. Methods Chemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy. Results We firstly examined the endocytosis pathway of the challenge virus standard (CVS) -11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MβCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy. Conclusions Our results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway. Electronic supplementary material The online version of this article (10.1186/s12985-019-1186-9) contains supplementary material, which is available to authorized users.

Background Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection. Methods B cell hybridoma technique was used to generate hybridoma cells that secrete specific antibodies. E.coli prokaryotic expression system and Bac-to-Bac expression system were used to identify the target protein of each monoclonal antibody. qPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody. Results We identified 10 monoclonal antibodies using hybridoma technology. Remarkably, 4 mAbs (designed 2G8, 2B11, 3D9, 1E3) neutralized virus infection potently, of which 2B11 and 1E3 targeted the conformational epitope of the PEDV S protein. qPCR results showed that both 2B11 and 2G8 blocked virus entry into Vero cells. Conclusion The data suggested that PEDV neutralizing antibody inhibited virus infection by binding to infectious virions, which could work as a tool to find the receptor-binding domains. Electronic supplementary material The online version of this article (10.1186/s12985-018-1042-3) contains supplementary material, which is available to authorized users.

Background Grass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV). The frequent outbreaks of this disease have suppressed development of the grass carp farming industry. GCRV104, the representative strain of genotype III grass carp (Ctenopharyngodon idella) reovirus, belongs to the Spinareovirinae subfamily and serves as a model for studying the strain of GCRV which encodes an outer-fiber protein. There is no commercially available vaccine for this genotype of GCRV. Therefore, the discovery of new inhibitors for genotype III of GCRV will be clinically beneficial. In addition, the mechanism of GCRV with fiber entry into cells remains poorly understood. Methods Viral entry was determined by a combination of specific pharmacological inhibitors, transmission electron microscopy, and real-time quantitative PCR. Results Our results demonstrate that both GCRV-JX01 (genotype I) and GCRV104 (genotype III) of GCRV propagated in the grass carp kidney cell line (CIK) with a typical cytopathic effect (CPE). However, GCRV104 replicated slower than GCRV-JX01 in CIK cells. The titer of GCRV-JX01 was 1000 times higher than GCRV104 at 24 h post-infection. We reveal that ammonium chloride, dynasore, pistop2, chlorpromazine, and rottlerin inhibit viral entrance and infection, but not nystatin, methyl-β-cyclodextrin, IPA-3, amiloride, bafilomycin A1, nocodazole, and latrunculin B. Furthermore, GCRV104 and GCRV-JX01 infection of CIK cells depended on dynamin and the acidification of the endosome. This was evident by the significant inhibition following prophylactic treatment with the lysosomotropic drug ammonium chloride or dynasore. Conclusions Taken together, our data have suggested that GCRV104 enters CIK cells through clathrin-mediated endocytosis in a pH-dependent manner. We also suggest that dynamin is critical for efficient viral entry. Additionally, the phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase C inhibitor rottlerin block GCRV104 cell entry and replication.

Background Hendra virus and Nipah virus are zoonotic viruses that have caused severe to fatal disease in livestock and human populations. The isolation of Cedar virus, a non-pathogenic virus species in the genus Henipavirus, closely-related to the highly pathogenic Hendra virus and Nipah virus offers an opportunity to investigate differences in pathogenesis and receptor tropism among these viruses. Methods We constructed full-length cDNA clones of Cedar virus from synthetic oligonucleotides and rescued two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that expresses a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. Replication kinetics of both viruses and stimulation of the interferon pathway were characterized in vitro. Cellular tropism for ephrin-B type ligands was qualitatively investigated by microscopy and quantitatively by a split-luciferase fusion assay. Results Successful rescue of recombinant Cedar virus expressing a green fluorescent protein did not significantly affect virus replication compared to the recombinant wild-type Cedar virus. We demonstrated that recombinant Cedar virus stimulated the interferon pathway and utilized the established Hendra virus and Nipah virus receptor, ephrin-B2, but not ephrin-B3 to mediate virus entry. We further characterized virus-mediated membrane fusion kinetics of Cedar virus with the known henipavirus receptors ephrin-B2 and ephrin-B3. Conclusions The recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals. Moreover, these experiments can be conducted safely under BSL-2 conditions.

Background Plant viruses in agricultural crops are of great concern worldwide, and over 75% of them are transmitted from infected to healthy plants by insect vectors. Tomato yellow leaf curl virus (TYLCV) is a begomovirus, which is the largest and most economically important group of plant viruses, transmitted by the whitefly Bemisia tabaci. The circulation of TYLCV in the insect involves complex insect-virus interactions, whereas the molecular mechanisms of these interactions remain ambiguous. The insect gut as a barrier for viral entry and dissemination is thought to regulate the vector specificity. However, due to its tiny size, information for the responses of whitefly gut to virus infection is limited. Methods We investigated the transcriptional response of the gut of B. tabaci Middle East-Asia Minor 1 species to TYLCV infection using Illumina sequencing. Results A total of 5207 differentially expressed genes (DEGs) between viruliferous and non-viruliferous whitefly guts were identified. Enrichment analyses showed that cargo receptor and ATP-binding cassette (ABC) transporters were enriched in DEGs, and might help the virus to cross gut barrier. TYLCV could perturb cell cycle and DNA repair as a possible result of its replication in the whitefly. Our data also demonstrated that TYLCV can activate whitefly defense responses, such as antimicrobial peptides. Meanwhile, a number of genes involved in intracellular signaling were activated by TYLCV infection. Conclusions Our results reveal the complex insect-virus relationship in whitefly gut and provide substantial molecular information for the role of insect midguts in virus transmission. Electronic supplementary material The online version of this article (10.1186/s12985-018-0926-6) contains supplementary material, which is available to authorized users.

Background Human enterovirus 71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways. However, we observed chlorpromazine (CPZ) or dynasore (DNS), which inhibit clathrin and dynamin mediated endocytosis, did not suppress EV71 cell entry in particular cell types. So the current knowledge of entry mechanisms by EV71 is not complete. Methods Viral infection was examined by flow cytometry or end-point dilution assays. Viral entry was monitored by immunofluorescence or pseudoviral infections. Various inhibitors were utilized for manipulating endocytic pathways. Cellular proteins were knockdown by siRNA. Results CPZ and DNS did not inhibit but rather enhance viral infection in A549 cells, while they inhibited infections in other cells tested. We further found CPZ did not affect EV71 binding to target cells and failed to affect viral translation and replication, but enhanced viral entry in A549 cells. Immunofluorescence microscopy further confirmed this increased entry. Using siRNA experiment, we found that the enhancement of EV71 infection by CPZ did not require the components of clathrin mediated endocytosis. Finally, CPZ also enhanced infection by Coxackivirus A16 in A549 cells. Conclusions CPZ and DNS, previously reported as EV71 entry inhibitors, may rather lead to increased viral infection in particular cell types. CPZ and DNS increased viral entry and not other steps of viral life cycles. Therefore, our study indicated an unknown dynamin-independent entry pathway utilized by enteroviruses that cause Hand-Foot-and-Mouth Diseases. Electronic supplementary material The online version of this article (10.1186/s12985-017-0913-3) contains supplementary material, which is available to authorized users.

Herpes simplex virus 1 (HSV-1) is composed of complex structures primarily characterized by four elements: the nucleus, capsid, tegument and envelope. The tegument is an important viral component mainly distributed in the spaces between the capsid and the envelope. The development of viral genome editing technologies, such as the identification of temperature-sensitive mutations, homologous recombination, bacterial artificial chromosome, and the CRISPR/Cas9 system, has been shown to largely contribute to the rapid promotion of studies on the HSV-1 tegument protein. Many researches have demonstrated that tegument proteins play crucial roles in viral gene regulatory transcription, viral replication and virulence, viral assembly and even the interaction of the virus with the host immune system. This article briefly reviews the recent research on the functions of tegument proteins and specifically elucidates the function of tegument proteins in viral infection, and then emphasizes the significance of using genome editing technology in studies of providing new techniques and insights into further studies of HSV-1 infection in the future.

Background In mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens (herpes simplex virus, vaccinia virus, human respiratory syncytial virus, human immunodeficiency virus) by activating natural killer cells (NK), cytotoxic T lymphocytes and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such it might have the potential to affect replication and pathogenesis of Newcastle disease virus (NDV). Methods To assess the effect of IL-2 during NDV infection in chickens, we produced a recombinant virulent NDV strain expressing chicken IL-2 (rZJ1-IL2). The effects of IL-2 expression were investigated in vivo using the intracerebral pathogenicity index (ICPI) in day-old chicks and pathogenesis experiments in 4-week-old chickens. In these studies, rZJ1-IL2 was compared to a control virus expressing the green fluorescent protein (rZJ1-GFP). Assessed parameters included survival curves, detailed histological and immunohistochemical grading of lesions in multiple organs, and virus isolation in blood, spleen and mucosal secretions of infected birds. Results At the site of infection (eyelid), expression of IL-2 was demonstrated in areas of rZJ-IL2 replication, confirming IL-2 production in vivo. Compared to rZJ1-GFP strain, rZJ1-IL2 caused milder lesions and displayed decreased viral load in blood, spleen and mucosal secretions of infected birds. In the rZJ1-IL2-infected group, virus level in the blood peaked at day 4 post-infection (pi) (103.46 EID50 /0.1 ml) and drastically decreased at day 5 pi (100.9 EID50/0.1 ml), while in the rZJ1-GFP-infected group virus levels in the blood reached 105.35 EID50/0.1 ml at day 5. However, rZJ1-IL2-infected groups presented survival curves similar to control birds infected with rZJ1-GFP, with comparable clinical signs and 100 % mortality. Further, expression of IL-2 did not significantly affect the ICPI scores, compared to rZJ1-GFP strain. Conclusions Increased expression of chicken IL-2 during virulent NDV replication in naïve chickens decreased viral titers in blood, spleens, oral and cloacal secretions on day 4–5 post infection. This is consistent with the previously described role of IL-2 in enhancing the clearance of viruses in mammals, such as human respiratory syncytial virus. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0353-x) contains supplementary material, which is available to authorized users.

The parvoviruses are widely spread in many species and are among the smallest DNA animal viruses. The parvovirus is composed of a single strand molecule of DNA wrapped into an icosahedral capsid. In a viral infection, the massy capsid participates in the entire viral infection process, which is summarized in this review. The capsid protein VP1 is primarily responsible for the infectivity of the virus, and the nuclear localization signal (NLS) of the VP1 serves as a guide to assist the viral genome in locating the nucleus. The dominant protein VP2 provides an "anti-receptor", which interacts with the cellular receptor and leads to the further internalization of virus, and, the N-terminal of VP2 also cooperates with the VP1 to prompt the process of nucleus translocation. Additionally, a cleavage protein VP3 is a part of the capsid, which exists only in several members of the parvovirus family; however, the function of this cleavage protein remains to be fully determined. Parvoviruses can suffer from the extreme environmental conditions such as low pH, or even escape from the recognition of pattern recognition receptors (PRRs), due to the protection of the stable capsid, which is thought to be an immune escape mechanism. The applications of the capsid proteins to the screening and the treatment of diseases are also discussed. The processes of viral infection should be noted, because understanding the virus-host interactions will contribute to the development of therapeutic vaccines.

Background: Millions of human infections caused by arthropod-borne pathogens are initiated by the feeding of an infected mosquito on a vertebrate. However, interactions between the viruses and the mosquito vector, which facilitates successful infection and transmission of virus to a subsequent vertebrate host, are still not fully understood. Finding: Here we describe early chikungunya virus (CHIKV) infectious events in cells derived from one of the most important CHIKV vectors, Aedes albopictus. We demonstrated that CHIKV infection of mosquito cells depended on acidification of the endosome as indicated by significant inhibition following prophylactic treatment with the lysosomotropic drugs chloroquine, ammonium chloride, and monensin, which is consistent with observations in mammalian cells. While all three agents inhibited CHIKV infection in C6/36 cells, ammonium chloride was less toxic to cells than the other agents. Conclusion: The observation of similar mechanisms for inhibition of CHIKV infection in mosquito and mammalian cell lines suggests that conserved entry pathways are utilized by CHIKV for vertebrate and invertebrate cell types.

Background Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. Methods ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. Results Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. Conclusions The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

Background Bovine rotavirus (BRV) is a non-enveloped dsRNA virus that cause neonatal calf diarrhea. Lipid rafts are cholesterol-enrich membrane mircodomains that play a vital role in many cellular processes. In this study, the effect of cellular cholesterol depletion on infection of MA-104 cells with bovine rotavirus was investigated. Results We demonstrated that cholesterol depletion of the plasma membrane by MβCD had no effect on BRV binding to cells but significantly impaired BRV entry in a dose-dependent manner and the effect was partially reversed by addition of exogenous cholesterol, suggesting the reduction of BRV infection by MβCD was specifically due to cholesterol depletion. Cholesterol depletion after virus entry did not reduce BRV replication, whereas affected virus assembly. Conclusions Taken together, our results demonstrate that cell membrane cholesterol is essential to BRV infectivity.

Most viruses take advantage of endocytic pathways to gain entry into host cells and initiate infections. Understanding of virus entry via endocytosis is critically important for the design of antiviral strategies. Virus entry via endocytosis is a complex process involving hundreds of cellular proteins. The entire process is dictated by events occurring at multiple time and length scales. In this review, we discuss and evaluate the available means to investigate virus endocytic entry, from both experimental and theoretical/numerical modeling fronts, and highlight the importance of multiscale features. The complexity of the process requires investigations at a systems biology level, which involves the combination of different experimental approaches, the collaboration of experimentalists and theorists across different disciplines, and the development of novel multiscale models.

BackgroundUpon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear.MethodsA panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells.ResultsInhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production.ConclusionsOur results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production.

Background The function and kinetics of some herpsvirus UL16 gene have been reported. But there was no any report of duck enteritis virus (DEV) UL16 gene. Findings The kinetics of DEV UL16 gene was examined in DEV CHv infected duck embryo fibroblasts (DEFs) by establishment of real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) and western-blotting. In this study, UL16 mRNA was transcript at a low level from 0–18 h post-infection (p.i), and peaked at 36 h p.i. It can’t be detected in the presence of acyclovir (ACV). Besides, western-blotting analysis showed that UL16 gene expressed as an apparent 40-KDa in DEV infected cell lysate from 12 h p.i, and rose to peak level at 48 h p.i consistent with the qRT-PCR result. Conclusions These results provided the first evidence of the kinetics of DEV UL16 gene. DEV UL16 gene was a late gene and dependent on viral DNA synthesis.

Background Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Results Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A) sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. Conclusions NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.

Background Human metapneumovirus (hMPV) is a significant viral respiratory pathogen of infants and children, the elderly and immunocompromised individuals. Disease associated with hMPV infection resembles that of human respiratory syncytial virus (RSV) and includes bronchiolitis and pneumonia. The glycosylated G attachment protein of hMPV is required for viral entry in vivo and has also been identified as an inhibitor of innate immune responses. Findings We designed and validated two siRNA molecules against the G gene using A549 cells and demonstrated consistent 88-92% knock-down for one siRNA molecule, which was used in subsequent experiments. Significant reduction of G mRNA in A549 cells infected with hMPV did not result in a reduction in viral growth, nor did it significantly increase the production of type I interferon (α/β) in response to infection. However, there was a moderate increase in IFN-β mRNA expression in response to infection in siG-transfected cells compared to untransfected and si-mismatch-transfected cells. Expression of G by recombinant adenovirus did not affect type I IFN expression. Conclusion G has been previously described as a type I interferon antagonist, although our findings suggest it may not be a significant antagonist.

Background Herpes simplex virus type-1 (HSV-1) enters into cells via membrane fusion of the viral envelope with plasma or endosomal membranes mediated by viral glycoproteins. HSV-1 virions attach to cell surfaces by binding of viral glycoproteins gC, gD and gB to specific cellular receptors. Here we show that the human ocular and highly neurovirulent HSV-1 strain McKrae enters substantially more efficiently into cells via the gB-specific human paired immunoglobulin-like type-2 receptor-α (hPILR-α). Comparison of the predicted amino acid sequences between HSV-1(F) and McKrae strains indicates that amino acid changes within gB, gC, gH and gL may cause increased entry via the hPILR- α receptor. Results HSV-1 (McKrae) entered substantially more efficiently than viral strain F in Chinese hamster ovary (CHO) cells expressing hPIRL-α but not within CHO-human nectin-1, -(CHO-hNectin-1), CHO-human HVEM (CHO-hHVEM) or Vero cells. The McKrae genes encoding viral glycoproteins gB, gC, gD, gH, gL, gK and the membrane protein UL20 were sequenced and their predicted amino acid (aa) sequences were compared with virulent strains F, H129, and the attenuated laboratory strain KOS. Most aa differences between McKrae and F were located at their gB amino termini known to bind with the PILRα receptor. These aa changes included a C10R change, also seen in the neurovirulent strain ANG, as well as redistribution and increase of proline residues. Comparison of gC aa sequences revealed multiple aa changes including an L132P change within the 129-247 aa region known to bind to heparan sulfate (HS) receptors. Two aa changes were located within the H1 domain of gH that binds gL. Multiple aa changes were located within the McKrae gL sequence, which were preserved in the H129 isolate, but differed for the F strain. Viral glycoproteins gD and gK and the membrane protein UL20 were conserved between McKrae and F strains. Conclusions The results indicate that the observed entry phenotype of the McKrae strain is most likely due to a combination of increased binding to heparan sulfate receptors and enhanced virus entry via gB-mediated fusion of the viral envelope with plasma membranes.

Background The aquatic birnavirus infectious pancreatic necrosis virus (IPNV) causes infectious pancreatic necrosis (IPN), a severe disease in farmed salmonid fish. IPNV has a very broad host range and infects many different species of fish as well as molluscs and crustaceans. Investigation of the host reservoir of a virus may reveal important molecular mechanisms governing the infection processes such as receptors and entry mechanisms. In the present work we have studied whether IPNV is able to infect cells with different mammalian origin. Results IPNV bound in a specific manner to a membrane protein of the rabbit kidney cell line RK-13 as shown by the use of a virus overlay protein binding assay (VOPBA). Six different mammalian cell lines were inoculated with IPNV and incubated in parallels at different temperatures. At 7 days post inoculation (dpi), IPNV was detected by indirect immunofluorescent antibody test (IFAT) in all the cell lines. Confocal microscopy confirmed intracellular presence of the virus. No apparent cytopathic effect (cpe) was observed in any of the cultures, and no viral replication was demonstrated with real-time RT-PCR. Conclusion Our results show that IPNV is able to enter into a wide range of mammalian cells, and virus entry is most likely receptor mediated. We found no indication of IPNV replication in any of the mammalian cell lines tested.

Human cytomegalovirus (HCMV), a member of the Betaherpesvirinae sub-family of Herpesviridae family, is a widespread pathogen that infects a majority of the world's population by early adulthood. In individuals whose immune systems are immature or weakened, HCMV is a significant pathogen causing morbidity and mortality. There is no effective vaccine and only limited antiviral treatments against HCMV infection to date. A possible target for novel antiviral treatments is the HCMV proteins that localize to the tegument of the virion, since they play important roles in all stages of the viral life cycle, including, viral entry, gene expression, immune evasion, assembly, and egress. The most likely tegument protein candidates are pp65 (immune evasion), pp71 (gene expression), and pp150 and pp28 (assembly and egress). Although the subcellular localization of these proteins has been identified during HCMV infections in vitro, their localization patterns have not been determined when each protein is expressed individually in living cells. Thus, the objective of this review is elucidate the HCMV tegument as well as present current research findings concerning the subcellular localization of the tegument proteins pp65, pp71, pp150, and pp28 as fusions to one of several fluorescent proteins.

Herpes simplex virus (HSV) type-1 and type-2 have evolved numerous strategies to infect a wide range of hosts and cell types. The result is a very successful prevalence of the virus in the human population infecting 40-80% of people worldwide. HSV entry into host cell is a multistep process that involves the interaction of the viral glycoproteins with various cell surface receptors. Based on the cell type, HSV enter into host cell using different modes of entry. The combination of various receptors and entry modes has resulted in a virus that is capable of infecting virtually all cell types. Identifying the common rate limiting steps of the infection may help the development of antiviral agents that are capable of preventing the virus entry into host cell. In this review we describe the major features of HSV entry that have contributed to the wide susceptibility of cells to HSV infection.

Adenovirus has a wide tissue tropism. The virus attaches to the surface of cells via the fiber protein knob binding to the Coxsackie and Adenovirus receptor known as CAR. Virus entry inside cells is facilitated by integrins αVβ3 and αVβ5. Mice platelets are shown to be the predominant Ad binding blood cell type and the virus is documented inside platelets. CAR was identified on human platelets in one study yet contradicted in another. The presence of CAR appears to be the most reasonable initial step for virus entry into platelets and is a key to the understanding of platelet adenovirus interaction. This study aimed to re investigate the presence of CAR on human platelets. Platelets were tested by indirect immune-fluorescence using rabbit H-300 polyclonal anti-CAR antibody and goat anti-rabbit IgG F(ab')2 Texas Red antibodies, alongside with CAR positive and negative controls. Platelets were found to express CAR on their surface and in contrast to the previous study only 3.5 ± 1.9% of the tested platelets did express CAR. In addition, CAR was seen within intracellular aggregates localized at the sites of cell-cell contacts indicating that CAR expression might be upregulated in response to platelet stimulation. We confirm the presence of CAR on human platelets, we provide explanation to some of the discrepancies in this regards and we add that this receptor is localized at the sites of intercellular interaction.

Background Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach. Results The cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipμ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses. Conclusions The proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.

Background Marek's disease virus (MDV), which is widely considered to be a natural model of virus-induced lymphoma, has the potential to cause tremendous losses in the poultry industry. To investigate the structural basis of MDV membrane fusion and to identify new viral targets for inhibition, we examined the domains of the MDV glycoproteins gH and gB. Results Four peptides derived from the MDV glycoprotein gH (gHH1, gHH2, gHH3, and gHH5) and one peptide derived from gB (gBH1) could efficiently inhibit plaque formation in primary chicken embryo fibroblast cells (CEFs) with 50% inhibitory concentrations (IC50) of below 12 μM. These peptides were also significantly able to reduce lesion formation on chorioallantoic membranes (CAMs) of infected chicken embryos at a concentration of 0.5 mM in 60 μl of solution. The HR2 peptide from Newcastle disease virus (NDVHR2) exerted effects on MDV specifically at the stage of virus entry (i.e., in a cell pre-treatment assay and an embryo co-treatment assay), suggesting cross-inhibitory effects of NDV HR2 on MDV infection. None of the peptides exhibited cytotoxic effects at the concentrations tested. Structural characteristics of the five peptides were examined further. Conclusions The five MDV-derived peptides demonstrated potent antiviral activity, not only in plaque formation assays in vitro, but also in lesion formation assays in vivo. The present study examining the antiviral activity of these MDV peptides, which are useful as small-molecule antiviral inhibitors, provides information about the MDV entry mechanism.

The serum-free medium from Japanese encephalitis virus (JEV) infected Baby Hamster Kidney-21 (BHK-21) cell cultures was analyzed by liquid chromatography tandem mass spectrometry (LC-MS) to identify host proteins that were secreted upon viral infection. Five proteins were identified, including the molecular chaperones Hsp90, GRP78, and Hsp70. The functional role of GRP78 in the JEV life cycle was then investigated. Co-migration of GRP78 with JEV particles in sucrose density gradients was observed and co-localization of viral E protein with GRP78 was detected by immunofluorescence analysis in vivo. Knockdown of GRP78 expression by siRNA did not effect viral RNA replication, but did impair mature viral production. Mature viruses that do not co-fractionate with GPR78 displayed a significant decrease in viral infectivity. Our results support the hypothesis that JEV co-opts host cell GPR78 for use in viral maturation and in subsequent cellular infections.

Background Host determinants of HIV-1 viral tropism include factors from producer cells that affect the efficiency of productive infection and factors in target cells that block infection after viral entry. TRIM5α restricts HIV-1 infection at an early post-entry step through a mechanism associated with rapid disassembly of the retroviral capsid. Topoisomerase I (TOP1) appears to play a role in HIV-1 viral tropism by incorporating into or otherwise modulating virions affecting the efficiency of a post-entry step, as the expression of human TOP1 in African Green Monkey (AGM) virion-producing cells increased the infectivity of progeny virions by five-fold. This infectivity enhancement required human TOP1 residues 236 and 237 as their replacement with the AGM counterpart residues abolished the infectivity enhancement. Our previous studies showed that TOP1 interacts with BTBD1 and BTBD2, two proteins which co-localize with the TRIM5α splice variant TRIM5δ in cytoplasmic bodies. Because BTBD1 and BTBD2 interact with one HIV-1 viral tropism factor, TOP1, and co-localize with a splice variant of another, we investigated the potential involvement of BTBD1 and BTBD2 in HIV-1 restriction. Results We show that the interaction of BTBD1 and BTBD2 with TOP1 requires hu-TOP1 residues 236 and 237, the same residues required to enhance the infectivity of progeny virions when hu-TOP1 is expressed in AGM producer cells. Additionally, interference with the expression of BTBD2 in AGM and human 293T target cells increased their permissiveness to HIV-1 infection two- to three-fold. Conclusions These results do not exclude the possibility that BTBD2 may modestly restrict HIV-1 infection via colocation with TRIM5 variants in cytoplasmic bodies.

The hemagglutinin (HA) protein of influenza virus mediates essential viral functions including the binding to host receptor and virus entry. It also has the antigenic sites required for virus neutralization by host antibodies. Here, we characterized an H3N2 triple reassortant (TR) influenza virus (A/turkey/Ohio/313053/04) with a mutation at the receptor binding domain (Asp190Ala) that occurred upon virus transmission from turkeys to pigs in an experimental infection study. The mutant virus replicated less efficiently than the parental virus in human, pig and turkey primary tracheal/bronchial epithelial cells, with more than 3-log10 difference in virus titer at 72 hours post infection. In addition, the mutant virus demonstrated lower binding efficiency to plasma membrane preparations from all three cell types compared to the parental virus. Antisera raised against the parental virus reacted equally to both homologous and heterlogous viruses, however, antisera raised against the mutant virus showed 4-8 folds lower reactivity to the parental virus.

Background Viruses bind to specific cellular receptors in order to infect their hosts. The specific receptors a virus uses are important factors in determining host range, cellular tropism, and pathogenesis. For adenovirus, the existing model of entry requires two receptor interactions. First, the viral fiber protein binds Coxsackie and Adenovirus Receptor (CAR), its primary cellular receptor, which docks the virus to the cell surface. Next, viral penton base engages cellular integrins, coreceptors thought to be required exclusively for internalization and not contributing to binding. However, a number of studies reporting data which conflicts with this simple model have been published. These observations have led us to question the proposed two-step model for adenovirus infection. Results In this study we report that cells which express little to no CAR can be efficiently transduced by adenovirus. Using competition experiments between whole virus and soluble viral fiber protein or integrin blocking peptides, we show virus binding is not dependent on fiber binding to cells but rather on penton base binding cellular integrins. Further, we find that binding to low CAR expressing cells is inhibited specifically by a blocking antibody to integrin αvβ5, demonstrating that in these cells integrin αvβ5 and not CAR is required for adenovirus attachment. The binding mediated by integrin αvβ5 is extremely high affinity, in the picomolar range. Conclusions Our data further challenges the model of adenovirus infection in which binding to primary receptor CAR is required in order for subsequent interactions between adenovirus and integrins to initiate viral entry. In low CAR cells, binding occurs through integrin αvβ5, a receptor previously thought to be used exclusively in internalization. We show for the first time that integrin αvβ5 can be used as an alternate binding receptor.

HSV-1 viral capsid maturation and egress from the nucleus constitutes a self-controlled process of interactions between host cytoplasmic membrane proteins and viral capsid proteins. In this study, a member of the tetraspanin superfamily, CTMP-7, was shown to physically interact with HSV-1 protein VP26, and the VP26-CTMP-7 complex was detected both in vivo and in vitro. The interaction of VP26 with CTMP-7 plays an essential role in normal HSV-1 replication. Additionally, analysis of a recombinant virus HSV-1-UG showed that mutating VP26 resulted in a decreased viral replication rate and in aggregation of viral mutant capsids in the nucleus. Together, our data support the notion that biological events mediated by a VP26 - CTMP-7 interaction aid in viral capsid enveloping and egress from the cell during the HSV-1 infectious process.

Background Geminiviruses encode few viral proteins. Most of the geminiviral proteins are multifunctional and influence various host cellular processes for the successful viral infection. Though few viral proteins like AC1 and AC2 are well characterized for their multiple functions, role of AC3 in the successful viral infection has not been investigated in detail. Results We performed phage display analysis with the purified recombinant AC3 protein with Maltose Binding Protein as fusion tag (MBP-AC3). Putative AC3 interacting peptides identified through phage display were observed to be homologous to peptides of proteins from various metabolisms. We grouped these putative AC3 interacting peptides according to the known metabolic function of the homologous peptide containing proteins. In order to check if AC3 influences any of these particular metabolic pathways, we designed vectors for assaying DNA replication and virus induced gene-silencing of host gene PCNA. Investigation with these vectors indicated that AC3 enhances viral replication in the host plant tomato. In the PCNA gene-silencing experiment, we observed that the presence of functional AC3 ORF strongly manifested the stunted phenotype associated with the virus induced gene-silencing of PCNA in tomato plants. Conclusions Through the phage display analysis proteins from various metabolic pathways were identified as putative AC3 interacting proteins. By utilizing the vectors developed, we could analyze the role of AC3 in viral DNA replication and host gene-silencing. Our studies indicate that AC3 is also a multifunctional protein.

Background Papillomaviruses (PVs) establish a persistent infection in the proliferating basal cells of the epithelium. The viral genome is replicated and maintained as a low-copy nuclear plasmid in basal keratinocytes. Bovine and human papillomaviruses (BPV and HPV) are known to utilize two viral proteins; E1, a DNA helicase, and E2, a transcription factor, which have been considered essential for viral DNA replication. However, growing evidence suggests that E1 and E2 are not entirely essential for stable replication of HPV. Results Here we report that multiple HPV16 mutants, lacking either or both E1 and E2 open reading frame (ORFs) and the long control region (LCR), still support extrachromosomal replication. Our data clearly indicate that HPV16 has a mode of replication, independent of viral trans-factors, E1 and E2, which is achieved by origin activity located outside of the LCR.

Background The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication. Results Using a motif-based search strategy for antiviral targets we identified caveolin-1 (Cav-1) as a putative cellular interaction partner of human influenza A viruses, including the pandemic influenza A virus (H1N1) strains of swine origin circulating from spring 2009 on. The influence of Cav-1 on human influenza A/PR/8/34 (H1N1) virus replication was determined in inhibition and competition experiments. RNAi-mediated Cav-1 knock-down as well as transfection of a dominant-negative Cav-1 mutant results in a decrease in virus titre in infected Madin-Darby canine kidney cells (MDCK), a cell line commonly used in basic influenza research as well as in virus vaccine production. To understand the molecular basis of the phenomenon we focussed on the putative caveolin-1 binding domain (CBD) located in the lumenal, juxtamembranal portion of the M2 matrix protein which has been identified in the motif-based search. Pull-down assays and co-immunoprecipitation experiments showed that caveolin-1 binds to M2. The data suggest, that Cav-1 modulates influenza virus A replication presumably based on M2/Cav-1 interaction. Conclusion As Cav-1 is involved in the human influenza A virus life cycle, the multifunctional protein and its interaction with M2 protein of human influenza A viruses represent a promising starting point for the search for antiviral agents.

The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV), Vaccinia (VACV), monkeypox (MPV) and Variola (VARV) viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi) as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA). Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R) or an important gene in viral entry (E8L), inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.