Microbiology

Co-immunoprecipitation or pull-down assays are frequently used to analyze protein–protein interactions. In these experiments, western blotting is commonly used to detect prey proteins. However, sensitivity and quantification problems remain in this detection system. Recently, the HiBiT-tag-dependent NanoLuc luciferase system was developed as a highly sensitive detection system for small amounts of proteins. In this report, we introduce the method of using HiBiT technology for the detection of prey protein in a pull-down assay. Using this protocol, we demonstrate the formation of a ternary complex consisting of Japanese encephalitis virus NS4B and two host factors, namely valosin-containing protein, and nuclear protein localization protein 4, which is a critical biological event during flavivirus replication in cells.

Profiling the specificities of antibodies can reveal a wealth of information about humoral immune responses and the antigens they target. Here, we present a protocol for VirScan, an application of the phage immunoprecipitation sequencing (PhIP-Seq) method for profiling the specificities of human antiviral antibodies. Accompanying this protocol is a video of the experimental procedure. VirScan and, more generally, PhIP-Seq are techniques that enable high-throughput antibody profiling by combining high-throughput DNA oligo synthesis and bacteriophage display with next-generation sequencing. In the VirScan method, human sera samples are screened against a library of peptides spanning the entire human viral proteome. Bound phage are immunoprecipitated and sequenced, identifying the viral peptides recognized by the antibodies. VirScan Is a powerful tool for uncovering individual viral exposure histories, mapping the epitope landscape of viruses of interest, and studying fundamental mechanisms of viral immunity.

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Although herpes simplex virus 1 (HSV-1) is a well-studied virus, how the virus invades its human host via skin and mucosa to reach its receptors and initiate infection remains an open question. For studies of HSV-1 infection in skin, mice have been used as animal models. Murine skin infection can be induced after injection or scratching of the skin, which provides insights into disease pathogenesis but is clearly distinct from the natural entry route in human tissue. To explore the invasion route of HSV-1 on the tissue level, we established an ex vivo infection assay using skin explants. Here, we detail a protocol allowing the investigation of how the virus overcomes mechanical barriers in human skin to penetrate in keratinocytes and dermal fibroblasts. The protocol includes the preparation of total skin samples, skin shaves, and of separated epidermis and dermis, which is followed by incubation in virus suspension. The ex vivo infection assay allows the visualization, quantification, and characterization of single infected cells in the epidermis and dermis prior to viral replication and the virus-induced tissue damage. Hence, this experimental approach enables the identification of primary viral entry portals.

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The absence of long term, primary untransformed in vitro models that support hepatitis B virus (HBV) infection and replication have hampered HBV pre-clinical research, which was reflected in the absence of a curative therapy until recently. One of the limitations for in vitro HBV research has been the absence of high titer and pure recombinant HBV stocks, which, as we describe here, can be generated using simple, and reproducible protocols. In addition to infection of more conventional in vitro and in vivo liver model systems, recombinant high titer purified HBV stocks can also be used to efficiently infect differentiated human liver organoids, whose generation, maintenance, and infection is discussed in detail in a companion organoid protocol. Here, we also describe the protocols for the detection of specific viral read-outs, including HBV DNA in the supernatant of the cultures, covalently closed circular DNA (cccDNA) from intracellular DNA preparations, and HBV viral proteins and viral RNA, which can be detected within the cells, demonstrating the presence of a complete viral replication cycle in infected liver organoids. Although an evolving platform, the human liver organoid model system presents great potential as an exciting new tool to study HBV infection and progression to hepatocellular carcinoma (HCC) in primary cells, when combined with the use of high-titer and pure recombinant HBV stock for infection.

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Hepatitis B virus (HBV) infection represents a major public health problem infecting approximately 400 million people worldwide. Despite the availability of a preventive vaccine and anti-viral therapies, chronic HBV infection remains a major health issue because it increases the risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). The lack of a relevant in vitro model for the study of the molecular mechanisms that drive HBV replication and latency, as well as HBV-related carcinogenesis, has been one of the major obstacles to the development of curative strategies. Here, we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. Human liver organoids can be seeded from both healthy and cirrhotic liver biopsies. They can be expanded in vitro when culturing in a medium containing a specific set of growth factors. When the culture medium is changed into a new medium containing growth factors that promote differentiation, organoids differentiate into functional hepatocytes, which makes them susceptible to infection with recombinant HBV. The novel in vitro primary model system described in this protocol can be utilized as a platform to study HBV pathogenesis and drug screening. Organoids generated from cirrhotic liver biopsies can be a potential tool for personalized medicine, and for modeling HCC and other liver diseases.

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The ubiquitous and cancer-associated Epstein-Barr virus (EBV) is associated with nearly all cases of nasopharyngeal carcinoma (NPC). Nasopharyngeal tissue is comprised of both pseudostratified and stratified epithelium, which are modeled in three-dimensional (3-D) cell culture. The cellular origin of EBV-associated NPC is as yet unknown, but both latent and lytic infections are likely important for preneoplastic mechanisms and replenishing the compartmentalized viral reservoir. Conventional 2-D cultures of nasopharyngeal epithelial cells (as primary cells or immortalized cell lines) are difficult to infect with EBV and cannot mimic the tissue-specific biology of the airway epithelium, which can only be captured in 3-D models. We have shown that EBV can infect the pseudostratified epithelium in air-liquid interface (ALI) culture using primary conditionally reprogrammed cells (CRCs) derived from the nasopharynx. In this protocol, we provide a step-by-step guide for the (i) conditional reprogramming of primary nasopharyngeal cells, (ii) differentiation of CRCs into pseudostratified epithelium in ALI culture (known as pseudo-ALI), and (iii) EBV infection of pseudo-ALI cultures. Additionally, we show that nasopharyngeal CRCs can be grown as organotypic rafts and subjected to EBV infection. These nasopharyngeal-derived 3-D cell cultures can be used to study EBV latent and lytic infection in relation to cell type and donor variation, by immunostaining and single-cell RNA-sequencing methods (Ziegler et al., 2021). These methods are useful for studies of EBV molecular pathogenesis, and can overcome many of the limitations associated with conventional 2-D cell cultures.

Graphic abstract:

Workflow of nasopharyngeal-derived conditionally reprogrammed cells grown into pseudostratified-ALI and organotypic rafts in 3-D cell culture. Created with Biorender.com.

Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation or pull-downs, are helpful in investigating intricate viral-host proteins interactions. However, such assays may miss low-affinity and favour non-specific interactions. We have recently incorporated photoreactive amino acids at defined residues of a viral protein in vivo, by introducing amber stop codons (TAG) and using a suppressor tRNA. This is followed by UV-crosslinking, to identify interacting host proteins in live mammalian cells. The affinity-purified photo-crosslinked viral-host protein complexes are further characterized by mass spectrometry following extremely stringent washes. This combinatorial site-specific incorporation of a photoreactive amino acid and affinity purification-mass spectrometry strategy allows the definition of viral-host protein contacts at single residue resolution and greatly reduces non-specific interactors, to facilitate characterization of viral-host protein interactions.

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Schematic overview of the virus-host interaction assay based on an amber suppression approach.

Mammalian cells grown in Bpa-supplemented medium are co-transfected with plasmids encoding viral sequences carrying a Flag tag, a (TAG) stop codon at the desired position, and an amber suppressor tRNA (tRNA_CUA)/aminoacyl tRNA synthetase (aaRS) orthogonal pair. Cells are then exposed to UV, to generate protein-protein crosslinks, followed by immunoprecipitation with anti-Flag magnetic beads. The affinity-purified crosslinks are probed by western blot using an anti-Flag antibody and the crosslinked host proteins are characterised by mass spectrometry.

Aedes aegypti mosquitoes are the main vectors of many medically relevant arthropod-borne (arbo) viruses, including Zika (ZIKV), dengue (DENV), and yellow fever (YFV). Vector competence studies with Ae. aegypti often involve challenging mosquitoes with an artificial bloodmeal containing virus and later quantifying viral titer or infectious plaque-forming units (PFU) in various mosquito tissues at relevant time points post-infection. However, Ae. aegypti mosquitoes are known to exhibit midgut infection and escape barriers (MIB and MEB, respectively), which influence the prevalence and titer of a disseminated infection and can introduce unwanted variability into studies analyzing tissues such as the salivary glands. To surmount this challenge, we describe herein a protocol for the intrathoracic inoculation of ZIKV in Ae. aegypti. This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge, and mosquitoes become infected with a consistent dose of virus. Our protocol is advantageous for studies that need a large sample size of infected mosquitoes, need to bypass the midgut, or are analyzing salivary gland infection or escape barriers.

Graphic abstract:

Cartoon depiction of Aedes aegypti intrathoracic inoculation. Figure made with Biorender.com.

The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike (CoV-2 S) proteins and green fluorescent protein (GFP) reporters. The virus titer is determined by the GFP reporter (fluorescent) expression with a flow cytometer. High titers (>1.00 E+06 infectious units/ml) are produced using codon-optimized CoV-2 S, harbouring the prevalent D614G mutation and lacking its ER retention signal. Enhanced and consistent cell entry is achieved by using permissive HEK293T/17 cells that were genetically engineered to stably express the SARS-CoV-2 human receptor ACE2 along with the cell surface protease TMPRSS2 required for efficient fusion. For the widespread use of this protocol, its reagents have been made publicly available.

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Production and quantification of lentiviral vectors pseudotyped with the SARS-CoV-2 Spike glycoprotein

Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity in vitro, methods that report changes in tyrosine kinase activity in single cells have the potential to provide information about kinase responses at the cell population level. In this protocol, we combined bimolecular fluorescence complementation (BiFC), an established method for the assessment of protein-protein interactions, and immunofluorescence staining with phosphospecific antibodies to characterize changes in host cell tyrosine kinase activity in the presence of an HIV-1 virulence factor, Nef. Specifically, two Tec family kinases (Itk and Btk) as well as Nef were fused to complementary, non-fluorescent fragments of the Venus variant of YFP. Each kinase was expressed in 293T cells in the presence or absence of Nef and immunostained for protein expression and activity with anti-phosphotyrosine (pTyr) antibodies. Multi-color confocal microscopy revealed the interaction of Nef with each kinase (BiFC), kinase activity, and kinase protein expression. Strong BiFC signals were observed when Nef was co-expressed with both Itk and Btk, indicative of interaction, and a strong anti-pTyr immunoreactivity was also seen. The BiFC, pTyr, and kinase expression signals co-localized to the plasma membrane, consistent with Nef-mediated kinase activation in this subcellular compartment. Image analysis allowed calculation of pTyr-to-kinase protein ratios, which showed a range of responses in individual cells across the population that shifted upward in the presence of Nef and back down in the presence of a kinase inhibitor. This method has the potential to reveal changes in steady-state non-receptor tyrosine kinase activity and subcellular localization in a cell population in response to other protein-kinase interactions, information that is not attainable from immunoblotting or other in vitro methods.