Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.
20 Related JoVE Articles!
Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Institutions: University of Texas Medical Branch.
Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1
, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus
in the family Bunyaviridae
and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3
as well as wild-type RVFV strains 4-6
, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3
, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8
and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10
IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11
, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14
. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.
Immunology, Issue 57, Rift Valley fever virus, reverse genetics, NSs, MP-12, vaccine development
Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells
Institutions: University of Rochester School of Medicine and Dentistry, The Scripps Research Institute.
The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4
. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11
. In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5
. Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16
. The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19
, which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.
Virology, Issue 78, Infection, Infectious Diseases, Microbiology, Molecular Biology, Cellular Biology, Medicine, Biomedical Engineering, Viruses, arenaviruses, plasmid transfection, recombinant virus, reverse genetics techniques, vaccine/vaccine vector seed development, clinical applications
An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus
Institutions: Georgetown University, The Ohio State University.
Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus
, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus.
Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus
due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.
Genetics, Issue 93, Aedes albopictus Asian tiger mosquito, photoperiodic diapause, RNA-Seq de novo transcriptome assembly, mosquito husbandry
Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems
Institutions: German Cancer Research Center (DKFZ), German Cancer Research Center (DKFZ).
Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1
. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2
. NS1 is the major effector of viral cytotoxicity3
. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g.
a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4
. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1
. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6
) and the VA RNA
pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5
. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g.
NB324K) (Fig. 2
). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5
. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.
Immunology, Issue 62, Recombinant parvovirus, adenovirus, virus production, pXX6, virus helper, virology, oncology
Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus
Institutions: UT Southwestern Medical Center, UT Southwestern Medical Center.
In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2
. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine γHV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4
. γHV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo
, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in γHV68 lytic replication both in vivo
and ex vivo
. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during γHV68 acute infection in vivo
. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during γHV68 lytic replication ex vivo
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant γHV68 carrying these mutations can be used to recapitulate the phenotypes of γHV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo
and ex vivo
Recently, we have discovered that γHV68 usurps an innate immune signaling pathway to promote viral lytic replication5
. Specifically, γHV68 de novo infection activates the immune kinase IKKβ and activated IKKβ phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, γHV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.
Immunology, Issue 56, herpesvirus, gamma herpesvirus 68, γHV68, signaling pathways, host-virus interaction, viral lytic replication
Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining
Institutions: CNRS UMR 5534, Université de Lyon 1, LabEX DEVweCAN, CNRS UPR 3296, CNRS UMR 5286.
Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ
in animal models. We describe a DNA-fluorescent in situ
hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.
Neuroscience, Issue 83, Life Sciences (General), Virology, Herpes Simplex Virus (HSV), Latency, In situ hybridization, Nuclear organization, Gene expression, Microscopy
Reverse Genetics Mediated Recovery of Infectious Murine Norovirus
Institutions: Imperial College London .
Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2
. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3
. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4
but which can be propagated in cells 5
has opened new avenues for the investigation of these pathogens 6, 7
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1
). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1
). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11
. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14
. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches.
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro
synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17
Virology, Issue 64, Immunology, Genetics, Infection, RNA virus, VPg, RNA capping, T7 RNA polymerase, calicivirus, norovirus
A Protocol for Analyzing Hepatitis C Virus Replication
Institutions: Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA.
Hepatitis C Virus (HCV) affects 3% of the world’s population and causes serious liver ailments including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae
. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla
luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro
transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.
Infectious Diseases, Issue 88, Hepatitis C Virus, HCV, Tumor-virus, Hepatitis C, Cirrhosis, Liver Cancer, Hepatocellular Carcinoma
High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses
Institutions: Institut Pasteur, CNRS UMR3569, Institut Pasteur, CNRS UMR3523, Institut Pasteur.
RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.
Immunology, Issue 87, Viral infections, high-throughput screening assays, broad-spectrum antivirals, chikungunya virus, measles virus, luciferase reporter, chemical libraries
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Institutions: Institut Pasteur .
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3
. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7
Immunology, Issue 52, Polymerase fidelity, RNA virus, mutation frequency, mutagen, RNA polymerase, viral evolution
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Rescue of Recombinant Newcastle Disease Virus from cDNA
Institutions: Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai, University of Rochester.
Newcastle disease virus (NDV), the prototype member of the Avulavirus
genus of the family Paramyxoviridae1
, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1)
with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5
. Viral cDNA can be conveniently modified in vitro
by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.
Immunology, Issue 80, Paramyxoviridae, Vaccines, Oncolytic Virotherapy, Immunity, Innate, Newcastle disease virus (NDV), MVA-T7, reverse genetics techniques, plasmid transfection, recombinant virus, HA assay
Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus
Institutions: Johns Hopkins University School of Medicine.
A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro
phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.
Stem Cell Biology, Issue 86, Induced pluripotent stem cells, Human embryonic stem cells, Sendai-virus
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Generation of Recombinant Influenza Virus from Plasmid DNA
Institutions: University of Rochester School of Medicine and Dentistry, Mount Sinai School of Medicine .
Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2
plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3
Microbiology, Issue 42, influenza viruses, plasmid transfection, recombinant virus, reverse genetics techniques, HA assay
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Institutions: Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg.
HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.
Medicine, Issue 16, HIV, Cell Biology, Recombinase, provirus, HeLa Cells
Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato
Institutions: Cornell University, Boyce Thompson Institute for Plant Research.
RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1
. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4
. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens
to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens
strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7
. Inoculation of Nicotiana benthamiana
and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase
) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana
. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana
can be used to silence their respective orthologs in tomato and vice versa8
Plant Biology, Issue 28, Virus-induced gene silencing (VIGS), RNA interference (RNAi), Tobacco Rattle Virus (TRV) vectors, Nicotiana benthamiana, tomato
Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection
Institutions: Cornell University, Cornell University.
Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.
Plant Biology, Issue 15, Annual Review, Aphids, Plant Virus, Potato Leaf Roll Virus, Microinjection Technique
Titration of Human Coronaviruses Using an Immunoperoxidase Assay
Institutions: INRS-Institut Armand-Frappier.
Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.
Microbiology, Issue 14, Springer Protocols, Human coronavirus, HCoV-229E, HCoV-OC43, cell and tissue sample, titration, immunoperoxidase assay, TCID50
Institutions: University of California, San Francisco - UCSF.
RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.
Microbiology, Issue 32, Lentivirus, RNAi, viral titration, transfection, retrovirus, flow cytometry, split vector system, shRNA.