Name: confocal imaging of double-stranded rna and pattern recognition receptors in negative-sense rna virus infection
Uploaded: 2019-01-26T15:00:00
Description: Watch this Scientific Journal Video about Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection at JoVE.com

We would like to thank the UTMB imaging core facilities and Maxim Ivannikov for microscope assistance.
This work was supported by Public Health Service grant RO1AI093445 and RO1AI129198 awarded to SP, UTMB Commitment Fund P84373 to CH, and T32 AI007526 to EM.

1. Preparation of A549 cells and JUNV infection
<ol>
	<li>Seed 2 x 105 human lung epithelial A549 cells onto poly-D-lysine (PDL) coated glass coverslips in 12-well plates at 24 hours prior to infection.</li>
	<li>Prepare aliquots of 150 &#956;L of JUNV13 at a multiplication of infection (MOI) of 1.0 plaque forming unit per cell diluted in Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) media supplemented with 2% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S).<...

<ol>
	<li>Jensen, S., Thomsen, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+RNA+viruses:+a+review+of+innate+immune+receptors+involved+in+recognizing+RNA+virus+invasion.">Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion.</a> Journal of Virology. 86, 2900-2910 (2012).</li><li>Weber, F., Wagner, V., Rasmussen, S. B., Hartmann, R., Paludan, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+is+produced+by+positive-strand+RNA+viruses+and+DNA+viruses+but+not+in+detectable+amounts+by+negative-strand+RNA+viruses.">Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses.</a> Journal of Virology. 80, 6 (2006).</li><li>Triantafilou, K., Vakakis, E., Kar, S., Richer, E., Evans, G. L., Triantafilou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+of+direct+interaction+of+MDA5+and+the+dsRNA+replicative+intermediate+form+of+positive+strand+RNA+viruses.">Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses.</a> Journal of Cell Science. 125, 4761-4769 (2012).</li><li>Onomoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+an+antiviral+stress+granule+containing+RIG-I+and+PKR+in+viral+detection+and+innate+immunity.">Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.</a> PLoS One. 7, e43031 (2012).</li><li>Son, K. N., Liang, Z., Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+is+detected+by+immunofluorescence+analysis+in+RNA+and+DNA+virus+infections,+including+those+by+negative-sense+RNA+viruses.">Double-stranded RNA is detected by immunofluorescence analysis in RNA and DNA virus infections, including those by negative-sense RNA viruses.</a> Journal of Virology. 89, 9383-9392 (2015).</li><li>Mateer, E. J., Paessler, S., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+double-stranded+RNA+colocalizing+with+pattern+recognition+receptors+in+arenavirus+infected+cells.">Visualization of double-stranded RNA colocalizing with pattern recognition receptors in arenavirus infected cells.</a> Frontiers Cellular and Infection Microbiology. 8, 251 (2018).</li><li>Buchmeier, M. J., de la Torre, J. C., Peters, C. J., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arenavirdae:+The+viruses+and+their+replication.">Arenavirdae: The viruses and their replication.</a> Fields Virology. 2, (2006).</li><li>Levis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+interferon+in+Argentine+hemorrhagic+fever.">Endogenous interferon in Argentine hemorrhagic fever.</a> The Journal of Infectious Diseases. 149, 428-433 (1984).</li><li>Levis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+endogenous+interferon+and+the+clinical+evolution+of+patients+with+Argentine+hemorrhagic+fever.">Correlation between endogenous interferon and the clinical evolution of patients with Argentine hemorrhagic fever.</a> Journal of Interferon Research. 5, 383-389 (1985).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+pathogenic+new+world+and+old+world+human+arenaviruses+induce+distinct+interferon+response+in+human+cells.">Highly pathogenic new world and old world human arenaviruses induce distinct interferon response in human cells.</a> Journal of Virology. 89, 7079-7088 (2015).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junin+virus+infection+activates+the+type+I+interferon+pathway+in+a+RIG-I-dependent+manner.">Junin virus infection activates the type I interferon pathway in a RIG-I-dependent manner.</a> PLoS Neglected Tropical Diseases. 6, e1659 (2012).</li><li>Huang, C., Kolokoltsova, O. A., Mateer, E. J., Koma, T., Paessler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+pathogenic+new+world+arenavirus+infection+activates+the+pattern+recognition+receptor+protein+kinase+R+without+attenuating+virus+replication+in+human+cells.">Highly pathogenic new world arenavirus infection activates the pattern recognition receptor protein kinase R without attenuating virus replication in human cells.</a> Journal of Virology. 91, 20 (2017).</li><li>Emonet, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+from+cloned+cDNAs+and+in+vivo+characterization+of+recombinant+pathogenic+Romero+and+live-attenuated+Candid#1+strains+of+Junin+virus,+the+causative+agent+of+Argentine+hemorrhagic+fever+disease.">Rescue from cloned cDNAs and in vivo characterization of recombinant pathogenic Romero and live-attenuated Candid#1 strains of Junin virus, the causative agent of Argentine hemorrhagic fever disease.</a> Journal of Virology. 85 (4), 1473-1483 (2011).</li><li>Child, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonism+of+the+protein+kinase+R+pathway+in+human+cells+by+Rhesus+Cytomegalovirus.">Antagonism of the protein kinase R pathway in human cells by Rhesus Cytomegalovirus.</a> Journal of Virology. 92, 6 (2018).</li><li>Hobro, A. J., Smith, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li></ol>

For positive-sense RNA viruses and dsDNA viruses, dsRNA is readily detected with the widely-used J2 anti-dsRNA antibody. However, negative-sense RNA viruses are believed to produce dsRNA at a low or below the detection level using the same antibody2. Thus, many aspects of viral RNA and PRR interaction are largely unclear for negative-sense RNA viruses. We attempted to stain for dsRNA in arenavirus infection using the J2 antibody but the fluorescence signals were not differentiable compared to the ...

The initial step of the induction of the innate immune response is host recognition of double-stranded (ds) RNA by the pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5)1. For positive-sense RNA viruses, dsRNA can usually be readily detected using the J2 or K1 anti-dsRNA monoclonal antibodies (Mab)2. Interaction between dsRNA and PRRs in positive-strand RNA viruses, such as picornavirus, has been characterized using confocal microscopy3. However, for negative-sense RNA virus, visualization and characterization of PRR and dsRNA interaction has been hindered by the lack of sensitive antibodies to dsRNA. RNA fluorescent in situ hybridization (FISH) has been applied to the visualization of viral RNA and PRRs4. Nevertheless, the FISH methodology requires the knowledge of the target RNA sequence and may not be compatible with PRR co-staining. Recently, the 9D5 Mab, which was originally developed for the diagnosis of pan-enterovirus infection, was found to be more sensitive than the J2 Mab and can readily detect dsRNA in negative-sense RNA virus infection5,6. Thus, Mab 9D5 is a novel and useful tool to study viral replication and the interaction between PRR and viral RNA for negative-sense RNA virus.
Arenaviruses are a family of single-stranded, negative-sense RNA viruses, which include several human pathogens, such as Lassa virus (LASV), Jun&#237;n virus (JUNV) and Machupo virus (MACV), that cause severe hemorrhagic fever diseases in humans7. Clinical data from severe and fatal cases of Argentine hemorrhagic fever caused by the New World arenavirus JUNV exhibit unusually high levels of serum IFN-&#945;8,9. We have shown that the pathogenic NW arenaviruses (JUNV and MACV), but not the pathogenic Old World arenavirus, LASV, induce a type I interferon (IFN) response in human monocyte-derived dendritic cells10. Furthermore, RIG-I is one of the sensors mediating type I IFN response in JUNV-infected cells11. We also found that the protein kinase R (PKR) receptor, which is traditionally known for dsRNA recognition, is activated in pathogenic NW arenavirus infection12. To further understand the mechanism of virus-specific IFN response during arenavirus infection, we aimed to develop a protocol to visualize the interaction between viral dsRNA and the cytoplasmic PRRs.
Infection experiments with pathogenic JUNV, MACV and LASV have to be performed in biosafety level 4 (BSL-4) facilities. Thus, in addition to the presumably low level of dsRNA formed in arenavirus infection, meeting the biosafety requirements is another technique challenge when performing imaging studies for these highly pathogenic viruses. By utilizing the 9D5 antibody and the Candid1# vaccine strain of JUNV, a confocal microscopy-based protocol is described in this report, which has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in cells infected by arenavirus in BSL2 labs. The protocol is also suitable for visualization of intracellular distribution of dsRNA and PRR during pathogenic arenavirus infection in BSL4 facilities.

<table>
	<tbody>
		<tr>
			<td>APEX Alexa Fluor 647 antibody labeling kit</td>
			<td>Invitrogen</td>
			<td>A10475</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Cell Signaling</td>
			<td>4083</td>
			<td>1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Donkey-anti rabbit Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-11058</td>
			<td>1:2,000 dilution; Lot #: 1454437</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Bio</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti mouse Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td>1:2000 dilution; Lot #: 1874804</td>
		</tr>
		<tr>
			<td>Human lung epithelial A549 cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mouse MAb anti-JUNV NP</td>
			<td>BEI</td>
			<td>NA05-AG12</td>
			<td>Conjugated to Alexa-647 at 1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Mouse MAb pan-Enterovirus 9D5 Reagent</td>
			<td>Millipore Sigma</td>
			<td>3361</td>
			<td>ready for use; diluted to 1:2; Lot #: 3067445</td>
		</tr>
		<tr>
			<td>PBS supplemented with Ca and Mg</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PDL Coated coverslips</td>
			<td>Neuvitro</td>
			<td>H-12-1.5-pdl</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit MAb MDA-5</td>
			<td>Abcam</td>
			<td>ab126630</td>
			<td>1:250 dilution; Lot #: GR97758-7</td>
		</tr>
		<tr>
			<td>recombiant Candid#1 strain of JUNV</td>
			<td>Lab generated</td>
			<td>Lab generated</td>
			<td>As previously described in reference 13.</td>
		</tr>
		<tr>
			<td>RIG-I mouse MAb conjugated to Alexa-594</td>
			<td>Santa Cruz</td>
			<td>sc-376845</td>
			<td>1:1000 dilution; Lot #: AO218</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

confocal imaging of double-stranded rna and pattern recognition receptors in negative-sense rna virus infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

This protocol was applied to study the distribution and colocalization between the RLRs (RIG-I and MDA-5) and dsRNA in JUNV-infected cells. As shown in Figure 1 and Figure 2, the accumulation of dsRNA increases over time as viral infection progresses. Concentrated MDA-5 (Figure 1) and RIG-I (Figure 2) signals were found colocalized with the punctate structures of the NP ...

Watch this Scientific Journal Video about Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection at JoVE.com

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Traditional double-strand RNA detection assay usually is not sensitive enough to detect double-strand RNA formed in negative-sense RNA virus infection.Therefore the interaction between double-stranded RNA and host pattern recognition receptors has remained largely elusive for these viruses.Utilizing this protocol we are able to visualize and characterize the interaction between viral nuclear protein, double-strand RNA and host a PRRs at a single-cell level for negative-sense RNA virus.This is a multiplex assay that allows for simultaneous staining of double-stranded RNA and two viral or host protein targets in individual cells.Unlike the RNA-FISH assay this technique is RNA sequence independent and usually compatible with antibody detection of protein targets.Begin 24 hours prior to infection by seeding 200, 000 human lung epithelial A549 cells onto Poly-D-Lysine coated glass cover slips in 12-well plates and growing them at 37

confocal-imaging-double-stranded-rna-pattern-recognition-receptors

Research

JoVE Journal

Immunology and Infection

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

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.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

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 ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...