This work was supported in whole or in part by the National Institutes of Health (R01 AI121315, R01 AI146017, R01 AI148382, R01 AI120860, R37 AI076119, and U54 AI150472). We thank Dr. Raymond F. Schinazi and Sadie Amichai for providing cells infected with influenza A Virus.

1. Seed cells (Suspension vs. Adherent Cells) on coverslips or chamber slides (protocol presented uses coverslips).
<ol>
	<li>Seeding of suspension cells
	<ol>
		<li>Prepare poly-D-lysine (PDL) coated coverslips (facilitates adherence of suspension cells to coverslip) by first incubating coverslips in ethanol (EtOH) for 5 minutes (sterilizes coverslips and removes any residue). Then wash 2x in phosphate buffered saline (PBS) before incubating coverslips in PDL (20 &#181;g/mL) for 30 minutes (min) at room temperature (RT).</li>
		<li>Remove PDL and wash 2x in PBS. Pellet cells (previously infected/treated based on desired imaging conditions) and resuspend 106cells in 50 &#181;L of PBS. Spot 50 &#181;L of cells on glass (PDL-coated) and incubate at RT for 30 min.</li>
	</ol>
	</li>
	<li>Seeding of adherent cells
	<ol>
		<li>Culture cells on sterile coverslip placed in 6-well dish and infect cells with viral particles or treat with compound of interest. Allow cells to reach 50-70% confluency prior to sample preparation for imaging experiments.</li>
	</ol>
	</li>
</ol>
2. Cellular fixation: To preserve cellular morphology for fluorescence imaging studies
NOTE: Keep 4% PFA and PBS at RT for at least 30 minutes before cellular fixation.
<ol>
	<li>Aspirate the cellular media, wash cells on coverslips 3x in PBS, and fix cells in 4% paraformaldehyde (PFA) for 30 min. Aspirate PFA and wash cells in PBS 2x. 
	NOTE: Cells can now be dehydrated and stored, if the experimenter wishes to resume the experiment on another date. Fixed cells can be dehydrated in EtOH prior to storage.
	<ol>
		<li>Remove PBS following wash after cellular fixation and replace with 500 &#181;L of 50% EtOH (v/v in water). Incubate at RT for 5 min.</li>
		<li>Remove 50% EtOH and replace with 500 &#181;L of 70% EtOH. Incubate at RT for 5 min.</li>
		<li>Remove 70% EtOH and replace with 500 &#181;L of 100% EtOH. Incubate at RT for 5 min.</li>
		<li>Remove 100% EtOH and replace with fresh 100% EtOH. 
		NOTE: Dehydrated cells can be stored at -20 &#176;C for 6 months. Seal plates with tape or parafilm prior to storage to prevent evaporation of EtOH.</li>
		<li>Rehydrate cells to move onto multiplexed fluorescence labeling of cells/virus.</li>
		<li>Remove 100% EtOH and replace with 500 &#181;L of 70% EtOH. Incubate at RT for 2 min. 
		NOTE: Do not let cells dry out at any time. Always use enough solution to submerge all the cells.</li>
		<li>Remove 70% EtOH and replace with 500 &#181;L of 50% EtOH. Incubate at RT for 2 min.</li>
		<li>Remove 50% EtOH and replace with 1x PBS. 
		NOTE: Prepare protease dilution, hybridization probes, and wash buffer in advance of protease treatment (Step 4) and target probe application (Step 5). Specific instructions on reagent preparation are presented at the beginning of each respective step.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Other Notes</td>
		</tr>
		<tr>
			<td>Protease solution</td>
			<td>Prepare in 1X PBS</td>
		</tr>
		<tr>
			<td>Target oligonucletodie probe(s)</td>
			<td>Dilute in hybridization buffer</td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>Washes following target hybridization steps</td>
		</tr>
		<tr>
			<td>Hybridization buffer</td>
			<td>Recipe presented in Table 3</td>
		</tr>
	</tbody>
</table>
Table 1: Key reagents in MICDDRP protocol.
3. Cell permeabilization: Increases access into the cell and cellular organelles for entry of large molecules (antibodies, nucleic acid hybridization probes)
<ol>
	<li>Remove 1x PBS following cellular fixation or rehydration and replace with 500 &#181;L of 0.1% (v/v) Tween-20 in 1x PBS. Incubate at RT for 10 min. Replace one by one. Wash once with 500 &#181;L of 1x PBS and add fresh 1x PBS.</li>
</ol>
4. Coverslip immobilization on glass slide and protease treatment to remove nucleic acid binding proteins from fixed viral/cellular nucleic acid to improve hybridization efficiency
NOTE: Prepare the diluted Protease III (see specifics of reagent in Table of Materials) solution during cellular permeabilization. Let protease reach RT for 10 min before permeabilization.
<ol>
	<li>Place a small drop of nail polish on a sterilized glass slide. Dry back (side with no cell layer) and place the edge of coverslip on the nail polish drop, with the side with the adhered cells facing upwards. Add few drops of PBS on the immobilized coverslip to prevent drying.
	<ol>
		<li>Draw a circle (about 3 mm away from the coverslip) around the perimeter of the coverslip now adhered to the slide using hydrophobic barrier pen (water-repellant pen that keeps reagents localized on cells).</li>
	</ol>
	</li>
	<li>Dilute Protease III in 1x PBS (100 &#181;L/coverslip). 
	NOTE: Protease concentration may need to be adjusted depending on differences in cell types, probes, or target nucleic acid(s) through empirical optimization (See Discussion). For most efficient RNA/DNA labeling across different viral systems, we had success with the following dilutions presented in Table 2 below with dilutions ranging from (1 to 2)-(1 to 15) (protease to 1x PBS).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Probe Targets</td>
			<td>Protease Dilution in 1X PBS (Protease III to 1X PBS)</td>
		</tr>
		<tr>
			<td>HIV-1 DNA/RNA</td>
			<td>1 to 5</td>
		</tr>
		<tr>
			<td>HTLV-1 DNA/RNA</td>
			<td>1 to 5</td>
		</tr>
		<tr>
			<td>HBV pgRNA and total HBV RNA</td>
			<td>1 to 15</td>
		</tr>
		<tr>
			<td>IAV RNA</td>
			<td>1 to 15</td>
		</tr>
		<tr>
			<td>ZIKV RNA</td>
			<td>1 to 2</td>
		</tr>
	</tbody>
</table>
Table 2: Protease III dilutions in PBS for viral nucleic acid hybridization.
<ol start="3">
	<li>Decant the 1x PBS on the coverslip following immobilization and apply the diluted Protease III.</li>
	<li>Incubate in a humidified oven at 40 &#176;C for 15 min.</li>
	<li>Decant protease solution and submerge slides in 1x PBS. Agitate with a rocking dish for 2 min at RT. Repeat wash with new 1x PBS.
	<ol>
		<li>For only DNA detection, wash samples three times with nuclease-free water for 2 min each, followed by incubation with 5 mg/mL RNase A diluted in PBS for 30 min at 37 &#176;C.</li>
		<li>Decant RNase A solution, and wash 3x for 2 min with ultrapure water. Continue with ISH of target probe(s). 
		NOTE: Hybridization buffer improves vDNA detection without affecting vRNA staining efficiency for the results presented (Representative Results). Dilute DNA Channel 1 (C1) probes 1:1 with hybridization probe. Dilute RNA C2 and C3 probes in hybridization buffer. The C2 and C3 probes used in our imaging studies are in 50X solutions (1:50; target probe to hybridization buffer).</li>
	</ol>
	</li>
	<li>Prepare hybridization buffer in nuclease-free water following the step-by-step procedure below:
	<ol>
		<li>In a 15 mL tube, add 700 &#181;L of nuclease free-water, 300 &#181;L of 50% (weight/volume (w/v)) dextran sulfate, 300 &#181;L of 5 M NaCl, 125 &#181;L of 200 mM sodium citrate (pH 6.2), and 375 mg (powder) of ethylene carbonate.</li>
		<li>Mix well using a vortex to dissolve ethylene carbonate (ensure all powder is dissolved and clear solution).</li>
		<li>Add 25 &#181;L of 10% (volume/volume (v/v)) Tween-20 and enough nuclease-free water to complete 2.5 mL (2x solution). 
		NOTE: The recipe for the hybridization buffer presented can be found in Table 3 below. Solution is stable for a week. Ensure sufficient mixing of Tween-20 detergent, while being careful to prevent bubbling of solution.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock Concentration</td>
			<td>Additional Notes</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>50% (w/v)</td>
			<td>Very viscous</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>5 M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate (pH 6.2)</td>
			<td>200 mM</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Ethylene carbonate</td>
			<td>NA</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>10% (v/v)</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3: Hybridization buffer list of reagents.
5. Incubation with DNA/RNA target hybridization probes: Target oligonucleotide probes bind to region(s) of interest, creating a molecular scaffold for pre-amplifiers, amplifiers, and fluorescent probes to bind.
NOTE: Warm DNA/RNA probes at 40 &#176;C for 10 min (during Cell Permeabilization) and cool down to RT for at least 10 min, if no RNase-treatment is included. If RNase-treatment or further sample treatment is needed prior to target probe hybridization, warm probes accordingly. Spin down C2 and C3 probes after warming and dilute in hybridization buffer. After dilution, C2 and C3 probes can be briefly warmed. Warm 50x wash buffer (See Table of Materials for more detail) at 40 &#176;C for 10-20 min and dilute to 1x in molecular biology grade water.
<ol>
	<li>Incubate 200 &#181;L of the hybridization buffer at 67 &#176;C for 10 min prior to addition of hybridization probe(s). Dilute probe in hybridization buffer (recipe listed in Table 2). Add 50 &#181;L/coverslip.</li>
	<li>Incubate in humidified oven at 40 &#176;C for 2 hours (h). Decant probes and submerge slides in 1x wash buffer. Agitate by rocking dish for 2 min at RT. Repeat wash with new 1x wash buffer.</li>
</ol>
6. Amplifier (Amp) 1-FL Hybridization: Addition of pre-amplifier that is complementary to the tail sequence of the target DNA/RNA probes (Step 5)
NOTE: Amplifiers should be at RT before use. Get each individual amplifier out of the fridge 30 min before use and leave on the bench at RT.
<ol>
	<li>Remove slides from 1x wash buffer and tap/absorb to remove excess liquid.</li>
	<li>Add 1 drop of Amp 1-FL on the coverslip. Incubate in humidified oven at 40 &#176;C for 30 min.</li>
	<li>Decant Amp 1-FL and submerge in 1x wash buffer. Agitate by rocking dish 2 min at RT. Repeat wash with new 1x wash buffer.</li>
</ol>
7. Amp 2-FL Hybridization: Incubation with signal amplifier with cognate recognition sequence to pre-amplifiers (Amp 1-FL)
<ol>
	<li>Remove slides from 1x wash buffer and tap/absorb to remove excess liquid.</li>
	<li>Add 1 drop of Amp 2-FL on the coverslip. Incubate in humidified oven at 40 &#176;C for 15 min.</li>
	<li>Decant Amp 2-FL and submerge in 1x wash buffer. Agitate by rocking dish 2 min at RT. Repeat wash with new 1x wash buffer.</li>
</ol>
8. Amp 3-FL Hybridization: Incubation with second signal amplifier
<ol>
	<li>Remove slides from 1x wash buffer and tap/absorb to remove excess liquid.</li>
	<li>Add 1 drop of Amp 3-FL on the coverslip. Incubate in humidified oven at 40 &#176;C for 30 min.</li>
	<li>Decant Amp 3-FL and submerge in 1x wash buffer. Agitate by rocking dish 2 min at RT. Repeat wash with new 1x wash buffer.</li>
</ol>
9. Amp 4-FL Hybridization: Fluorescent label and final hybridization step
NOTE: First, see Table 4 to choose the suitable Amp 4 (A,B, or C)- FL based on the channels of the target probe(s). Assess what Amp 4-FL is needed to label DNA/RNA of interest. An example is provided by the table below:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>A</td>
			<td>B</td>
			<td>C</td>
		</tr>
		<tr>
			<td>DNA Channel 1 (C1)</td>
			<td>488</td>
			<td>550</td>
			<td>550</td>
		</tr>
		<tr>
			<td>DNA/RNA Channel 2 (C2)</td>
			<td>550</td>
			<td>488</td>
			<td>647</td>
		</tr>
		<tr>
			<td>RNA Channel 3 (C3)</td>
			<td>647</td>
			<td>647</td>
			<td>488</td>
		</tr>
	</tbody>
</table>
Table 4: Selection of Amp 4 (A, B, or C)-FL fluorescent probe for multiplexed ISH.
NOTE: For multiplexed FISH (multiple targets), choosing the correct Amp 4 (A, B, or C)- FL is critical for properly labeling your target of interest(s). Target probes (Step 5) have different color channels (C1, C2, or C3), which dictate their respective fluorescent label (Alexa 488, Atto 550, or Alexa 647), based on the Amp 4-FL chosen. Examples of choosing fluorophore combinations for multiplexed imaging are provided in the legends of Figure 2 and Figure 3. As an additional example, selection of Amp 4B-FL will selectively label DNA C1 probes with Atto 550 and RNA C3 probes with Alexa 647.
<ol>
	<li>Remove slides from 1x wash buffer and tap/absorb to remove excess liquid.</li>
	<li>Add 1 drop of Amp 4-FL on the coverslip. Incubate in humidified oven at 40 &#176;C for 15 min. 
	NOTE: Following step 9.2, keep samples covered, protected from the light.</li>
	<li>Decant Amp 4-FL and submerge in 1x wash buffer. Agitate by rocking dish for 2 min at RT. Repeat wash with new 1x wash buffer. Wash with 1x PBS (2 min) and store in PBS.</li>
</ol>
10. Protein immunostaining: To label protein(s) of interest
<ol>
	<li>Decant PBS and add 200 &#181;L of blocking buffer (1% w/v BSA, 10% v/v FBS in PBS with 0.1% v/v Tween-20 (PBST)) to the coverslip. Incubate 1 h at RT.</li>
	<li>Decant blocking buffer and apply 200 &#181;L of primary antibody diluted in PBST + 1% w/v BSA. Incubate 1 h at RT.</li>
	<li>Wash the slide twice with PBST for 10 min at RT with shaking.</li>
	<li>Apply secondary antibody of choice for 1 h at RT in PBST + 1% w/v BSA.</li>
	<li>Wash the slide with PBST for 10 min at RT with shaking.</li>
</ol>
11. Nuclear staining: Counter-stain nuclei following immunostaining
<ol>
	<li>Decant PBST and apply DAPI or nuclear stain of choice for 1 min at RT.</li>
	<li>Wash the slide twice with PBS for 10 min at RT with shaking.</li>
</ol>
12. Mounting
<ol>
	<li>Place 1 drop of antifade solution (e.g., Prolong Gold) on new sterile glass slide (First, clean slide with EtOH and let dry to ensure no residues are on glass). With the same tip, spread the antifade solution drop to cover an area approximately the size of the coverslip. 
	NOTE: The antifade solution is very viscous and may be difficult to pipette. Cutting the tip off a 200 &#181;L tip prior to pipetting may mitigate these issues.</li>
	<li>Remove coverslip with cell sample from the slide and submerge in PBS to remove residual nail polish at the back using the forceps and PBS. Dry forceps and back of the coverslip using a Kimwipe.</li>
	<li>Gently imbed coverslip in the drop of antifade solution, placing sample side (side with cell layer) of coverslip on drop).</li>
	<li>Let samples dry overnight.</li>
</ol>
13. Imaging
<ol>
	<li>Image with an epifluorescent microscope.</li>
</ol>

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The authors have nothing to disclose.

Simultaneous visualization of RNA, DNA, and protein often requires extensive optimization. Two commonly used methods are 5-ethynyl-2-deoxyuridine (EdU) labeling and DNA FISH. EdU labeling has been applied to visualize viral DNA and protein simultaneously, as EdU is incorporated in nascent DNA and subsequently labeled with azide-containing fluorescent dyes via click chemistry. EdU labeling can thus be used to monitor native virus replication kinetics of DNA viruses or viruses with DNA templates for replication<su...

While thousands of commercial antibodies are available to specifically label proteins via conventional immunostaining approaches, and while fusion proteins can be engineered with photo-optimized fluorescent tags for tracking multiple proteins in a sample1, microscopic visualization of protein is often not compatible with conventional DNA fluorescence in situ hybridization (FISH)2. Technical limitations in simultaneous visualization of DNA, RNA, and protein using fluorescence-based approaches have hindered in-depth understanding of virus replication. Tracking both viral nucleic acid and protein during the course of infection allows virologists to visualize fundamental processes that underly virus replication and assembly3,4,5,6.
We have developed an imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and Protein (MICDDRP)3, which utilizes branched DNA (bDNA) in situ technology to improve the sensitivity of nucleic acid detection7,8,9. In addition, this method utilizes paired probes for enhanced specificity. bDNA sequence-specific probes use branching preamplifier and amplifier DNAs to produce an intense and localized signal, improving upon previous hybridization methods that relied on targeting repeated regions in the DNA9. Infected cells in a clinical context often do not contain abundant viral genetic material, providing a commodity for a sensitive method for fluorescent nucleic acid detection in diagnostic settings. The commercialization of bDNA technology through approaches such as RNAscope7 and ViewRNA10 have filled this niche. The sensitivity of bDNA fluorescence imaging also has important utility in cell biology, allowing detection of scarce nucleic acid species in cell culture models. The vast improvement of sensitivity makes bDNA-based imaging methods suitable for studying viruses. A potential shortcoming, however, is that these methods focus on visualizing RNA or RNA and protein. All replicating cells and many viruses have DNA genomes or form DNA during their replication cycle, making methods capable of imaging both RNA and DNA, as well as protein, highly desirable.
In the MICDDRP protocol, we perform bDNA FISH for detection of viral nucleic acid using the RNAscope method, with modifications7. One of the major modifications to this protocol is optimization of protease treatment following chemical fixation. Protease treatment facilitates removal of proteins bound to nucleic acid to improve probe hybridization efficiency. Protease treatment is followed by incubation with branched oligonucleotide probe(s). After application of bDNA probe(s), samples are washed and subsequently incubated with signal pre-amplifier and amplifier DNAs. Multiplexed in situ hybridization (ISH), labeling of multiple gene targets, requires target probes with different color channels for spectral differentiation7. Incubation with DNA amplifiers is followed by immunofluorescence (IF).
bDNA ISH imparts improvements in signal-to-noise by amplification of target-specific signals, with a reduction to background noise from non-specific hybridization events7,11. Target probes are designed using software programs publicly available that predict the probability of non-specific hybridization events, as well as calculate melting temperature (Tm) of the probe-target hybrid7,11. Target probes contain an 18- to 25-base region complementary to the target DNA/RNA sequence, a spacer sequence, and a 14-base tail sequence. A pair of target probes, each with a distinct tail sequence, hybridize to a target region (spanning ~50 bases). The two tail sequences form a hybridization site for the pre-amplifier probes, which contain 20 binding sites for the amplifier probes, which, in addition contain 20 binding sites for the label probe. As an example, a one kilobase (kb) region on the nucleic acid molecule is targeted by 20 probe pairs, creating a molecular scaffold for sequential hybridization with the preamplifier, amplifier, and label probe. This can thus lead to a theoretical yield of 8000 fluorescent labels per nucleic acid molecule, enabling detection of single molecules and vast improvements over conventional FISH approaches7 (See Figure 1A for schematic of bDNA signal amplification). To set up probes for multiplexed ISH, each target probe must be in a different color channel (C1, C2, or C3). These target probes with different color channels possess distinct 14-base tail sequences. These tail sequences will bind distinct signal amplifiers with different fluorescent probes, thus enabling facile spectral differentiation across multiple targets. In the presented Protocol, Table 4 in Step 9, provides further information on fluorescently labeling target probes. In addition, Figure 2 and Figure 3 provide examples of how we chose the appropriate Amplifier 4-FL (A, B, or C) (fluorescent probe &#38; the final hybridization step) to achieve specific fluorescent labeling of multiple viral nucleic acid targets following HIV-1 and HTLV-1 infections.
We have demonstrated several applications of simultaneous fluorescence visualization of RNA, DNA, and proteins, observing critical stages of virus replication with high spatiotemporal resolution3,4,5. For example, simultaneous single-cell visualization of viral RNA, cytoplasmic and nuclear DNA, and protein have allowed us to visualize key events during HIV-1 infection, including following RNA containing cores in the cytoplasm prior to nuclear entry and integration of proviral DNA3. In addition, we have applied MICDDRP to characterize the effects of host factors and drug treatment on viral infection and replication4,5. In Ukah et al., we tracked reactivation of HIV-1 transcription in latency cell models treated with different latency-reversing agents to visualize HIV transcription and latency reversal4. In addition, MICDDRP can allow us to visualize phenotypic changes associated with antiviral inhibition attributed to small molecule treatment or host factor restriction. As a proof of concept to the robustness and broad applicability of our approach, we have demonstrated that we can use modifications of our protocol to efficiently label viral nucleic acid to follow infection not only in human immunodeficiency virus (HIV)-1, but also human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZIKV), and influenza A virus (IAV). As the HIV-1 life cycle consists of both viral DNA and RNA species, we have performed the majority of our optimization of MICDDRP following HIV-1 replication kinetics. However, in addition, we have demonstrated that we can track synthesis of different viral RNA transcripts of either or both sense (+) and antisense (-) strandedness in viruses such as ZIKV, IAV, HBV, and HCV to monitor viral transcription and replication3,4,5,6. The studies aim to improve the mechanistic understanding of several viral processes and serve as a guideline to implement this fluorescence imaging technology to a broad range of cellular models.

<table>
	<tbody>
		<tr>
			<td>4% PFA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50% dextran sulfate</td>
			<td>Amreso</td>
			<td>198</td>
			<td>For DNA hybridization buffer</td>
		</tr>
		<tr>
			<td>50X wash buffer</td>
			<td>ACD Bio</td>
			<td>320058</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier 1-FL</td>
			<td>ACD Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier 2-FL</td>
			<td>ACD Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier 3-FL</td>
			<td>ACD Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier 4-FL</td>
			<td>ACD Bio</td>
			<td></td>
			<td>Consult Amp-4 table in protocol</td>
		</tr>
		<tr>
			<td>Anti-HCV NS5a antibody</td>
			<td>Abcam</td>
			<td>ab13833</td>
			<td>Mouse monoclonal; works with HCV genotypes 1a, 1b, 3, and 4</td>
		</tr>
		<tr>
			<td>Anti-HIV-1 p24 monoclonal antibody</td>
			<td>NIH AIDS Reagent Program</td>
			<td>3537</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mov10 antibody</td>
			<td>Abcam</td>
			<td>ab80613</td>
			<td>Rabbit polyclonal</td>
		</tr>
		<tr>
			<td>Anti-PB1 antibody</td>
			<td>GeneTex</td>
			<td>GTX125923</td>
			<td>Antibody against flu protein</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td></td>
			<td></td>
			<td>Blocking reagent for immunostaining</td>
		</tr>
		<tr>
			<td>Cell media with supplements</td>
			<td></td>
			<td></td>
			<td>Media appropriate for cell model</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ACD Bio</td>
			<td></td>
			<td>Nuclear stain (RNAscope kit from ACD Bio)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (1X PBS)</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td>No calcium and magnesium</td>
		</tr>
		<tr>
			<td>Ethylene carbonate</td>
			<td>Sigma</td>
			<td>E26258</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td></td>
			<td></td>
			<td>Use specific FBS based on what serum secondary antibody was raised in (e.g goat FBS)</td>
		</tr>
		<tr>
			<td>Fisherbrand colorfrost plus microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-17/18/19</td>
			<td>Precleaned</td>
		</tr>
		<tr>
			<td>HCV-GT2a-sense-C2 probe</td>
			<td>ACD Bio</td>
			<td>441371</td>
			<td>HCV(+) sense RNA probe</td>
		</tr>
		<tr>
			<td>HIV-gagpol-C1</td>
			<td>ACD Bio</td>
			<td>317701</td>
			<td>HIV-1 cDNA probe</td>
		</tr>
		<tr>
			<td>HIV-nongagpol-C3</td>
			<td>ACD Bio</td>
			<td>317711-C</td>
			<td>HIV-1 RNA probe</td>
		</tr>
		<tr>
			<td>HybEZ hybridization oven</td>
			<td>ACD Bio</td>
			<td>321710/321720</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td></td>
			<td></td>
			<td>For immobolizing coverslip to slide prior to protease treatment</td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-d-lysine (PDL)</td>
			<td></td>
			<td></td>
			<td>Coat coverslips in 20 &#181;g/mL of PDL for 30 minutes</td>
		</tr>
		<tr>
			<td>Probe diluent</td>
			<td>ACD Bio</td>
			<td>300041</td>
			<td>For diluting RNA C2 or C3 probes</td>
		</tr>
		<tr>
			<td>Prolong gold antifade</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease III</td>
			<td>ACD Bio</td>
			<td>322337</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope&#174;&#160;Probe- V-Influenza-H1N1-H5N1-NP</td>
			<td>ACD Bio</td>
			<td>436221</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td></td>
			<td></td>
			<td>For DNA hybridization buffer</td>
		</tr>
		<tr>
			<td>Sodium citrate, pH 6.2</td>
			<td></td>
			<td></td>
			<td>For DNA hybridization buffer</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td></td>
			<td></td>
			<td>For DNA hybridization buffer and PBS-T</td>
		</tr>
		<tr>
			<td>V-HBV-GTD</td>
			<td>ACD Bio</td>
			<td>441351</td>
			<td>Total HBV RNA</td>
		</tr>
		<tr>
			<td>V-HBV-GTD-01-C2</td>
			<td>ACD Bio</td>
			<td>465531-C2</td>
			<td>HBV pgRNA probe</td>
		</tr>
		<tr>
			<td>V-HCV-GT2a probe</td>
			<td>ACD Bio</td>
			<td>441361</td>
			<td>HCV(-) sense RNA probe</td>
		</tr>
		<tr>
			<td>V-HTLV-HBZ-sense-C3</td>
			<td>ACD Bio</td>
			<td>495071-C3</td>
			<td>HTLV-1 (-) sense RNA probe targetting HBZ</td>
		</tr>
		<tr>
			<td>V-HTLV1-GAG-C2</td>
			<td>ACD Bio</td>
			<td>495051-C2</td>
			<td>HTLV-1 DNA probe</td>
		</tr>
		<tr>
			<td>V-HTLV1-GAG-POL-sense</td>
			<td>ACD Bio</td>
			<td>495061</td>
			<td>HTLV-1 (+) sense RNA probe</td>
		</tr>
		<tr>
			<td>V-Influenza-H1N1-H5N1-NP</td>
			<td>ACD Bio</td>
			<td>436221</td>
			<td>IAV RNA probe</td>
		</tr>
		<tr>
			<td>V-ZIKA-pp-O2</td>
			<td>ACD Bio</td>
			<td>464531</td>
			<td>Zika(+) sense RNA probe</td>
		</tr>
		<tr>
			<td>V-ZIKA-pp-O2-sense-C2</td>
			<td>ACD Bio</td>
			<td>478731-C2</td>
			<td>Zika(-) sense RNA probe</td>
		</tr>
	</tbody>
</table>

single-cell multiplexed fluorescence imaging to visualize viral nucleic acids and proteins and monitor hiv, htlv, hbv, hcv, zika virus, and influenza infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

A schematic of MICDDRP is depicted in Figure 1. Labeling of DNA and RNA is followed by immunostaining. The use of branching amplifiers increases signal, allowing detection of single nucleic acid molecules.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61843/61843fig1v5.jpg" /> 
Figure 1: Schematic of MICDDRP and step-by-step workflow.&#160;(A) bDNA signal is amplified via branch...

Watch this Scientific Journal Video about Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infectio

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

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Biology

2.6K Views.  Emory University School of Medicine. Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Video: Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...