This work was supported by the Swiss National Science Foundation (grants 31003A_166412 and 314730_188877).

<ol>
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The authors have nothing to disclose.

The role of RNA modifications in viral infection is still largely unknown. A better understanding of the role of epitranscriptomic modifications in the context of viral infection could contribute to the quest for new antiviral treatment targets.
In this work, we provide a complete workflow that allows investigation of the m6A and m5C epitranscriptomes of infected cells. Depending on the biological question, we advise to use poly-A-selected RNA as starting material. Although optional, as the pipeline could be used with total RNA, it is important to keep in mind that rRNAs as well as small RNAs are highly modified and contain an important number of methylated residues. This could result in a decreased quality and quantity of meaningful sequencing data.
However, if the focus of the study is non-poly-adenylated RNA, the RNA extraction step should be adapted in order to avoid discarding small RNA (in case of column-based RNA extraction) and to privilege ribosome-depletion techniques rather than poly-A selection to enter the pipeline.
In order to ensure high quality RNA, correct fragmentation and suitable m6A-enriched and BS converted RNA quality for library preparation we strongly advise to use a fragment analyzer or a bioanalyzer. However, this equipment is not always available. As an alternative, quality of RNA, mRNA and size of fragmented RNA could also be assessed by visualization on agarose gel. Alternatively, library preparation can be performed without previous assessment of RNA quantity.
We used the antibody-based MeRIP-Seq16 technique to explore the m6A epitranscriptomic landscape. This technique is based on RNA immunoprecipitation and is successful; however, some steps need careful optimization and can be critical. Although m6A methylation has been described to occur mainly within the consensus sequence RRA*CH, this motif is highly frequent along mRNA molecules and does not allow precise identification of the methylated site. It is thus critical to achieve a reproducible and consistent RNA fragmentation, generating small RNA fragments, to improve the RIP-based resolution. In this protocol, we recommend an optimized procedure, providing reproducible and consistent results in our experimental setting; however, this fragmentation step may need further optimization according to specific sample features.
Recently a new technique allowing m6A direct sequencing was described. It is based on the use of specific reverse transcriptase variants that exhibit unique RT-signatures as a response to encountering m6A RNA modification24. This technology, upon careful optimization, could circumvent the major limitation faced with MeRIP-Seq (decreasing the amount of initial material and allowing a higher resolution). To explore the m5C modification we decided to use the bisulfite conversion technique in order to detect at nucleotide resolution the modified C residues. In order to reduce the false positive rate due to the presence of RNA secondary structures, we performed 3 cycles of denaturation/bisulfite conversion and further control bisulfite conversion rate performance thanks to the use of ERCC spike-in controls. One of the limitations linked to this technique is that bisulfite conversion is very harsh and three cycles of denaturation/bisulfite conversion could degrade some RNA and hence reduce resolution. However, in our setting, we chose to settle for a potentially slightly lower resolution in order to increase the quality of the dataset.
Thanks to these optimizations and controls, we were able to provide a reliable and sound workflow that can be exploited to investigate the epitranscriptomic landscape and its alteration in the context of viral infections, host-pathogen interactions, or any exposure to specific treatments.

It&#160;is long known that RNA molecules can be modified, and more than 150 post-transcriptional modifications have been described to date1. They consist in the addition of chemical groups, mainly methyl groups, to virtually any position of the pyrimidine and purine rings of RNA molecules2. Such post-transcriptional modifications have already been shown to be highly enriched in transfer RNA (tRNA) and ribosomal RNA (rRNA) and have recently been described on mRNA molecules as well.
The rise of new technologies, such as Next Generation Sequencing (NGS), and the production of specific antibodies recognizing definite chemical modifications allowed, for the first time, the investigation of the location and the frequency of specific chemical modifications at a transcriptome-wide level. These advancements have led to a better understanding of RNA modifications and to the mapping of several modifications on mRNA molecules3,4.
While epigenetics investigates the role of DNA and histone modifications in transcriptome regulation, epitranscriptomics in a similar fashion focuses on RNA modifications and their role. The investigation of epitranscriptomic&#160;modifications provides new opportunities to highlight novel mechanisms of regulation that may tune a variety of cellular processes (i.e., RNA splicing, export, stability and translation)5. It was thus no great surprise that recent studies uncovered many epitranscriptomic modifications upon viral infection in both cellular and viral RNAs6. Viruses investigated so far include both DNA and RNA viruses; among them, HIV can be considered as a pioneering example. Altogether, the discovery of RNA methylation in the context of viral infections may allow the investigation of yet undescribed mechanisms of viral expression or replication, thus providing new tools and targets to control them7.
In the field of HIV epitranscriptomics, modifications of viral transcripts have been widely investigated and have shown that the presence of this modification was beneficial for viral replication8,9,10,11,12,13. To date various techniques can be used to detect epitranscriptomic marks at the transcriptome-wide level. The most used techniques for m6A identification rely on immune precipitation techniques such as MeRIP-Seq and miCLIP. While MeRIP-Seq relies on RNA fragmentation to capture fragments containing methylated residues, miCLIP is based on the generation of &#945;-m6A antibody specific signature mutations upon RNA-antibody UV crosslinking, thus allowing a more precise mapping.
Detection of m5C modification can be achieved either by antibody-based technologies similar to m6A detection (m5C RIP), or by bisulfite conversion or by AZA-IP or by miCLIP. Both Aza-IP and m5C miCLIP use a specific methyltransferase as bait to target RNA while going through RNA methylation. In Aza-IP, target cells are exposed to 5-azacytidine, resulting in the random introduction of cytidine analog 5-azacytidine sites into nascent RNA. In miCLIP, the NSun2 methyltransferase&#160;is genetically modified to harbor the C271A mutation14,15.
In this work, we focus on the dual characterization of m6A and m5C modifications in infected cells, using HIV as a model. Upon methodological optimization, we have developed a workflow that combines methylated RNA immunoprecipitation (MeRIP) and RNA bisulfite conversion (BS), allowing the simultaneous exploration of m6A and m5C epitranscriptomic marks at a transcriptome-wide level, in both cellular and viral contexts. This workflow can be implemented on cellular RNA extracts as well as on RNA isolated from viral particles.
The Methylated RNA ImmunoPrecipitation (MeRIP)16 approach allowing investigation of m6A at the transcriptome-wide level is well established and an array of m6A-specific antibodies are commercially available to date17. This method consists in the selective capture of m6A-containing RNA pieces using an m6A-specific antibody. The two major drawbacks of this technique are (i) the limited resolution, which is highly dependent on the size of RNA fragments and thus provides an approximated location and region containing the methylated residue, and (ii) the large amount of material needed to perform the analysis. In the following optimized protocol, we standardized the fragment size to about 150 nt and reduced the amount of starting material from 10 &#181;g of poly-A-selected RNA, which is currently the advised amount of starting material, to only 1 &#181;g of poly-A-selected RNA. We also maximized the recovery efficiency of m6A RNA fragments bound to specific antibodies using an elution by a competition approach with an m6A peptide instead of more conventional and less specific elution methods using phenol-based techniques or proteinase K. The main limitation of this RIP-based assay, however, remains the suboptimal resolution that does not allow the identification of the precise modified A nucleotide.
Analysis of the m5C mark can be currently performed using two different approaches: a RIP-based method with m5C-specific antibodies and RNA bisulfite conversion. As RIP offers only limited resolution on the identification of the methylated residue, we used bisulfite conversion that can offer single nucleotide resolution. RNA exposure to bisulfite (BS) leads to cytosine deamination, thereby converting the cytosine residue into uracil. Thus, during the RNA bisulfite conversion reaction, every non-methylated cytosine is deaminated and converted to uracil, while the presence of a methyl group in position 5 of the cytosine has a protective effect, preventing the BS-induced deamination and preserving the cytosine residue. The BS-based approach allows for the detection of a m5C modified nucleotide at single base resolution and for assessment of the methylation frequency of each transcript, providing insights into m5C modification dynamics18. The main limitation of this technique however relies on the false positive rate of methylated residues. Indeed, BS conversion is effective on single-stranded RNA with accessible C residues. However, the presence of a tight RNA secondary structure could mask the N5C position and hamper BS conversion, resulting in non-methylated C residues that are not converted to U residues, and thus false positives. To circumvent this issue and minimize the false positive rate, we applied 3 rounds of denaturation and bisulfite conversion cycles19. We also introduced 2 controls in the samples to enable estimation of bisulfite conversion efficiency: we spike-in ERCC sequencing controls (non-methylated standardized and commercially available sequences)20 as well as poly-A-depleted RNAs to assess bisulfite conversion rate on one hand, and to verify by RT-PCR the presence of a known and well conserved methylated site, C4447, on 28S ribosomal RNA on the other hand21.
In the field of virology, coupling these two epitranscriptomic investigation methods with next generation sequencing and accurate bioinformatic analysis allows for the in-depth study of m6A and m5C dynamics (i.e., RNA modification temporal changes that could occur upon viral infection and could uncover an array of new therapeutically relevant targets for clinical use).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AccuPrime Pfx SuperMix</td>
			<td>Invitrogen</td>
			<td>12344-040</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-m6A antibody _Clone 17-3-4-1</td>
			<td>Millipore</td>
			<td>MABE1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>ERCC</td>
			<td>Invitrogen</td>
			<td>4456740</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ RNA Methylation Kit</td>
			<td>Zymo Research</td>
			<td>EZR5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fragment analyzer RNA Kit - HS RNA Kit</td>
			<td>Agilent</td>
			<td>DNF-472-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fragment analyzer RNA Kit - RNA Kit</td>
			<td>Agilent</td>
			<td>DNF-471-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystem</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina TruSeq Stranded mRNA</td>
			<td>Illumina</td>
			<td>20020594</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Beads A/G Blend</td>
			<td>Merck</td>
			<td>16-663</td>
			<td></td>
		</tr>
		<tr>
			<td>N6-Methyladenosine, 5&#8242;-monophosphate sodium salt (m6A)</td>
			<td>Sigma Aldrich</td>
			<td>M2780-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse IgG</td>
			<td>Merk</td>
			<td>12371</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo(dT)25</td>
			<td>Life Technologies</td>
			<td>61005,</td>
			<td></td>
		</tr>
		<tr>
			<td>PCRapace</td>
			<td>Stratec</td>
			<td>1020220300</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick RNA Viral Kit</td>
			<td>Zymo Research</td>
			<td>1034</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>R1015</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Fragmentation Reagent</td>
			<td>Ambion</td>
			<td>AM8740</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor</td>
			<td>Ambion</td>
			<td>AM2684</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>TRIzol Reagent</td>
			<td>15596026</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This workflow has proven useful to investigate the role of m6A and m5C methylation in the context of HIV infection. For this, we used a CD4+ T cell line model (SupT1) that we either infect with HIV or left untreated. We started the workflow with 50 million cells per condition and obtained an average of 500 &#181;g of total RNA with an RNA quality number of 10 (Figure 1A-B). Upon poly-A selection we retrieved between 10 and 12 &#181;g of mRNA per condition (representing about 2% of total RNA) (Figure 1B). At this point, we used 5 &#181;g of poly-A-selected RNA for the MeRIP-Seq pipeline and 1 &#181;g for the BS-Seq pipeline. Since HIV RNA is poly-adenylated, no further action is needed and MeRIP-Seq and BS-Seq procedures can be directly applied.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62426/62426fig01.jpg" /> 
Figure 1: RNA preparation for downstream applications. A) Workflow depicting RNA preparation and distribution for simultaneous MeRIP-Seq and BS-Seq pipelines. Every filled hexagonal shape represents an RNA modification type, such as m6A (green) or m5C (pink). Amounts of RNA material needed to carry out the experiment are indicated. B) Representative results depicting expected RNA distribution profiles (size and amount) upon total RNA extraction (upper panel) and poly-A selection (lower panel). Samples were loaded on the fragment analyzer with standard sensitivity kit in order to assess RNA quality before entering specific MeRIP-Seq and BS-Seq procedures. RQN: RNA quality number; nt: nucleotides.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62426/62426fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
MeRIP-Seq pipeline is an RNA immunoprecipitation-based technique that allows investigation of m6A modification along RNA molecules. For this, RNA is first fragmented and then incubated with m6A-specific antibodies coupled to magnetic beads for immunoprecipitation and capture. MeRIP-enriched RNA fragments and the untouched (input) fraction are then sequenced and compared to identify m6A-modified RNA regions and thus m6A-methylated transcripts (Figure 2A). The resolution of the technique relies on the efficiency of RNA fragmentation. Indeed, shorter fragments allow for a more precise localization of the m6A residue. Here, cellular poly-A-selected RNAs and viral RNAs were subjected to ion-based fragmentation with RNA fragmentation buffer during 15 min in a 20 &#181;L final volume to obtain RNA fragments of 100-150 nt. Starting with 5 &#181;g of mRNA, we recovered 4.5 &#181;g of fragmented RNA, corresponding to a recovery rate of 90% (Figure 2B). We used 100 ng of fragmented, purified RNA as input control, subjected directly to library preparation and sequencing. The remaining RNA (~4.4 &#181;g) was processed according to the MeRIP-Seq pipeline, which starts with incubation of fragmented RNA with beads bound either to anti-m6A specific antibodies or to anti-IgG antibodies as control. m6A-specific RIP (MeRIP) of 2.5 &#181;g of fragmented RNA allowed retrieving around 15 ng of m6A-enriched material that underwent library preparation and sequencing (Figure 2B). RIP with anti-IgG control, as expected, did not yield enough RNA to allow further analysis (Figure 2B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62426/62426fig02.jpg" /> 
Figure 2: MeRIP-Seq pipeline. A) Schematic representation of MeRIP-Seq workflow and input control. Upon poly-A selection, samples were fragmented into 120-150 nt pieces and, either directly subjected to sequencing (100 ng, input control), or used for RNA immunoprecipitation (2.5 &#181;g, RIP) with anti-m6A specific antibody or anti-IgG antibody as negative control prior to sequencing. B) Representative results showing expected RNA distribution profiles (size and amount) upon fragmentation (upper panel) and RIP (lower panels, MeRIP: left, IgG control: right). Samples were loaded on fragment analyzer to evaluate RNA quality and concentration before further processing to library preparation and sequencing. Fragmented RNA analysis was performed using the RNA standard sensitivity kit while immunoprecipitated RNA used the high sensitivity kit. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62426/62426fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
BS-Seq pipeline allows exploration of m5C RNA modification at nucleotide resolution and leads to the identification of m5C-methylated transcripts. Upon bisulfite conversion, non-methylated cytosines are converted into uracil, while methylated cytosines remain unchanged (Figure 3A). Due to the harsh conditions of bisulfite conversion procedure (i.e., high temperature and low pH), converted mRNAs are highly degraded (Figure 3B), however this does not interfere with library preparation and sequencing. Bisulfite conversion is efficient only on single-stranded RNA and can thus potentially be hindered by secondary double-stranded RNA structures. To evaluate the efficiency of C-U conversion we introduced two controls. As a positive control, we took advantage of the previously described presence of a highly methylated cytosine in position C4447 of the 28S rRNA23. Upon RT-PCR amplification and sequencing of a 200 bp fragment surrounding the methylated site we could observe that all cytosines were successfully converted to uracils, thereby appearing as thymidines in the DNA sequence, except the cytosine in position 4447 that remained unchanged. As a control for bisulfite conversion rate, we used commercially available synthetic ERCC RNA sequences. This mixture consists in a pool of known, non-methylated and poly-adenylated RNA sequences, with a variety of secondary structures and lengths. Upon library preparation and sequencing, we focused on these ERCC sequences to calculate the conversion rate, which can be performed by counting the number of converted C among the total C residues in all the ERCC sequences and in each sample. We obtained a conversion rate of 99.5%, confirming the efficiency and the success of the bisulfite conversion reaction (Figure 3D).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62426/62426fig03.jpg" /> 
Figure 3: BS-Seq pipeline. A) Schematic representation of BS-Seq workflow. Upon poly-A selection, samples are exposed to bisulfite, resulting in C to U conversion (due to deamination) for non-methylated C residues. In contrast, methylated C residues (m5C) are not affected by bisulfite treatment and remain unchanged. B) Representative result of bisulfite converted RNA distribution profile (size and amount) upon analysis on fragment analyzer with a standard sensitivity kit. C) Electropherogram showing representative sequencing result of RT-PCR amplicon of the region surrounding the 100% methylated C at position 4447 in 28S rRNA (highlighted in blue). In contrast, C residues of the reference sequence were identified as T residues in the amplicon sequence due to bisulfite conversion success. D) Evaluation of C-U conversion rate by analysis of ERCC spike-in sequences in HIV-infected and noninfected cells. The average conversion rate is of 99.5%. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62426/62426fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
M6A-enriched samples, bisulfite converted samples and input controls are further processed for library preparation, sequencing and bioinformatic analysis (Figure 4). According to the experimental design and biological question(s) addressed, multiple bioinformatic analyses can be applied. As proof of principle here, we show representative results from one potential application (i.e., differential methylation analysis), which focuses on the identification of differentially methylated transcripts induced upon HIV infection. Briefly, we investigated the m6A or m5C methylation level of transcripts, independently from their gene expression level, in both non-infected and HIV-infected cells, in order to further understand the role of RNA methylations during viral life cycle. Upon gene expression normalization, we identified that the ZNF469 transcript was differentially m6A-methylated according to the infection status, indeed this transcript was not methylated in non-infected cells while it displayed several methylated peaks upon HIV infection (Figure 5A). A similar differential methylation analysis on m5C revealed that the PHLPP1 transcript contained several methylated residues, which tend to be more frequently methylated in the HIV condition (Figure 5B). In this context, both analyses suggest that HIV infection impacts the cellular epitranscriptome.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62426/62426fig04.jpg" /> 
Figure 4: Schematic representation of the bioinformatic workflow for the analysis of m6A and m5C data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62426/62426fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62426/62426fig05.jpg" /> 
Figure 5: Example of differentially methylated transcripts upon infection. A) Representative result showing m6A methylation of ZNF459 transcript in HIV-infected (green) and non-infected (grey) cells. Peak intensity (upon input expression subtraction) is shown on the y-axis and position in the chromosome along the x-axis. Differential methylation analysis reveals that ZFN469 transcript is hypermethylated upon HIV infection. B) Representative result of m5C methylated gene in HIV-infected (upper lane) and non-infected (lower lane) cells. The height of each bar represents the number of reads per nucleotide and allows coverage assessment. Each C residue in represented in red, and the proportion of methylated C is represented in blue. The exact methylation rate (%) is reported above each C residue. Arrows highlight statistically significant differentially methylated C. Samples were visualized using IGV viewer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62426/62426fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV at JoVE.com

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.

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 ...

exploring-m6a-m5c-epitranscriptomes-upon-viral-infection-an-example

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3.1K Views.  Lausanne University Hospital and University of Lausanne. 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.

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

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies ...

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

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 Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

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.

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 ...