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><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&amp;prime;-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)&lt;sub&gt;25&lt;/sub&gt;</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 &amp;amp; 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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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 ...

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Universidad de Cartagena UDEC

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Biology

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

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Immunology and Infection

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Genetics

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Neuroscience

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

Studying ER Stress and UPR Activation During HIV-1 Infection in T-Cells

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. 
Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2&#945;, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

Author Spotlight: Exploring the Role of Unfolded Protein Response in HIV-1 Replication and Infectivity

Here, we describe some established methods to determine endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, with particular emphasis on HIV-1 infection. This article also describes a set of protocols to investigate the effect of ER stress/UPR on HIV-1 replication and virion infectivity.

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses ...

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

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Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4⁺ T Cells Isolated from Rhesus macaques

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication