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HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

Vongrad V, Imig J, Mohammadi P, Kishore S, Jaskiewicz L, Hall J, Günthard HF, Beerenwinkel N, Metzner KJ - PLoS ONE (2015)

Bottom Line: HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs.Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin.However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

View Article: PubMed Central - PubMed

Affiliation: University Hospital Zurich, Division of Infectious Diseases and Hospital Epidemiology, University of Zurich, Zurich, Switzerland; Institute of Medical Virology, University of Zurich, Zurich, Switzerland.

ABSTRACT

Background: MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM).

Methods: The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.

Results and conclusion: PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

No MeSH data available.


Related in: MedlinePlus

Characteristics of small RNAs derived from small RNA sequencing.Small RNA species detected in HIV-1JR-FL infected MDMs (n = 4) (A), and non-infected MDMs (n = 4) (B). (C) Read length distribution of HIV-1 sncRNAs (dark grey) and total small RNA (light grey). (D) Small RNA sequencing reads (n = 4) aligned to the HIV-1JR-FL genome. Transcripts aligned in antisense orientation are shown in red, predominantly representing tRNALys. Upper panel shows a diagram of the HIV-1 genome organization.
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pone.0132127.g001: Characteristics of small RNAs derived from small RNA sequencing.Small RNA species detected in HIV-1JR-FL infected MDMs (n = 4) (A), and non-infected MDMs (n = 4) (B). (C) Read length distribution of HIV-1 sncRNAs (dark grey) and total small RNA (light grey). (D) Small RNA sequencing reads (n = 4) aligned to the HIV-1JR-FL genome. Transcripts aligned in antisense orientation are shown in red, predominantly representing tRNALys. Upper panel shows a diagram of the HIV-1 genome organization.

Mentions: The absence of HIV-1 sncRNAs in the Ago2 PAR-CLIP and HITS-CLIP data prompted us to generate small RNA libraries from the MDM donor samples also used for the Ago2 PAR-CLIP. In addition, MDMs infected with HIV-1JR-FL from two additional donors were analyzed. Over 65 million and 85 million reads from HIV-1JR-FL infected (n = 4) and non-infected (n = 4) samples, respectively, were aligned to either the human or the HIV-1JR-FL genome by competitive alignment strategy (Table 3). Sequencing of the total small RNA fraction yielded 19.2% and 13.1% miRNAs, and 21.9% and 20.2% protein-coding transcripts in the infected and non-infected samples, respectively (Fig 1A and 1B). The size distribution of HIV-1 sncRNA ranged from 16 to 29 nucleotides (corresponding to ~2.5–97.5% quantiles), reaching a plateau between 17 and 26 nucleotides, showing a distinct pattern compared to other total small RNA species (Fig 1C). HIV-1 sncRNAs comprised 1.4% of all small RNAs in HIV-1JR-FL infected MDMs (n = 4) whereas they were represented only 0.01% in non-infected MDMs mainly represented by the host-derived tRNALys (Fig 1D). HIV-1 sncRNAs were aligned to all regions of the HIV-1JR-FL genome with hot spots in gag and env (Fig 1D). The majority of HIV-1 sncRNAs were derived from sense orientation (>99%), and the minority of antisense orientation was largely represented by tRNALys present in both infected and non-infected samples (Fig 1D). The presence of HIV-1 sncRNAs, sncRNA in LTR and an antisence sncRNA in env [28], with 3’OH ends was further confirmed by qPCR in HIV-1JR-FL infected samples (Fig D in S1 File). Both HIV-1 sncRNAs were present in all HIV-1JR-FL infected samples and were absent in controls. The cellular miRNAs, miR-21 and miR-223 were highly abundant miRNAs in macrophages (qPCR data not shown). The levels of HIV-1 sncRNAs were comparable with cellular miRNAs expressed at lower levels [45,46].


HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

Vongrad V, Imig J, Mohammadi P, Kishore S, Jaskiewicz L, Hall J, Günthard HF, Beerenwinkel N, Metzner KJ - PLoS ONE (2015)

Characteristics of small RNAs derived from small RNA sequencing.Small RNA species detected in HIV-1JR-FL infected MDMs (n = 4) (A), and non-infected MDMs (n = 4) (B). (C) Read length distribution of HIV-1 sncRNAs (dark grey) and total small RNA (light grey). (D) Small RNA sequencing reads (n = 4) aligned to the HIV-1JR-FL genome. Transcripts aligned in antisense orientation are shown in red, predominantly representing tRNALys. Upper panel shows a diagram of the HIV-1 genome organization.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520458&req=5

pone.0132127.g001: Characteristics of small RNAs derived from small RNA sequencing.Small RNA species detected in HIV-1JR-FL infected MDMs (n = 4) (A), and non-infected MDMs (n = 4) (B). (C) Read length distribution of HIV-1 sncRNAs (dark grey) and total small RNA (light grey). (D) Small RNA sequencing reads (n = 4) aligned to the HIV-1JR-FL genome. Transcripts aligned in antisense orientation are shown in red, predominantly representing tRNALys. Upper panel shows a diagram of the HIV-1 genome organization.
Mentions: The absence of HIV-1 sncRNAs in the Ago2 PAR-CLIP and HITS-CLIP data prompted us to generate small RNA libraries from the MDM donor samples also used for the Ago2 PAR-CLIP. In addition, MDMs infected with HIV-1JR-FL from two additional donors were analyzed. Over 65 million and 85 million reads from HIV-1JR-FL infected (n = 4) and non-infected (n = 4) samples, respectively, were aligned to either the human or the HIV-1JR-FL genome by competitive alignment strategy (Table 3). Sequencing of the total small RNA fraction yielded 19.2% and 13.1% miRNAs, and 21.9% and 20.2% protein-coding transcripts in the infected and non-infected samples, respectively (Fig 1A and 1B). The size distribution of HIV-1 sncRNA ranged from 16 to 29 nucleotides (corresponding to ~2.5–97.5% quantiles), reaching a plateau between 17 and 26 nucleotides, showing a distinct pattern compared to other total small RNA species (Fig 1C). HIV-1 sncRNAs comprised 1.4% of all small RNAs in HIV-1JR-FL infected MDMs (n = 4) whereas they were represented only 0.01% in non-infected MDMs mainly represented by the host-derived tRNALys (Fig 1D). HIV-1 sncRNAs were aligned to all regions of the HIV-1JR-FL genome with hot spots in gag and env (Fig 1D). The majority of HIV-1 sncRNAs were derived from sense orientation (>99%), and the minority of antisense orientation was largely represented by tRNALys present in both infected and non-infected samples (Fig 1D). The presence of HIV-1 sncRNAs, sncRNA in LTR and an antisence sncRNA in env [28], with 3’OH ends was further confirmed by qPCR in HIV-1JR-FL infected samples (Fig D in S1 File). Both HIV-1 sncRNAs were present in all HIV-1JR-FL infected samples and were absent in controls. The cellular miRNAs, miR-21 and miR-223 were highly abundant miRNAs in macrophages (qPCR data not shown). The levels of HIV-1 sncRNAs were comparable with cellular miRNAs expressed at lower levels [45,46].

Bottom Line: HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs.Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin.However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

View Article: PubMed Central - PubMed

Affiliation: University Hospital Zurich, Division of Infectious Diseases and Hospital Epidemiology, University of Zurich, Zurich, Switzerland; Institute of Medical Virology, University of Zurich, Zurich, Switzerland.

ABSTRACT

Background: MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM).

Methods: The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.

Results and conclusion: PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

No MeSH data available.


Related in: MedlinePlus