microRNA mediated host-virus interactions http://health.howstuffworks.com/light-virus.htm What I’m presenting today is the highlights of a literature review that I’m doing for this class on microRNAs and the role that microRNA-mediated pathways play in host-virus interactions. Essentially I’ve been reading up on different ways that microRNA’s are used by both the host and the virus to exercise some control over gene expression, and I’ve also been surveying what kind of experiments were conducted to shed light on these roles of microRNAs in infected cells.

miRNA Appear in non-coding regions Primary miRNAs are transcribed by RPII or RPIII Drosha cleaves off cap and poly-A tail to form pre-miRNA After export to cytoplasm, Dicer cleaves stem-loop to form mature miRNA duplex Dicer also ‘selects’ the guide strand for incorporation into the RISC complex -microRNAs are short segments of RNA, 22-25 nucleotides long and initially they’re coded for just within the genome, and they’re found in non-coding regions, specifically, so they can be found in -intron regions of protein coding genes or exon regions of non-protein coding genes -The first step to producing microRNAs is just for them to be transcribed by RP RP II in most eukaryotes, RPIII in some viruses -Upon transcription, the mRNA takes on a secondary structure. It forms a stem-loop precursor with a complementary region which is referred to as the primary microRNA. At this stage, the pri-miRNA has a cap and a poly-A tail. -The primary-microRNA is recognized by the Rnase Drosha, which recognizes the stem-loop, and cleaves off the cap and poly A tail, leaving a stem loop intermediate referred to as the pre-miRNA. The pre-miRNA has a 3’ overhang which comes into play a bit later. -In the meantime, the pre-miRNA gets exported from the nucleus, and then in the cytoplasm, the Rnase Dicer recognizes the 3’ overhang and cleaves it further, taking the stem loop off, leaving a mature microRNA duplex. -Dicer is also involved with selecting one of the strands, the one with lower 5’ thermodynamic stability, to be the guide strand in the RISC complex -RISC = RNA induced silencing complex http://en.wikipedia.org/wiki/MicroRNA

PTGS / RNAi binds 3’ UTR translational repression or target degradation -Now once the RISC complex is formed, you’re getting into what’s called the PTGS, or Post transcriptional Gene Silencing pathway in plants or the RNAi or RNA interference pathway in animals. -In the RISC complex, the microRNA acts as a probe to guide the complex to its target. It binds in the 3’ un-translated region and causes either translational repression or mRNA degradation of the target. -The outcome of the binding is, in fact, dictated by the degree of complementarity between the miRNA and the target; perfect complementarity results in target degradation whereas imperfect complementarity results in translational repression. -And that’s your crash course on miRNA. After that it’s just a matter of taking their function and mechanism of action and putting them in the context of an infected cell and sort piecing together how that function comes into play in terms of the virus life cycle and the host cell’s survival

Roles of miRNA -When you do so, things can get a little complicated. -It’s been known for a while that cells produce microRNAs and that these can function as negative feedback inhibitors of gene expression just within the cell. -It’s also been known that, particularly in plants, since they don’t have an immune system per se, host microRNAs have served as a defense mechanism against viruses -More recently, however, it’s been shown that viruses also encode microRNAs, which can be processed by the virus’s own machinery or by the cell’s machinery if they can sneak in with the cell’s miRNAs for processing. -part of the process of identifying putative viral miRNAs has been through the use of computer programs that use structural predictions to identify regions that may correspond to microRNAs -and then the other part has been taking these hypothesized miRNAs to the bench and verifying whether or not they actually exist and what they act on.

Roles of miRNA Examples… Host miRNA Viral miRNA In an infected cell you’ll have Host miRNA Regulate host gene expression Antiviral defense and is looking to suppress viral gene expression Viral miRNA Regulate viral gene expression Regulate host gene expression by suppressing host defense mechanisms Add into the mix that some viruses have been found to produce proteins that act as counterdefense by suppressing the RNAi pathway…so they’re effectively suppressing the suppressors SO you end up with a very dynamic system and the outcome at any given time is a net effect of these opposing effectors. Roles of miRNA Host miRNA Regulating host gene expression Antiviral defense Viral miRNA Regulating viral gene expression Targets for silencing suppressors Examples… 5

HIV-1 HIV-encoded miRNA suppresses transcription of HIV’s nef gene , acting as a negative regulator of transcription the microRNA is acting as a negative regulator of transcription -This was investigated by taking a virus that doesn’t have the nef gene, inserting a plasmid with nef in it, and then coupling the transcription of the nef gene to the production of beta gal using a reporter plasmid. -The results here are showing the relative activity of the reporter gene when the cell is transduced with a plasmids containing either the specific miRNA, or some other gene. -What they found was that the samples transduced with the miRNA had the lowest relative activity, showing that a) that miRNA is suppressing the gene and b) it’s doing so at the transcriptional level. 6

HIV-1 HIV-encoded miRNA suppresses transcription of HIV’s nef gene , acting as a negative regulator of transcription the microRNA is acting as a negative regulator of transcription -This was investigated by taking a virus that doesn’t have the nef gene, inserting a plasmid with nef in it, and then coupling the transcription of the nef gene to the production of beta gal using a reporter plasmid. -The results here are showing the relative activity of the reporter gene when the cell is transduced with a plasmids containing either the specific miRNA, or some other gene. -What they found was that the samples transduced with the miRNA had the lowest relative activity, showing that a) that miRNA is suppressing the gene and b) it’s doing so at the transcriptional level. 7

EBV EBV-encoded miRNAs suppress expression of an interferon-inducible cellular protein that attracts T-cells -An instance of miRNAs acting as immunosuppressants by downregulating expression of a host gene -Just some quick background info on EBV, infection by EBV results in viral latency. And viral latency can be one of a few different types, and these different types of latency are defined by the gene expression patterns they exhibit. -HOW: So these researchers first showed that one of the EBV-expressed miRNAs in particular was present at elevated levels in cells lines that had one of the latent forms of the virus. This was quantified by a Rnase protection assay using P32 labeled RNA probes made from the target sequence. -HOW: they then investigated the effect of this miRNA on its target protein by taking latent cells known to express high amounts of that particular miRNA and transfecting them with increasing amounts of an oligonucleotide antisense to miRNA, and this was found to induce expression of the target gene. PICTURE figure 4B lanes 6-9

Silencing Suppressors A number of plant viruses produce proteins that target various steps in the miRNA-induced RNA silencing pathway A number of plant viruses produce proteins that target various steps in the miRNA-induced RNA silencing pathway Host defense (Ryu)

Silencing Suppressors A number of plant viruses produce proteins that target various steps in the miRNA-induced RNA silencing pathway -HOW: this was established by inserting ORFs for the suppressor proteins into a system in which a gene called Chalcone Synthase is constitutively silenced by miRNA. -The chalcone synthase gene encodes a protein involved in the production of purple dyes, making the activity of the gene indirectly measurable, and they also did RNA gel blot analysis. In this artificial system, it was shown that these proteins did alter the expression of the Chalcone Synthase gene. Ruh roh! 10

References Dunoyer, Patrice, Charles-Henri Lecellier, Eneida Abreu Parizotto, Christophe Himber, and Olivier Voinnet. "Probing the MicroRna and Small Interfering RNA Pathways with Virus-encoded Suppressors of RNA Silencing." The Plant Cell. 16(2004): 1235-50. Omoto, Shinya, and Yoichi Fujii. "Regulation of human immunodeficiency virus 1." Journal of General Virology 86(2005): 751-5. Provost, Patrick, Corinna Barat, Isabelle Plante, and Michel J. Tremblay. "HIV-1 and the microRNA-guided silencing pathway: An intricate and multifaceted encounter." Virus Research. 121(2006): 107-115. Scaria, Vinod, Manoj Hariharan, Souvik Maiti, Beena Pillai and Samir K. Brahmachari. "Host-virus interaction: a new role for microRNAs." Retrovirology. 3(2006): Triboulet, Robinson, Bernard Mari, Yea-Lih Lin, Christine Chable-Bessia, Yamina Benasser, Kevin Lebrigand, et.al. "Suppresion of MicroRNA-Silencing Pathway by HIV-1 During Virus Replication." Science. 315(2007): 1579-82. Xia, Tianli, Andrea O'Hara, Iguaracyra Araujo, Jose Barreto, Eny Carvalho and Jose Bahia Sapucaia. "EBV MicroRNAs in Primary Lymphomas and Targeting of CXCL-11 by ebv-mir-BHRF1-3." Cancer Research. 68(2008): 1436-42.