Antje Ksienzyk 18.01.2010

miRNA Regulatory RNAs of 22 nucleotides in length Found in plants and metazoans Involved in cellular processes like proliferation, regulation and development, apoptosis, homeostasis and tumor formation Found in humans: More than 500 different miRNAs Expressed in a developmental or tissue specific manner 30% of mammalian mRNAs are regulated by miRNAs

miRNA: regulatory RNAs of 22 nucleotides in length miRNAs are first transcribed as pri-miRNA (capped and polyadenylated) Pri-miRNA are cleaved by Drosha (and its cofactor DGCR8) -> pre-miRNA hairpin with 2nt 3’ overhang -> recognized by Exportin-5: transport to cytoplasm Dicer and cofactor TRBP recognize same 2nt 3’ overhang -> binding and cleavage of pre-miRNA two complementary short RNA molecules are formed, but only one is integrated into the RISC (RNA induced silencing complex): forms miRNA miRNA guides RISC to complementary mRNA .

miRNA development: the exceptions The normal way: The exception: miRNA from short excised introns (mirtrons); resemble pre-miRNA hairpins; no Drosha but Dicer essential for developing (found in Drosophila) “tailed pre-miRNAs: miRNAs which are transcribed as shRNAs: Drosha independent; Dicer essential .

RISC: The RNA induced silencing complex RISC bound to partially complementary mRNA induces translational repression: results in destabilization of target mRNA The more RISC bound the greater the inhibitory effect RISC bound to perfectly complementary mRNA (to miRNA) are cleaved and degraded by RISC in a process similar to RNAi

RNAi: innate antiviral mechanism Infection with virus leads to development of dsRNA during virus life cycle Viral dsRNA is generally long and perfectly complementary -> outcome: cleavage by Dicer ->siRNA duplexes are generated One strand of siRNA duplex is loaded into RISC -> RISC is guided to complementary viral mRNAs -> RISC binding leads to cleavage and degradation-> inhibiting virus replication In plants: 2. wave of siRNA generated by RNA-dependent RNA polymerases (RdRPs): more siRNA available to RISC .

Strategies of viruses against RNAi Suppressor of RNA silencing (SRS) Examples: Inhibition of siRNA loading into RISC (p19 protein of tomato bushy stunt virus) Interaction with RISC and inhibition of its cleavage activity Inhibits delivery of systemic siRNA signals Inhibits production of RdRP-derived viral siRNA and thereby the spread of RNAi response (2b protein of cucumber mosaic cucumovirus) Prevention of DICER processing Inhibition of viral siRNA production (B2 protein of flock house virus) Inhibition of antiviral siRNA response is an important requirement for propagation of these viruses

Mammalian innate antiviral defenses Recognized by MDA-5 and RIG-I (cytoplasmic) and TLR3 (endosomes) OAS recognizes viral dsRNA and tags them with 2’-5’ adenosine oligomers -> activation of RnaseL which degrades these RNAs 1 If PKR recognizes viral dsRNA; eIF2a will be phosphorylated, causing translational arrest and apoptosis of the infected cell 2 Counteractive measures of the virus: E1A protein of Adenovirus interferes with JAK-STAT signaling pathway NS5A (HCV) and ICP34.5 (HSV1) block PKR activation 1 2

RNAi response in mammalian cells? No siRNA of viral origin in infected cells HIV-1 and HCV produce neither viral miRNA nor siRNA in infected cells But Dicer of mammalian cells is capable of generating siRNA through cleavage of dsRNAs Do mammalian viruses produce Suppressor of RNA silencing (SRS)? YES?! Adenoviral VA1 RNA has SRS properties: Inhibits PKR and Dicer VA1 is short RNA with 3’ overhang which is recognized and bound by Exportin 5; VA1 is highly present in viral infection: saturates Exportin pathway and so the nuclear transport of pre-miRNA .

Cellular machinery in plants and invertebrates required to generate an antiviral siRNA response In mammalian cells not used Mammalian cells do not have RdRPs (2. wave of siRNA generated by RNA-dependent RNA polymerases (RdRPs): more siRNA available to RISC Mammals don’t use RNAi as an antiviral innate immune response It could be possible that the RNAi response is replaced by an more effective system like the IFN system

Viral miRNA miRNA

Features of miRNA which are useful for viruses Down-regulation of specific genes -> establishment of virus positive environment Evolution of miRNA complementary to a new target gene can be accomplished much easier than regulation of novel regulatory protein miRNA are very small: perfect for the tight space characteristic for the viral genome miRNAs are not antigenic in contrast to proteins A lot of viruses encode miRNAs (herpesviridae) All DNA viruses (cause Drosha which is essential for initial pre-miRNA excision is located in the nucleus) Viruses which invariable replicate in the cytoplasm can’t use the machinery Viruses which encode miRNA are nuclear DNA viruses capable of persistent or latent infections

Viral mRNA targets of viral miRNA Most of the viral mRNA target of viral miRNAs are targets which have influence on the host immune response or are viral regulatory proteins Examples: SV40 miR-S1: down regulation of viral T-antigen (TAg) production SV40 TAg is essential for viral transcription and replication (at the beginning of infection) At the end not useful but leads to CTL responses against SV40 infected cells RBV miR-BART-2: down regulation of BALF-5 (EBV DNA Polymerase) Leads to stabilization of the viral latency hCMV miR-UL112-1: target the viral gene IE1 (leads to activation of hCMV early gene transcription) miR-UL112-1 leads to establishment and maintenance of latency Viral miRNA regulation of viral gene expression is a common strategy of DNA viruses; leads to advancement of viral replication by inhibition of antiviral immune responses of the host

Cellular targets of viral miRNA Viral miRNA target cellular miRNA which are involved in regulation of apoptosis or modulation of the host antiviral immune response Examples: EBV: mir-BART5 Down regulation of PUMA (pro-apoptotic factor): protects EBV infected cells against apoptosis KSVH: miR-K1, miR-K3-3p, miR-K6-3p and miR-K11 Down regulation of THBS1 (regulates cell adhesion, migration, angiogenesis, chemo attractant for monocytes and T-cells): aid infected cells in avoiding detection by the host immune system EBV: mir-BHRF1-3: Down regulation of CXCL-11 (T cell chemo attractant): no T cell detection and killing of infected cells hCMV: miR-UL112-1 Inhibits MICB (up regulated in stressed cells; MICB is recognized by NKG2D of NK cells): down regulation of MICB inhibits recognition through NK cells

Conservation of viral miRNAs Lack of viral miRNA sequence conservation between related viruses do not necessarily imply an evolution of distinct functions Other meaning: viral miRNA could target different regions of same target mRNA or different gene products in the same cellular pathway

Antiviral cellular miRNA? It has been suggested that mammalian cells inhibit virus infections by targeting viral transcripts with cellular miRNA Cons: miRNAs are conserved from bird to human: it is unlike that one miRNA specifically acts on one virus; cause viral host ranges are more limited Viruses have short lifecycles and high mutation rates: one mutation can block down regulation through specific miRNA Many viruses appear to have evolved long after their current host species; cellular miRNA encoded by that host could not have evolved to inhibit these virus Pros: Cellular miRNA have antiviral effects Mice with reduced Dicer level and so reduced miR-24 and miR-93 are hyper susceptible to VSV miR-28, miR-125b, miR-150 can inhibit replication of HIV-1 via binding sites in its genome miR-32 leads to antiviral defense against PFV (retrovirus in Hela cells) Cellular miRNAs can act as antiviral defense mechanism if target sites are present; relevance must be proven

Influence of cellular miRNA on viral tropism Viruses have evolved “selectively avoid” binding sites for inhibitory miRNA that are expressed in their target tissues but still remain target sites for inhibitory miRNAs present in cells which they not infect under normal conditions Example: HCV : contains two adjacent binding sites for cellular miR-122 within its 5’UTR; binding of miR-122 to the viral 5’UTR facilitates HCV replication miR-122 is exclusively expressed in the liver, the primary replication site of HCV Seems to be that the miRNA plays a key role in determining the tropism of HCV in this tissue

Viral induction of cellular miRNAs many publications show changes in miRNA expression after virus infection; reason: represent host cell innate immune response triggered by virus infection and can inhibit virus replication Can be induced by virus to create a favorable intracellular microenvironment for viral replication Example: EBV induces up regulation of miR-155 Increased expression of miR-155 is found in some cancer types KSHV encodes viral miR-K11 which have the same seed as miR-155 and down regulates the same cellular mRNAs (important for lifecycle of these viruses) Virus induced expression of miR-155 or its viral orthologs is the reason for the oncogenic transformation of EBV and KSHV infected cells

UPSHOT miRNA and siRNA pathway are used by viruses and host cells and play an important role in antiviral defense of the host but also in the replication of the virus

The end

Second paper: additional things HIV: TAR Element (transcription response element): promotes transcription of HIV-1 proviral DNA through cellular RNA polymerase II (RNAP II) Tat interacts with cyclin T1(cellular factor) which build up together with CDK9: P-TEFb; P-TEFb and Tat bind at HIV TAR: initiation of transcription Has initial viral transcripts in different spliced forms: Unspliced gag and pol Partial spliced env, vif, vpr, vpu Fully spliced tat rev nef Problem: eukaryotic cells do not permit transport of intron-containing mRNAs. Therefore rev interact with cellular factor CRM1; Ran:GTB binds CRM1 in the nucleus and activate the binding of CRM1 to nuclear export signal; complex of rev; Ran:GTP, CRM1 directs incomplete splice variants to the nuclear pore complex and the cytoplasm; Rev=nuclear mRNA export factor Next problem translation: recruiting of ribosomes: poliovirus exhibit IRES (internal ribosome entry site: 450nt structured RNA element); IRES recruits eIFs and 40S subunit to the translation initioncodon without a cap: poliovirus is independent of host cell cap recognition factor eIF4E

Second paper: additional things Programmed ribosomal frame shifting translational phenomenon in retroviruses and all corona viruses Prevents some ribosomes from terminating translation at the end of ORF Production of large gag-pol polyprotein Viral non-coding RNA: Done by DNA viruses: encode long noncoding RNAs that influence viral replication and pathogenesis Example: LAT (8,3kb capped polyadenylated RNA)of HSV-1; LAT is spliced in unstable 6.3kb exonic RNA (processed in several viral miRNAs) and stable 2 kb introns (modulating mRNA translation)