Nucleic Acid Activated Viral Defence Pathways Flashcards
How are dsRNA, structured RNA and modified RNA recognised by other systems across the phyla?
These structures in these systems interact with proteins that serve to either process these into smaller, defined length processed RNA’s called short RNA’s or small RNA’s. There is an RdRp sometimes involved to amplify the small RNA’s and these serve as the genetic information to feed into the protein complex called the RNA induced silencing complex (RISC). This pulls apart the short dsRNA to have a guide sequence that is then able to identify viral RNAs that are subsequently targeted and cleaved for degradation.
How does this system differ from normal adaptive and immune recognition systems?
Many of these ancient recognition systems of this ancient adaptive immune system based on genetic sequences also exist. Chordates (humans) have a protein mediated response pathway to virus-encoded PAMPs leading to the expression of type I IFNs and ISG (inteferon stimulated genes). In contrast, nematodes, arthropods and plants use RNA PAMPs directly to generate virus derived interfering RNAs (viRNAs), which can be loaded in an RNA-induced silencing complex (RISC) to mediate targeted silencing and cleavage of viral RNA.
How is genetic immunity (interference) provided in plants?
Plant genomes have inverted repeats that allows for complementarity to form. Once in the cytoplasm, the RNA dependent RNA polymerase creates a replicative intermediate (perfectly duplicated) that interact with the Dicer RNase III complex in the cell. The perfectly duplexed RNAs are cut into smaller pieces called siRNAs and these are engaged in a protein complex called RISC. RISC is able to separate off one of the strands and use the other as a guide to interact with homologous target viral RNAs. It is then able to cleave the viral RNA and degrade it.
Is a similar genetic immunity to plants also observed in invertebrate animals?
Yes.
Are siRNAs common to both plants and invertebrates and if so, what is their structure?
Common to both types of silencing is the production of 21 nucleotide siRNA, which contains a perfectly duplexed 19 base pair double helix with 2 nucleotide, 3’ single stranded tails. These are called short interfering RNAs or siRNAs, because they interfere with the expression of genes or RNA transcripts which have complementary sequence.
Is the DICER complex and RISC complex the same in both plants and invertebrates?
Yes. In both, long dsRNA starting material is processed by an enzyme known as Dicer. One strand becomes the guide strand and is assembled into the RISC complex. The strand with the less-tighly base paired 5’ end is incorporated into the RISC and becomes the guide strand. The argonaut proteins in the RISC complex inhibit gene expression:
- Ago2 is an RNA endonuclease (Slicer)
- Ago1 is an RNA transcription inhibitor (Silencer)
What is the difference in mechanism between siRNA and miRNA?
The foreign (virus) dsRNA as well as cell derived mRNA transcripts such as short hairpins (shRNA) or pre-miRNA can also serve as substrates for the DICER complex. The foreign dsRNA and shRNA can be cleaved by DICER into perfectly duplexed siRNAs that can interact with the RISC complex and lead to splicing and elimination of the mRNA. The pre-miRNA on the other hand contains mismatches in the stem loop and when processed by DICER it forms imperfectly duplexed miRNAs that also assemble onto the RISC complex. It does not lead to the cleavage of the RNA but leads to a block of expression of the mRNA instead.
Can the siRNA silencing mechanism guide the targeting of DNA methylation in both plants and invertebrates? What is an example of a virus that can do this?
Yes. This process is called RNA-induced transcriptional silencing (RITS) and it is an epigenetic method of controlling gene transcription activity. Short RNAs are processed by DICER and interact in the nucleus with RITS, which directs the methylation of the complementary DNA sequence. Methylation adds bulk to the DNA, shuttin down the activity of the promoter so it is unable to transcribe.
HIV is an example as short RNAs can be to utilised to methylate proviral sequences and shut down the expression.
How do plants and invertebrates differ from mammals (humans) in the expression of short dsRNA?
In plants and invertebrates, long dsRNA is the main substrate for the production of small effector RNAs. But in mammals, RNA silencing pathways are only triggered by small RNAs of 21-27 nt. It includes siRNAs (artificially produced), shRNA (artifically produced), microRNAs (produced naturally) and others (rasi RNAs and piRNA).
How do human cells control their own gene expression using miRNA? Have DNA viruses evolved to this?
miRNAs are 22nt long imperfectly duplexed RNAs that generally repress mRNA translation, but also other activities. The mechanisms for miRNA suppression of gene expression include protein translation repression by miRNA, co-translational degradation of the nascent peptide and mRNA degradation occur only when a highly level of duplex is formed (by siRNA). Viruses have also encoded these miRNA structures to their own benefit. It is a system that many DNA viruses have utilised (herpesviridae, adenoviridae and polyomaviridae) because they are not perfectly duplexed (are not going to degrade their own DNA in that way). They are going to interact with the human genes to set up an environment within the cell that favours viral replication.
What are the three main ways that miRNAs can affect viral replication?
- The viral dsDNA genome can produce either dsRNA or more likely an imperfectly duplexed miRNA that is processed out and can feed into the RISC complex. This can prevent expression either through the degradation of mRNA or blockage of translation of mRNA.
- The viral genome might express a microRNA that is structured and processed.It feeds into the RISC complex and that can control the expression of some host genes and turn the cell into a much more favourable environment - host cell mRNA degradation of translation inhibition.
- The host cell might make miRNA itself to block the expression or replication of viral RNA.
What is an example of cellular miRNA silence expression of viral RNA?
Primate Foamy Virus - this is a retrovirus and humans are protected against this by the miR-32 sequence that has binding sites at the 3’ end of the RNA. The miRNA is able to inhibit the expression of all the spliced and genome length RNAs by blocking translation.
The virus has also evolved to counteract this by expressing a protein called Tas that is able to block and interact with the activity of the RISC complex loaded up with the mir-32. It is a defence system against the cellular defence system that is protecting humans.
What is an example of a miRNA that can enhance viral replication?
Liver specific cellular miR-122 binds the 5’UTR of HCV RNA, stabilising it and enhancing expression. The miR-122 feeds into the RISC complex and targets the 5’ UTR of HCV. It is a key determinant in the tropism of HCV and the replication of that within hepatocytes. This facilitates viral replication.
How can viruses produce RNAs that can block the RNA defence pathways? This is another example where the viruses can cross-talk on host cell expression.
It produces a VA1 pol III RNA that is highly structured and is able to interact with PKR and bind it in an inactive complex. It binds more strongly to PKR than perfectly duplexed dsRNA, which normally locks down and blocks PKR activation.
This is also able to engage with the biosynthesis pathways of miRNAs. We have identified that the VA1 blocks the processing of the cellular RNAs and changes the gene expression within infected cells because you are no longer producing miRNAs through the Dicer pathway.
How can viruses utilise the miRNA pathway to control their own gene expression?
SV-40 miRNA target T proteins to promote the switch from early to late gene expression and reduce apoptosis of CTL killing. The late genes of SV40 contain these structured elements - there are introns that are spliced out and they contain the pre-miRNAs that are processed into functional SV miRNAs. Their function is to target the early mRNAs, block their translation and degrade and cleave the RNA. T antigens are also good cytotoxic T cell targets and if you shut down their expression of the peptides, you are also going to escape CTL detection.
