Virus Genomes and Cellular Antiviral Defenses PDF

Virus Genomes and Cellular Antiviral Defenses Craig McCormick MICI 2100  Bacteriophage Infection What is the fate of the incoming bacteriophage genome? What is the fate of the cell?  Bacteriophage Replication (really...

Virus Genomes and Cellular Antiviral Defenses Craig McCormick MICI 2100  Bacteriophage Infection What is the fate of the incoming bacteriophage genome? What is the fate of the cell?  Bacteriophage Replication (really fast) After penetration, viral DNA is delivered into host E. coli Host gene expression is arrested (immediately) - host DNA/RNA degradation - inhibition of protein synthesis Enzyme synthesis (starting after 5 minutes) DNA replication (starting after 10 minutes) Formation of new virus particles (starting after 12 min) Lysis of host and release of viral particles (at 30 min)  Bacteria are not defenseless! Two well-described forms of antiviral defense: - Restriction Endonucleases - CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)  Restriction Endonucleases - an enzyme that cuts dsDNA at specific nucleotide sequences known as restriction sites. - provides a defense mechanism against invading viruses - the restriction enzyme specifically cuts up foreign viral DNA - host DNA is methylated by a host cell enzyme, which prevents cutting by host restriction endonuclease - to cut DNA, the enzyme makes two incisions at each sugar phosphate backbone.  Restriction Endonucleases - Useful tools for molecular biology - different bacteria make distinct restriction endonucleases: These enzymes are‘molecular scissors’ that can cut DNA fragments in a sequence-specific manner. Cut-and-paste gene cloning using restriction endonucleases  CRISPR – immune memory for prokaryotes CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats DNA Sequencing of a variety of bacteria revealed many viral short viral DNA sequences integrated into the genome (red, blue, green above) Viral DNA separated by non-viral repetitive sequences Always nearby a cluster of genes encoding DNA cutting enzymes (cas genes)  1. Cutting of foreign viral DNA 2. Insertion of DNA as a novel spacer in array 4. Cleavage of incoming viral DNA 3. Transcription of array, creation of RNAs that can target incoming viral DNA in a sequence-specific manner  RNA-guided cleavage of foreign viral DNA 1. Guide RNA is transcribed from CRISPR array on bacterial genome 2. Loaded onto Cas9 nuclease (dsDNA-cutting enzyme) 3. RNA guides Cas9 enzyme to cut homologous incoming viral dsDNA  Use of CRISPR as a tool to edit animal genomes Widespread adoption as a molecular biology technique to edit genomes (cell lines, model organisms). - Learn more at https://www.addgene.org/CRISPR/guide/  The 2020 Nobel Prize in Chemistry Dr. Jennifer Doudna, UC-Berkeley Dr. Emmanuelle Charpentier, Max Planck Unit for the Science of Pathogens https://www.nature.com/articles/d41586-020-02765-9  Use of CRISPR as a tool to edit animal genomes Hot ethical debate about editing of human germ line Francoise Baylis – Canada Research Chair in Bioethics and Philosophy (https://en.wikipedia.org/wiki/Francoise_Baylis)  Bacteria are not defenseless! Two well-described forms of antiviral defence: - Restriction Endonucleases - CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) These antiviral defense systems are complementary Their discovery has revolutionized molecular biology and medicine Solid argument for basic, curiosity-driven research (rather than applied research that is fashionable today)  How do eukaryotes combat infection? Eukaryotic cells do not have restriction enzymes or CRISPR arrays They do have an immune system, triggered by pattern recognition receptors (PRR) PRRs recognize molecular patterns that are foreign PRRs include molecules that recognize non-host nucleic acids, including viral dsRNA and 5’-triphosphate RNA (Why are these structures foreign to the host?) These viral targets are called “pathogen-associated molecular patterns”, or “PAMPs” PAMPs and PRRs Do not memorize this figure! But appreciate that there are: (open access source: Viruses 2013, 5(2), 470-527; doi:10.3390/v5020470). 1. multiple viral patterns (PAMPs) that the cell can recognize, including nucleic acids and proteins. 2. Multiple cellular pattern recognition receptors (PRRs) that do the sensing. How do viruses make gene products (RNA and protein) and replicate their genomes? Viruses are obligate intracellular parasites: - they depend on many parts of host biosynthetic machinery - therefore, viruses need to either: (i) conform to the machinery (e.g. use host ribosomes for translation), or (ii) alter that machinery for their own use.  The central dogma of molecular biology DNA-dependent DNA polymerase: - uses dsDNA as a template to make more dsDNA. RNA polymerase: - uses dsDNA as a template to make (+)-sense RNA Ribosomes: - translates (+)-sense RNA into an amino acid chain (protein) Viruses often break the central dogma of molecular biology  Virus genomes All virus genomes are either DNA are RNA (the Baltimore classification system – 7 different types) Their composition and structures are diverse Many possible tactics for encoding and decoding information in nucleic acid Viral genomes must make mRNA that can be decoded by host ribosomes  What kind of information is encoded in viral genomes? Gene products (RNA, protein) and regulatory signals required for: 1. Replication of the viral genome 2. Assembly and packaging of the genome into viral particles 3. Regulation and timing of the replication cycle 4. Defeating host defenses 5. Spread to other cells and hosts (+)ssRNA viruses get directly translated in the cytoplasm This generates polyproteins, which subsequently get cleaved by either viral or host proteases into the full complement of viral proteins  (+)ssRNA viruses ex. Poliovirus Q. How do they express their genes? A. Direct translation in the cytoplasm Q. How do they replicate their genomes? A. Viral RNA-dep RNA pol (RdRp) Q. Why don’t they use a host RNA-dep RNA pol? What would happen if you transfected (introduced) naked poliovirus RNA into a cell? Would you make progeny virions? Gene Expression from a (+)ssRNA genome – Poliovirus by host ribosomes Viral and host proteases Gene Expression from a (+)ssRNA genome – Poliovirus Gene Expression from a (+)ssRNA genome – Zika Virus Notice what happens when we switch from icosahedral to an enveloped virus – polyprotein synthesis at the ER and creation of transmembrane proteins Gene Expression from a (+)ssRNA genome – HCV Polyprotein processing is co-translational (+)ssRNA viruses get directly translated in the cytoplasm This generates polyproteins, which subsequently get cleaved by either viral or host proteases into the full complement of viral proteins These proteases make excellent drug targets. Why? RDRP is also an excellent drug target. Why?  Review Bacteriophage genomes are subject to attack by restriction endonucleases upon viral entry CRISPR provides a form of bacterial ‘memory’ of previous viral infections Eukaryotes have their own innate defenses that recognize foreign nucleic acids (pattern recognition receptors – PRRs) Viruses have a wide variety of gene expression and genome replication strategies +ssRNA virus genomes resemble mRNA and are directly translated by host ribosomes in the cytoplasm.