BIOC20 Lecture 7 Notes.pdf

Ø Final 1/3rd generally encoded for virion structure and non-structural proteins (nsp) - Family: Coronaviridae. Three genera based on genome homologies: Alpha-,beta-, and gammacoronaviruses Lecture 7 Key Points 1. Coronaviruses (+ sense RNA viruses) 1.1. History of infecGon in humans 1.2. COVID-1...

Ø Final 1/3rd generally encoded for virion structure and non-structural proteins (nsp) - Family: Coronaviridae. Three genera based on genome homologies: Alpha-,beta-, and gammacoronaviruses Lecture 7 Key Points 1. Coronaviruses (+ sense RNA viruses) 1.1. History of infecGon in humans 1.2. COVID-19 Gmeline and impact 1.3. Genes and proteins 1.4. Entry by fusion 1.5. Synthesis of viral proteins 1.6. Genome replicaGon 1.7. Assembly and release SecGon 1 - A newly emerged coronavirus caused a worldwide epidemic of severe acute respiratory syndrome (SARS) Ø Caused fever and progressive pneumonia which led to hypoxemia, and death Ø SARS coronavirus might have came from bats SecGon 1.1 - SARS found in bats because they are the most common natural reservoir of CoV and don’t fall ill - Zoonosis transfer from one animal to another SecGon 1.2 - Coronaviruses have enveloped virions containing helical nucleocapsids (which binds to ‘+’ sense RNA genome) - Spikes important for how the virus infect the cells, how it enter the cells. - Nucleocapsids formed from viral N-protein bound to viral RNA in a helical fashion Ø Most other ‘+’ strand RNA viruses have nucleocapsids with icosahedral symmetry Ø Covid resemble ‘-‘ strand RNA viruses with helical nucleocapsid - Some covid may have a spherical core structure (shell) formed via the M-protein> spherical core with internal helical nucleocapsid - Corona virions contain mulGple envelope proteins (E) - Corona virions contain mulGple envelope proteins; spike (S), membrane (M), envelope (E), nucleocapsid (N) and in some case HE (hemaggluGnin esterase) protein Ø Spike protein: Major surface transmembrane glycoprotein protruding from the surface; Responsible for viral entry and tropism; Targeted by neutralizing anGbodies and T-Cells in infecGon> possible vaccine/therapeuGc targe; Generally forms trimers >>Protein synthesized as a single polypepGde chain> cleaved by a cellular proteinase to yield an N-terminal S1 domain (or subunit) and a C-terminal S2 domain (subunit) S1 recognizes specific cellular receptors and iniGates aaachment (Receptor Binding Domain (RBD) is present within S1 for most corona); S2 forms the stalk with a short C-terminal tail, a hydrophobic transmembrane domain and exterior domain of interacGng alpha-helices >> Spike proteins bind to a variety of cellular receptors: 1) Alphacoronaviruses bind to aminopepGdase-N, a family of zinc binding metalloproteinases (use metal ions in catalyGc mechanism present on cell surface): 1.1 Broadly distributed on epithelial and fibroblast cells in small intesGne, kidneys, etc. 1.2 Sepcies specific 2) Covs that have HE can bind to sialic acid (9 carbon sugar), found on a variety of glycoproteins and glycolipids 3) Betacoronaviruses use a variety of receptors: 3.1 SARS-CoV binds to metalloproteinase Angiotensin-converGng enzyme (ACE2) and an co-receptor, L-SIGN (a LecGn-carbohydrate binding proteins that recognize specific sugar moGfs) 3.2 Transmembrane serine protease 2 (TMPRSS2), a cellular serine protease for host-cell entry> cleaves at S2 to acGvate viral fusion >>In Sar2, spike protein cleavage by TMPRSS2 or cathepsin L (endosomal route) is required 3.3 SARS CoV-2 also binds ACE2 and possible other receptors (this is sGll unknown) SecGon 1.4 - Spike protein generally mediates entry via fusion Ø External S1 subunit mediates aaachment Ø Stalk subunit S2 (a class I fusion protein) facilitates fusion Ø Series of conformaGonal changes> inserGon of S2 into target cell membrane> brings cell membrane and viral envelope into close contact Ø Some CoV S proteins can also cause formaGon of syncyGa (individual cells fused together) >>In some cases, fusion can be pH dependent Ø Fusion at plasma membrane is also required for egress(departure) from cells SecGon 1.5 - Aeer fusion, inside the cell, synthesis of viral proteins that organize and catalyze viral RNA synthesis - The replicase gene (gene 1) is translated from genomic RNA into a polyprotein that is processed by viral proteinases Ø Gene 1 is composed of ORF1a and ORF1b parGally overlapping reading frames >>ORF1a translated by ribosomes paused at pseudoknot (secondary structures formed in RNA via hydrogen bonds) and framshie (ribosomal frameshiR; ribosome ‘slipping’ by one nucleoGde in 5’(-1 nt) or 3’(+1nt)) aallowing translaGon of ORF1b> ieylds two polyproteins, pp1a and pp1ab (aeer frameshie) (pseudoknots) (ribosomal frameshie) Ø TranslaGon starts at the start codon (AUG)> progresses 5’-3’ - it tells me that the gene is extremely important RNA helicase and nucleoside triphosphatase acGvates assist with replicaGon and packaging of the viral genome (ORF1b, nsp13) RNA exonuclease (ORF1b, nsp14) has proofreading acGvity> rare for RNA viruses Various nsps may be involved in various roles/acGviGes SecGon 1.6 - Membrane associaGon of viral RNA synthesis is common among ‘+’ strand RNA viruses of eukaryotes - ReplicaGon complexes are the site of viral RNA synthesis (RNA factories)> consists of viral and cellular proteins associated with membrane in the cytoplasm of the infected cell Ø Rearrangement of subcellular architecture (cytoplasmic membranes) induced upon CoV infecGon Ø ReplicaGon complexes commonly observed on double membrane vesicles by EM - ReGculovesicular network- the virus induced membrane alteraGons. DMW= double membrane vesicles, CMs= convoluted membranes; DMSs= double membrane spherules Ø Networks are formed by a combinaGon of cell sGmuli to produce new membrane and modificaGon of exisGng membranes by virus proteins Ø Nucleocapsid protein can also be found in abundance at these sites> encapsidaGon of newly synthesized genome RNA can also occur at these sites - Genome replicaGon proceeds via a full-length negaGve-strand intermediate (anGgenome)> used to direct synthesis of full-lengths ‘+’ strand genome NegaGve-strand RNAs account for 1-2% of total viral RNA. Mostly are ‘+’ strand. These negaGve strand are the determinaGon of ‘+’ RNA strand in a replicaGve intermediate. ReplicaGve intermediate: RNA molecule on which - - one or several growing RNA strands are being synthesized. The growing strand typically forms base-pairing to template RNA only near their growing 3’ end. Aeer replicated genes, all the gene downstream on the replicase gene occurs from a series of subgenomic mRNAs (all have idenGcal “untranslated leader” (UTR) sequences at the 5’ end, poly (A) tails, and represent a nested set of mRNAs and all contain overlapping sequences on their 3’ end) Ø UTR is aaached to a unique mRNA ‘body’ sequence with one or more ORFs Subgenomic (sg) mRNAs are transcribed from subgenomic negaGve strand mRNA templates Ø NegaGve strand mRNA templates are made by disconGnuous transcripGon 1. Polyermase begins at 3’ end (because that’s where the template is being read) and stops at the end of a transcripGon-regulaGng sequence (TRS) 2. Polymerase then pauses and disassociates the nascent RNA chain from TRS> jumps to TRS located at the end of the leader sequence (template switching) - - Dissociated nascent RNA chain forms RNA-RNA hybrids of complementary sequences at 5’ TRS Ø RNA pol can pause and dissociate at any of the TRSs Ø Each of the subgenomic ‘-‘ strands are used to make a ‘+’ strand mRNA, then transcript it into proteins This disconGous model explain recombinaGon between viral genoms: 1. Viral RNa polymerase can switch between two different posiGve-strand genome RNAs if they are both in the same cell 2. Could be two different virus strains infecGng the same cell, or mutaGons in the virus genome during replicaGon 3. Template switching may help with genome repair and/or generate new viral strains/variants >>Aeer dissociaGon, polymerase has to find another template or transcripGon will abort> can lead to template switch SecGon 1.7 - Assembly of virions takes place at intracellular membrane structures- ERGIC (endoplasmic reGculum Golgi intermediate compartment); involves in transport, processing and modificaGon of proteins. Generally located in the perinuclear (around or near the nucleus) region of the cell 1. Helical nucleocapsids (curved blue lines) containing genome RNA are delievered from site of synthesis to these membranes packaging 2. Virus parGcles are formed by budding into the lumen of these membranes (virions acquire donut-shaped cores) 3. Progress to smaller and more uniformly dense cores as transit through Golgi membrane, envelope proteins also undergo glycosylaGon 4. Secretory vesicles transport virions to cell surface, for fusion with plasma membrane and release. - - M and E proteins play important roles in the formaGon of the virus envelops by budding Ø Enveloped virus-like parGcles can be formed in ERGIC when only M and E are expressed> which indicates that M and E proteins are sufficient in forming the parGcles C-terminal cytoplasmic tail of M is though to interact with packaging signals in N> ensures only full-length viral RNA gets packaged into virons HE (if present) and S are incorporated into the membrane through interacGons with the M protein As all these proteins transverse through Golgi, envelope proteins are glycosylated, mature virions are packaged into vesicles> targeted to plasma membrane for release