Module 7 Lecture 2 2022.pdf

The Cell Biology of Viruses -Introduction of virus types -Viral Entry -Viral replication -Viral exit (SARS-CoV-2) Credit: Veronica Falconieri Hays. Source: Lorenzo Casalino, Zied Gaieb and Rommie Amaro, U.C. San Diego (spike model with glycosylations). Bacteriophage - Bacterial Viruses Two types...

The Cell Biology of Viruses -Introduction of virus types -Viral Entry -Viral replication -Viral exit (SARS-CoV-2) Credit: Veronica Falconieri Hays. Source: Lorenzo Casalino, Zied Gaieb and Rommie Amaro, U.C. San Diego (spike model with glycosylations). Bacteriophage - Bacterial Viruses Two types of life cycles NOT ALL bacteriophages can go into LYTIC pathway - they go into lysogenic to wait until they are ready At this stage there is a split Figure 1. Bacteriophage Lifecycle: Lytic phages attach and infect a bacterial cell which results in the reproduction of phages and lysis of the cell host and this lysogenic cycle results in the integration of a phage genome into the bacterial genome. Some lysogenic phages do not integrate into the genome and remain in the cell as a circular or linear plasmid (not depicted here). Batinovic et al. Pathogens 8(3), 2019 Bacteriophage inject DNA through the Bacterial Wall - Phage are highly diversified and adapt constantly to bacterial anti-viral strategies - Can re-evolve a new receptor binding protein (RBP) - Can evolve enzymes to degrade any coats or modifications that impede access to receptors, like endosialidase or glycosidase enzymes. - Can express multiple receptor binding proteins to increase flexibility. -Bacterial anti-viral immune system resulted in the identification of Crispr-Cas9 gene editing system (Nobel) Phage as antibacterial treatments are very promising phage therapy- phages are HIGHLY specific, and they are very adaptive. Antibiotics are toxic and disrupt microbiome,but phages are EVERYWHERE so they won’t distrupt microbiome. Phage can self replicate, can lyse cells (lyric life cycle) and will propagate. Pretty efficient system phage therapy - can improve antibiotic potency Life Cycle of Viruses with Animal Hosts Are 75,000 viral genomes sequenced! Viral genomes can code for 2 to 2000 proteins Figure 4. In influenza virus infection, viral glycoproteins attach the virus to a host epithelial cell. As a result, the virus is engulfed. Viral RNA and viral proteins are made and assembled into new virions that are released by budding. Adapted from https://courses.lumenlearning.com/suny-microbiology/chapter/the-viral-life-cycle/ Architecture of animal viruses. Enveloped viruses -viral capsid enveloped in a membrane -DNA or RNA -single or double stranded -west Nile, influenza, HIV, Ebola, SARS-COV2 Non-enveloped viruses -capsids only -DNA or RNA -single or double stranded -rotavirus Animal viruses enter through endocytosis. Endocytosis allows viruses to infect - Viruses bind surface receptors. This can be cell specific of course. - Internalized in various ways, here showing Clathrin-mediated. - Clathrin uncoats, endosome acidifies. - Acidity generally triggers viral uncoating and/or fusion of the virus with endocytic membrane. - This fusion releases viral genome into the cytosol (nucleocapsid). - Some viruses can fuse right at cell surface. Features of viruses; genome types, entry, enveloped, receptors. ….continued….. still unknown Non-Enveloped Virus: Rotavirus as example. - Rotavirus causes acute gastroenteritis. In 2008, rotavirus infection led to the death of ~453,000 children younger than 5 worldwide. - 80–100-nm in diameter, three capsid layers with icosahedral symmetry, RNA(+) virus - Enters in clathrin and non-clathrin mediated endocytosis. - Release into cytosol (infection) requires Rab5, Rab7, Rab9, ESCRT, cathepsins. - Indicates some requirement for the late endosome, multivesicular body in viral exit from the endosome, but mechanisms unclear. Fig legend for previous slide… Working model for rotavirus cell entry into MA104 cells. (a) RVs attach to the cell surface through different glycans, depending on the virus strain. After initial binding, the virus interacts with several coreceptors concentrated at lipid rafts. All known coreceptors are represented as a single blue Y symbol. (b) RVs are internalized into cells by clathrin-dependent or -independent endocytic pathways, depending on the virus strain. (c) Regardless of the endocytic pathway used, all RV strains reach early endosome (EE)s in a process that depends on RAB5, EEA1, and probably on HRS and the vacuolar ATPase (v-ATPase) (11). (d) At the EE, the virus probably begins to be internalized into the endosomal lumen through the action of VPS4A. (e) EEs progress to MEs, with a progressive decrease in pH and intraendosomal calcium concentration through the function of the v-ATPase; during this process the formation of ILVs increases. (f) E-P rotaviruses RRV and SA11-4S reach the cytoplasm from MEs. (g) GTPase Rab7 participates in the formation of LE compartments; ILVs increase in number. (h) The stability and function of LEs depend on the arrival of cellular factors (e.g., cathepsins) from the trans-Golgi network, traffic that is mediated by M6PRs, and the GTPase Rab9, among other factors. (i) L-P RV strains reach late endosomes. RV nar3's exit from LEs requires the function of Rab9. (j) RV strains UK, Wa, WI61, DS-1, and YM require, in addition to Rab9, the function of the CD-M6PR and the activity of cathepsins to productively infect cells. (f, i, j) The cytosolic double-layered particles begin transcribing the RV genome to continue the replication cycle of the virus. The different colors of the viruses represent those strains that exit from MEs (orange) or from LEs that do not require cathepsins (blue) or form LEs, but do require the activity of CD-M6PR and cathepsins (green). HBGA, human histo-blood group antigen; LBPA, lysobisphosphatidic acid. Viral exit from endosome generally relies upon pH transitions. -pH drop can open helical proteins in capsid proteins, or activate viral proteases that cleave viral capsid proteins. -these proteins interact with the luminal side of the acidified endosome (late endosome) somehow disrupt the membrane (amphipathic peptides), allowing the capsid to escape. In some cases, only the viral genome needs to escape. “Carpet Model” -the “carpet model” suggests that the viral protein coats the membrane, and somehow solubilizes it like a detergent. -the “pore formation” model suggests that viral proteins directly embed into the membrane and generate a pore for viral escape. “Pore formation” Picornavirus RNA exits the capsid, and then through a pore. -RNA+ virus -examples: Rhinovirus, Hepatatis A, poliovirus, enterovirus -pH change (reconstituted with heat) allows uncoating of capsid, and RNA release. VP4 dissociates from VP3, will allow VP1 (blue to form a pore) -VP4 (shown green on left) is lost with low pH, allows VP1 (shown blue on left) access to the endosome, where it forms a pore. -RNA(thick green line) can then escape the capsid and exit this pore. Host genes required for Rotavirus infection. -genome-wide siRNA screen identified many host genes whose down-regulation either increased (blue) or decreased (red) rotavirus infection. -You see the integration of the viral life cycle with host cell biological processes. -This study (July 2016) identified AMPK and vacuolar ATP synthase as major determinants. Links to endosomal acidification and mTOR signaling. -But are obviously many more nodes here! Anatomy of an enveloped virus (SARS-CoV-2) Spike protein receptor binding (and mAB target) Lipid bilayer E protein pore forming N protein Helps pack the RNA How I Built a 3-D Model of the Coronavirus for Scientific American Author: Veronica Falconieri Hays Publication: Scientific American Publisher: SCIENTIFIC AMERICAN, a Division of Springer Nature America, Inc. Date: Jun 25, 2020 M protein membrane protein forming lattice ssRNA (30kbp) General schematic of the two types of binding and entry. -Viral proteins within the envelope bind to specific cell surface proteins for entry (again, making infection cell-type specific) -In addition to initial binding, SNARE-like fusion machinery sits in the viral envelope. either direct fusion with PM or fusion through endocytosis -Viral fusion proteins can be active upon docking to cell surface receptors, or require pH transitions to become activated. Are three classes of viral fusion proteins. similar to SNARES -Class I are mostly alpha-helical and require a cleavage event to reveal the fusogenic region. Single polypeptide chain. Activation of viral proteases occurs upon acidic pH. Influenza, retrovirus, coronavirus -Class II are mostly beta-sheets, and have an internal fusogenic region. Are bound to another membrane protein that is often cleaved, allowing activation of the fusion protein. Includes viral chaperones in a complex of proteins. Flavivirus, Alphavirus, Denguevirus -Class III does not require any cleavage events. Mix of alphahelix/beta-sheet. Conformational changes are induced by pH change. Viral SNARE-pins -Once viral fusion proteins are activated, the fusion process proceeds in a manner very similar to the SNARE pathway. -top shows a Class I fusion protein with alpha-helices from influenza virus where HA drives fusion. The conformational change allows cleavage of a fragment exposing the “red” fusion peptide. This enters the host endosomal membrane to drive fusion. -bottom shows a Class II fusion protein with beta sheets from Dengue virus. The glycoproteins (yellow) rearrange upon low pH to release the fusion peptides (orange FP) that embed into lumenal side of endosome membrane. Once fused, virus escapes. J. Virol. October 15, 2007 vol. 81 no. 20 11526-11531 SARS-COV2 entry pathways endosomal entry and cell surface entry BOTH require ACE2 receptor binding -ACE2 receptor binding -Cleavage event S2 essential for viral fusion. An S1 cleavage during biogenesis in Golgi (furin site) of previously infected cell -Protease TMPRSS2 on cell surface means it will fuse there, without this virus will be endocytosed. -Acidification alters conformation, cathepsin L cleavage allows the fusion -therapies shown in pink boxes to block viral binding or acidification of endosome. Latter therapies didn’t work in patients. Vaccine targets Spike protein Jackson et al. Nature Reviews Molecular cell Biology 23 , 2022. Viral genome replication can occur in the cytosol or nucleus. -A variety of mechanisms again! -Most RNA viruses replicate in cytosol, DNA viruses are replicated in nucleus (see tables earlier). -Can be true for both enveloped and nonenveloped viruses. Enveloped viruses fuse with endosome to release nucleocapsids into the cytosol. -Entire capsids can cross the nuclear pore (b,c,e), or capsids can disassemble at the pore (a,d), with only the genomes entering the nucleus -Some viral genomes are transported and cross into the nucleus without capsids (g,h). An example of HSV capsid docking and releasing content at the nuclear pore. Egress from the nucleus -While in the nucleus, some viral genomes integrate into the chromosomes, leading to cancer. Papilloma virus is an example. -Some viruses trigger apoptosis to exit the nucleus, but others have distinct mechanisms to leave the nucleus intact. -Some simpler viruses assemble in the nucleus and the capsids exit, others transport the genomes for cytosolic assembly. Herpes and bacculoviral egress as examples. 1 2 -Herpesvirus buds into the perinuclear space -these vesicles fuse with outer nuclear envelope, releasing capsid in cytosol. 3 -capsid binds Golgi (left) that is now expressing viral proteins in the membrane, and capsid is internalized or “wrapped”. This is how the virus becomes enveloped again. -The internalized/wrapped, enveloped virus is then secreted by the cell, releasing active virus. Broader view of the complex topology of enveloping a virus. TGN derived membranes can take up HSV -Capsid emerged from the nucleus -Bound to viral encoded proteins that had been translated in the cytosol and imported into the ER. -This protein recycles through TGN/PM -Naked capsids bind to this, and are engulfed by the TGN structure, thereby incorporating viral encoded proteins into their new envelopes. J. Virol. January 2005 vol. 79 no. 1 299-313 -This TGN derived structure fuses with surface, releasing the enveloped virus. Look at the various versions of enveloping! -many examples of viruses that stay within the perinuclear space and enter the ER. -These can carry into the Golgi and egress in different possible ways. -Newly made viruses cannot fuse back because the pH is wrong. They stay out of the (late) endocytic compartments! Many viruses have adopted the apoptotic pathway for transmission -apoptotic fragments are internalized to ensure cell death is immunologically silent. -viruses exit cells carrying markers that are interpreted as apoptotic fragments. Enveloped viruses shown here. -this is called “apoptotic mimicry”. Non-enveloped viruses can do this too. -Multiple exit pathways have adopted mechanisms for apoptotic mimicry -SV40 (top) has a capsid protein that mimics the GAS6 protein that binds phosphatidylserine receptors. -release of exosomes carrying viral capsids can effectively mimic an enveloped state enriched in PS. -Other types of membrane shedding will carry virus particles. This is all emerging….. Internalization of apoptotic fragments drives anti-inflammatory signaling. -engaging the apoptotic receptors activates transcriptional responses. -Some transcriptional targets inhibit host cell receptors that receive pro-inflammatory signals from neighbouring cells. Particularly within the innate immune pathways. -Other targets are secreted factors that are antiinflammatory for neighbouring cells. -This helps the virus to propagate without alerting the immune system. Summary. - Virology is primarily a cell biology problem! - Non-enveloped and enveloped viruses enter cells through endocytosis. - pH transitions are critical to uncoat or disassemble viruses - Exit from the endosome into the cytosol is achieved through multiple mechanisms - Viral replication and assembly occurs in the cytosol and nucleus, depending on the virus - Exit from the cell also occurs in various mechanisms, from induction of cell lysis to complex mechanisms of exocytosis and secretion - Viruses have adapted a “cloaking” mechanism to mimic apoptotic fragments, allowing efficient uptake and suppressing inflammatory signaling pathways in the host. - Understanding these mechanisms is leading to the development of antiviral therapies and vaccines.