Viruses Replication mechanism and pattern

Virus Replication

1. Course Name: Mechanistic Virology Course Code: PVMDMV-201

2. Viruses Replication Dr. Sana Iqbal Assistant Professor UIMLT MV(Mechanistic Virology)

3. Learning Objectives Viral Life Cycle Overview Overview of virus replication Mechanisms used by animal viruses to gain entry into host cells Evidence that a cell surface molecule is a virus receptor Two endocytic mechanism Initial stages of SV40 Internalization Via Caveolae

4. Figure 1. Pathways of virus entry into cells.  ( A ) Enveloped viruses can bind to cell surface receptors and directly fuse with the plasma membrane. Virus particles can also be internalized via endocytosis, with escape to the cytosol occurring either from the ( B ) early endosome or ( C ) late endosome and lysosome. The acidic environment and proteolytic enzymes in these compartments are required for fusion and cytosol entry by different viruses. Figure 2. Methods for viral genome release into host cells.  ( A ) Certain viruses, including rhinoviruses, expand to form pores in the endosome through which the viral genome can escape. ( B ) Influenza and other viruses induce fusion of the virion envelope with the endosomal membrane, releasing the viral genome. ( C ) Many viruses, including reoviruses, maintain a partially intact capsid in the cytosol that acts as a “home base” for replication. Figure 3. Several viruses must transport their genomes into the nucleus for viral transcription or replication to occur.  Influenza genome segments are transported through the nuclear pore into the nucleus. Herpesvirus capsids are transported along microtubules to the nuclear pore, where uncoating occurs. Adenovirus capsids disassemble at the nuclear pore, and the viral DNA is transported into the nucleus. Other viruses, including hepatitis B virus, are small enough that the entire capsid may pass through the nuclear pore. Figure 4. Nucleocapsid formation and formation of mature virions.  After the production viral structural proteins, nucleocapsids are assembled in the cytoplasm and followed by budding into the lumen of the endoplasmic reticulum (ER)–Golgi intermediate compartment. Virions are then released from the infected cell through exocytosis. Viral Life Cycle Overview

5. Overview of virus replication The aim of a virus is to replicate itself, and in order to achieve this aim it needs to enter a host cell, make copies of itself and get the new copies out of the cell. Seven steps: 1. Attachment of a virion to a cell 2. Entry into the cell 3. Transcription of virus genes into messenger RNA molecules (mRNAs) 4. Translation of virus mRNAs into virus proteins

6. 5. Genome replication 6. Assembly of proteins and genomes into virions 7. Exit of the virions from the cell.

7. Mechanisms used by animal viruses to gain entry into host cells Cell receptors and co-receptors A virion attaches via one or more of its surface proteins to specific molecules on the surface of a host cell. These cellular molecules are known as receptors. Some viruses need to bind to a second type of cell surface molecule (a co-receptor) in order to infect a cell.

8. Cont. Receptors and co-receptors are cell surface molecules, usually glycoproteins. Functions of Receptors and co-receptors include Acting as cell signaling mediator for chemokines and growth factors. Mediating cell-to-cell contact and adhesion.

9. Some examples of cell receptors, virus proteins involved in attachment and fusion proteins

10. Evidence that a cell surface molecule is a virus receptor If one of the antibodies blocks virus binding and infectivity, then this is strong evidence that the corresponding antigen is the receptor. Soluble derivatives of the molecule block virus binding/infectivity. The normal ligand for the molecule blocks virus binding/infectivity.

11. A. Virus attachment sites The virus attachment sites of some naked viruses are on specialized structures, such as the fibre and knobs of adenoviruses and the spikes of rotaviruses

12. The poliovirus receptor is the glycoprotein CD155 CD155 is a member of the immunoglobulin super family of molecules with three immunoglobulin-like domains. The virus attachment site is located in the outermost domain. CD155 is found only in humans and some other primate species. Transgenic mice expressing CD155 were developed. These animals were found to be susceptible to infection with all three serotypes of poliovirus and they have been used in studies of replication and pathogenesis.

13. B. Attachment of virions to receptors The forces that bind a virus attachment site to a receptor include Hydrogen bonds, Ionic attractions Van der Waals forces. No covalent bonds are formed between virions and receptors. At this bind, the attachment is bind, and the virion may detach. If sufficient receptors bind, then the attachment to the cell becomes irreversible.

14. C. Entry of animal viruses into cells Viruses may either: Cross the plasma membrane at the cell surface directly Or they may cross the membrane through Endosome This process (endocytosis) is used by cells for a variety of functions, including nutrient uptake and defense against pathogens. There are a number of endocytic mechanisms and most animal viruses hi-jack one or more of these mechanisms in order to gain access to their host cells.

15. Two endocytic mechanism Clathrin mediated endocytosis Caveolin-mediated endocytosis Clathrin and caveolin are proteins that are involved in endosome formation. When virion binds to a region of the plasma membrane the coat protein clathrin or caveolin protein molecules force the membrane to bend around the virion.

16. Clathrin -coated endosomes Many viruses, such as adenoviruses, are endocytosed at clathrin -coated regions of the plasma membrane. The virions end up in clathrin -coated endosomes, from which the clathrin is soon lost. An endosome may fuse with other vesicles such as lysosomes, which have a pH of 4.8–5.0, thus lowering the pH within the vesicle.

17. EM views - coated pit to coated vesicle Coated pits coated vesicles Coated pits coated vesicles

18. Protein coat is “geodesic” clathrin cage Clathrin “heavy chain” “Light chain” 3 clathrin “heavy chains” (~180-190 kDa )… …plus… 3 clathrin light chains (~40 kDa )… …form… “ Triskelions ”… Spontaneously assemble into “geodesic” vesicle coats…

19. clathrin adaptins membrane receptors “cargo” Components of a clathrin -coated vesicle Adaptins - adaptors that bind clathrin and cargo receptor, thereby regulating which cargo gets loaded into clathrin-coated vesicle

20. ECB 15-19 Coated pit “pinching off” ( dynamin) budding uncoating Pinching off the vesicles requires the protein dynamin Assembly of coat causes pit to form due to 3D shape of clathrin coat

21. dynamin GTP GDP GTPase that regulates pinching off Dynamin is a GTPase

22. GTP GDP + Pi Clathrin uncoating ATPase Naked transport vesicle Dynamin ATP ADP + Pi Clathrin-coated vesicles are rapidly uncoated By the “ clathrin -uncoating ATPase” a member of the HSP70 family of chaperones; requires ATP hydrolysis Naked transport vesicles targeted to endosome… Clathrin and adaptins recycled See ECB figure 15-19 “Clathrin-coated pit” To endosome… Clathrin Adaptin complexes

23. Caveolins are proteins essential for the formation and stability of caveolae ( signaling platforms ). Caveolae rounded plasma membrane invaginations of 50–80 nm in diameter. Caveolin -mediated endocytosis

24. After binding to the membrane, virus particles are mobile until trapped in caveolae , which are linked to the actin cytoskeleton (step 1). In the caveolae , SV40 particles trigger a signal transduction cascade that leads to local protein tyrosine phosphorylation and depolymerization of the cortical actin cytoskeleton (step 2). Initial stages of SV40 Internalization Via Caveolae

25. Actin monomers are recruited to the virus-loaded caveolae and an actin patch is formed (step 3). Concomitantly, Dynamin is recruited to the virus-loaded caveolae and a burst of actin polymerization occurs on the actin patch (step 4). Virus-loaded caveolae vesicles are now released from the membrane and can move into the cytosol (step 5).

26. After internalization, the cortical actin cytoskeleton returns to its normal pattern (step 6).

27. Learning Outcomes Mechanisms used by animal viruses to gain entry into host cells Evidence that a cell surface molecule is a virus receptor Initial stages of SV40 Internalization Via Caveolae

28. Recommended Literature Replication of RNA virus | How RNA virus replicate | Virology USMLE Review of Medical Microbiology and Immunology Fenner and White's Medical Virology (5th Edition) by Christopher J. Burrell, ‎Colin R. Howard, ‎Frederick A. Murphy. Human Virology (4th Edition) by Leslie Collier, John Oxford and Dr. Paul Kellam.

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