Summary of the last lecture

1. Summary of the last lecture • Virus classification schemes: • Classical • Recent: ICVT • Baltimore • Virus Structure: Virion • DNA or RNA, virus replication enzymes • Capsid, or nucleocapsid • Icosahedral and helical • Envelope(lipids and carbohydrates from cells) • Glycoproteins (Gp, G): spikes • Matrix protein • Assessory proteins • Virus assembly • Spontaneous • Protein-protein, protein nucleic acid and protein-lipid interaction (hydrophobic and electrostatic)

2. Assembly of poliovirus

3. Mechanism of packaging genome • Direct contact of genome with nucleocapsid or capsid proteins: small viruses such as poliovirus contain specific proteins that interact with RNA. • Specialized viral encoded nucleic acid binding proteins: these proteins, called ribonucleoproteins, nucleoproteins or core proteins, contain a nucleic acid binding motif in the sequence, or recognize a specific packaging signal sequence in RNA or DNA. These proteins are highly basic which react with negatively charged nucleic acids. • Packaging by cellular proteins: unique to papovaviruses, so the small viral genome doesn’t need to devote its limited genome to packaging.

4. Packaging sequence in Herpesvirus

5. Cell culture • Medium for cell growth • -- water, salts, minerals • -- amino acids • -- glucose • -- serum • -- antibiotics (penicillin and streptomycin) • -- special growth factors (for some cell types) • Cell culture • -- primary cell culture: prepared freshly from tissue • -- cell lines (immortalized cell culture):directly derived from tumor or primary cells transformed with tumor virus or chemical mutagen

6. Cell culture (cont’) • CO2 incubator: temperature 37oC, humidified, co2 • Long term storage: at liquid nitrogen (-140oC). Cells should be frozen quickly in -80oC freezer and thaw rapidly at 37oC waterbath

7. Primary cells vs. cell line

8. Cell types Fibroblast-like cells (BHK) Epithelial-like cells (Lung carcinoma)

9. Cytopathic effects (CPE) uninfected 0.01PFU of virus/cell infected 0.1PFU of virus/cell infected 10 PFU of virus/cell infected

10. CPE(cont’) Intranuclear inclusion body(LM) Syncytium Intranuclear inclusion body(EM)

11. Virus-cell interaction • Attachment (reversible) -- virus attachment proteins (capsids, or glycoproteins) -- receptors or co-receptors (cell surface molecules) -- virus tropism ( spectrum of cells that virus can infect) • 2. Entry (penetration cross the plasma membrane, irreversible) • -- Receptor mediated endocytosis (the most common) • -- Cell membrane fusion (usually enveloped virus) • -- Translocation: very rare • 3. Uncoating( release of nucleic acid from its protein coat) • -- uncoating at the plasma membrane (Paramyxovirus) • -- uncoating triggered by change in pH (Influenza) • -- uncoating in the cytoplama

12. Virus attachment

13. Viral receptors

14. Virus entry

15. Virus entry (cont’)

16. Virus entry inhibitors

17. Virus-cell interaction (cont’) • 4. Replication and protein synthesis • -- In general, early proteins ( viral enzymes, regulatory • proteins) are synthesized first, followed by virus • genome replication and late proteins (structural • proteins) synthesis • 5. Assembly and release • -- Non-enveloped virus become mature inside the cell • and are released by cell lysis • -- enveloped viruses mature by budding from the plasma • membrane of host cells

18. Virus release

19. Virus budding

20. One-step growth curve Pfu/cell(log)

21. Applications of cell culture