Applications of cell culture

1. Applications of cell culture

2. Virus-Cell Interaction • Attachment • Viral attachment proteins • capsid (naked virus) • Glycoproteins (spikes): enveloped virus • Cell surface molecules as receptors, or co-receptors • Entry • Receptor mediated endocytosis (most viruses) • Membrane fusion(some enveloped viruses) • Uncoating • Plasma membrane • Cytoplasma • Replication and protein synthesis • Assembly and release

3. Virus Replication • Virus doesn’t grow or divide ( living organism or not) • Virus reproduces itself by the assembly of individual components (DNA, or RNA, proteins) • Virus replication involves the synthesis of proteins and nucleic acids (DNA and RNA) • Virus uses cell’s machinery for protein synthesis • (non-free living organism) • The central dogma of protein synthesis: DNA > mRNA > Proteins • Baltimore virus classification scheme: How mRNA is made for protein synthesis?

4. Baltimore virus classification • Class I: ds DNA viruses • Same mechanism of protein synthesis as eukaryotic cells • Use DNA dependent RNA polymerase ( cellular) to make mRNA • Class II: ss DNA viruses • Use ds DNA as intermediate to synthesize mRNA and ss DNA progeny • Class III: ds RNA viruses with segmented genome • Viral encoded RNA dependent RNA polymerase (RdRp) to make mRNA from (-) RNA • Each segment is transcribed individually to produce monocistronic mRNA • Class IV: ss (+) RNA viruses • Polycistronic mRNA > polyprotein • Class V: ss (-) RNA viruses • Viral encoded RdRp to make mRNA from (-) RNA template • Class VI: ss(+) RNA virus with DNA intermediate (Retrovirus) • Viral encoded reverse transcriptase to make DNA from RNA, then DNA > mRNA > protein using cellular polymerase • Class VII: ds DNA virus (Pox virus) • Viral encoded DNA dependent RNA polymerase to make early mRNA

5. Replication Strategies Key molecule: mRNA

6. (+) ss RNA viruses (single polyprotein precursor)

7. (+) ss RNA viruses ( multiple proteins)

8. (-) RNA viruses

9. Ambisense RNA viruses

10. ds RNA viruses

11. Retroviruses

12. General features of RNA viruses • Encode RNA dependent RNA polymerase (RdRp): • --unique to virus (copy RNA from RNA template) • -- Genome replication • -- Generate mRNA from viral genome (-) RNA virus • Replicate in the cytoplasma, exceptions include • -- Orthomyxoviridae (Influenza virus) • -- Hepatitis delta virus • -- Retrovirus (HIV)

13. General features of DNA viruses • Encode their own DNA polymerase (copy DNA from DNA) • Exceptions: Parvo and Papillomavirues (cellular DNA polymerase, only replicate in dividing cells) • Replicate in the nucleus of cells, dependent on cellular factors • Exception: pox viruses, replicate in cytoplasma, encode all the factors necessary for replication • Protein expression occurs in a defined, sequential order • Immediate early-- early-- late (structural proteins)

14. Brief Review of Molecular Biology • Transcription • Make mRNA from DNA • Promoter • RNA polymerase • Transcription terminator • Transcription factors (regulators of transcription) • Enhancers and transactivators • mRNA: • Polycistronic: several genes share the same promoter • Monocistronic • Open reading frame(ORF)

15. DNA replication • Origin of replication (ori) • Termination of replication • Regulators of replication • DNA polymerase: • DNA synthesis, chain elongation • ss DNA binding protein • Bind and stablize ssDNA at forks • Primase • Synthesizes primers for lagging strand • Helicase • Unwinding the helix • Ligase • Close the nick left by PolI (make final phosphodiester bond) • Topoisomerase (Gyrase) • Unwind supercoils ahead of fork

16. DNA viruses