1. THE REPLICATION OF VIRUSES Virology Lecture 2 Three lectures dealing with (1) replication of DNA viruses (2) the culture, growth and recognition of virus infections (3) the replication of RNA viruses

2. A growth curve for a virus in cell culture, based upon the simultaneous infection of all cells in the culture

3. THE MODE OF REPLICATION OF VIRUSES -- WITH PARTICULAR REFERENCE TO DNA VIRUSES There are a number of sequential steps in replication of DNA viruses 1.Attachment or adsorption 2.Penetration 3.Uncoating of virus and transport of genome to site of replication 4.Early transcription (mRNA) 5.Early translation and early proteins 6.Viral DNA synthesis 7.Late transcription of further mRNA 8.Late translation and synthesis of structural proteins 9.Assembly 10.Release

4. These sequential steps in the replication of DNA viruses can be considered in groups 1.Attachment or adsorption 2.Penetration 3.Uncoating of virus.. transport of genome to site of replication 4.Early transcription (mRNA) 5.Early translation and early proteins 6.Viral DNA synthesis 7.Late transcription of further mRNA 8.Late translation and synthesis of structural proteins 9.Assembly 10.Release

5. ….. the red blood cells agglutinate (haemagglutination) If the virus attaches to red blood cells.... and not all viruses will attach to red blood cells

7. Step 2. Penetration Fusion of the envelope of the virus with the plasma membrane of the cell and the direct release of the nucleocapsid into the cytoplasm. The fusion process is mediated by specific proteins or glycoproteins OR Engulfment of the virus by the cell in a specific phagocytic process (adsorptive endocytosis) before the viral nucleic acid (genome) is released.

8. Step 3. Uncoating This is the general term applied to events after penetration which free the viral genome. –It can occur in the cytoplasm, or in the cases of viruses that replicate in the nucleus (and this includes most DNA viruses) it occurs in the nucleus following transport of the virion through the cytoplasm to the nucleus. Different viruses have evolved different strategies. –With poxviruses, which replicate in the cytoplasm, host factors induce the disruption of the virus. –In herpesviruses, after fusion and deposition of the viral nucleocapsid ….in the cytoplasm of the cell …. nucleocapsid migrates to the cell nucleus along the microtubules ….to the nuclear pore, and … is released into the nucleus. –With papillomaviruses, the virus is transported to the nucleus in vesicles –Circoviruses gain entry to the nucleus during mitosis

9. Next few slides demonstrate the initial stages in the replication of HIV –attachment –penetration –uncoating and release of virus components into the nucleus

10. CD4 antigen Co-receptor CD4+ cell HIV binds to CD4-positive cells

11. HIV Gp120 (SU) CD4 antigen Co-receptor gp41 (TM ) T4 cell

12. SU binds to CD4 and SU changes conformation CD4 antigen Co- receptor

13. Modified SU then binds to co-receptor CD4 antigen Co- receptor

14. CD4 antigen Co- receptor TM undergoes conformational change bringing membranes close together

15. TM is exposed and embeds in the membrane of the CD4 cell

16. HIV membrane and cell membrane begin to fuse

17. Membranes fuse

18. Nucleocapsid enters cytoplasm

19. Nuclear membrane Cell membrane Nuclear pore Nucleocapsid moves toward nucleus

20. Nuclear membrane Interacts with nuclear protein (nuclear pore?)

21. Reverse transcriptase protease integrase RNA Surface membrane Nuclear membrane tRNA primer Components enter the nucleus

22. Steps 4 and 5. Early transcription and translation of viral proteins The next step is the formation of a mRNA from the virus DNA (transcription) and formation of early viral ‑ coded proteins (translation). DNA ‑ dependent RNA ‑ polymerase (cell origin, except poxviruses) Viral DNAViral mRNA Nucleotide pool (ATP,TTP,CTP,GTP) using cell ribosomes Viral-coded proteins What are the early coded proteins? Enzymes necessary for the next step in the process --DNA synthesis (eg. thymidine kinase and a DNA-dependent DNA polymerase).

23. Where in the cell does this occur? With most DNA viruses (but not poxviruses, which replicate in the cytoplasm), the transcriptional events occur in the nucleus. From there mRNA is transported to the cytoplasm where translation of the coded proteins occurs. The proteins are then transported back to the nucleus where they are needed before further steps in the replication process can proceed.

25. Uncoating and release of virus genome in nucleus

26. Transcription of mRNA from DNA

27. Translation of mRNA and production of early virus-coded proteins

28. Some of these virus-coded proteins re-enter the nucleus where they are needed for DNA replication, eg. The virus encoded DNA-dependent DNA polymerase

29. Step 6. Viral DNA synthesis (DNA replication) Nucleotide pool in cell Viral DNA Progeny viral DNA DNA-dependent DNA polymerase

30. Progeny virus DNA

31. Steps 7 and 8. Late transcription and translation of proteins Late transcriptional events occur only after DNA synthesis has begun and does not occur if DNA synthesis is inhibited [by inhibitors such as IUDR] The late mRNAs which are transcribed are again transported to the cytoplasm and code for structural proteins which are later assembled to form the progeny virus particles. Some of the structural proteins formed may be glycosylated on the trans-Golgi network before they are transported back to the nucleus, where assembly of the proteins into viral particles occurs.

32. Late transcription of mRNA Late translation of virus proteins

33. Step 9. Assembly of viral particles After synthesis of viral nucleic acid and protein these are packaged together to form progeny virus. In assembly, the various proteins are assembled in a defined sequence. The inner `core' proteins are the first to associate with DNA, and the capsomers are laid down last. With the poxviruses and the iridoviruses ‑ which replicate in the cytoplasm of the cell ‑ progeny DNA and protein accumulate in a common cytoplasmic factory (inclusion) and there is little problem in assembly when the concentration of the two reach an adequate level. Other DNA viruses are assembled in the nucleus ‑ assembly occurs after structural proteins (synthesised in the cytoplasm) have migrated back to the nucleus where DNA replication and transcription have occurred.

34. Assembly Progeny virus DNA Structural virus proteins

35. Step 9. Assembly of viral particles After synthesis of viral nucleic acid and protein these are packaged together to form progeny virus. Most DNA viruses are assembled in the nucleus ‑ assembly occurs after structural proteins (synthesised in the cytoplasm) have migrated back to the nucleus where DNA replication and transcription have occurred. Poxviruses replicate in the cytoplasm of the cell ‑ progeny DNA and protein accumulate in a common cytoplasmic factory (inclusion) and assemble when the concentration of the two reach an adequate level.

36. Step 10. Release of the virus particles from the cell By autolysis of the cell - this is the method used by most DNA viruses –Virus-induced programmed cell death (apoptosis) may be involved Herpesviruses mature by a more complex process during which the viruses acquire an envelope

37. Primary envelopement of capsids by budding at the inner leaflet of the nuclear membrane and translocation of capsids into the cytoplasm after loss of the primary envelope by fusion with the outer leaflet of the nuclear membrane Tegumentation occurs in the cytoplasm Capsids obtain final envelope by budding into vesicles of the trans- Golgi network Final endocytosis from the plasma membrane and directional budding of virions results in release of virus from the cell. Glycosylation of envelope proteins and insertion into membrane of vesicles of the Golgi network