The Viruses Part II: Viral Molecular Biology

1. The Viruses Part II: Viral Molecular Biology Lecture #12 Bio3124

2. Must bind to host cell receptor Must cross a cell wall G hosts also cross 2 membranes Must not damage host cell initially Use host nucleotides, amino acids, ATP Replicate viral genome, build capsid, assemble new viruses Exit through cell wall Usually lyse host cells Prokaryotic Viruses: Bacteriophages

3. Bacteriophage T4 • T4 phage: virulent coliphage, order of Caudovirales(tailed viruses), family of Myoviridae • Host:enterobacteriacaeegE.coli • ~ 200 nm in length • ds linear genome ~170 kbp with a terminally redundant sequences, but not at circular stage • 43 phage proteins,16 are located in the head, and 27 form the tail • icosahedral head, contractile helical tail, a base plate and 6 tail fibers

4. early Genes late Genes: structural proteins Genes are functionally and temporally clustered • genes with • related • functions • are usually • found • clustered

5. Events during Binding and Entry of T4 phage • attachment of tail fibers to the surface lipopolysaccharides electrostatically • lowers the base plate, tail sheath is shortened • Conformational changes in base plate proteins opens it and allows the core tube to pass through the cell wall and cytoplasmic membrane • gp5 protein in base plate is a lysozyme that weakens the cell wall • Linear DNA genome is injected through the core tube into the cytoplasm

6. Brief life cycle of T4 phage • lytic cycle ~30 minutes • Host chromosome is degraded and nucleotides are used • Viral proteins synthesized and, genome replicated • Complete virions released

7. Early events • viral endonuclease digest host chromosome; nucleotides used for phage genome replication • make hydroxymethylcytosine (HMC) from CMP released from host DNA degradation • HMC replaces cytosine in T4 DNA • HMC glucosylated by phage enzymes • Glucosylation protects phage DNA from host restriction endonucleases • enzymes that cleave DNA at specific sequences • restriction endonucleases are host defense mechanism against viral infection

8. Linear genome circularize upon entry Replication by rolling circle Produce a concatamer Cleavage of concatamers Packaging in the head with 3% above genome size Results in terminally redundant ds-linear genomes Circularly permutated Genome replication

9. Late genes transcribed Capsid particles Head polymerizes around progeny DNA Tail fibers, long tail made Head, tail, tail fibers assemble Lysis and virion release two proteins involved Holincreates holes in membrane T4 lysozymeattacks the E. coli cell wall ~150 viral particles released Phage Particles Self-Assemble

10. Genome: 6.4 kb (+)SSC; codes for 11 proteins Simple capsid structure P8 protein wrapped around DNA P7, P9 at tip; P3 and P6 at the base Filamentous Phage M13

11. Phage M13 Infection • Binds to the pilus of F+ cells • and to coreceptorTolA • DNA released into cytoplasm • coat proteins disassemble • inserted to inner membrane

12. Host enzymes replicate SSC Replicative form (RF) RF Replicates via rolling circle Makes capsid proteins Self-assembles at inner membrane Exits without killing host Makes own export pore complex (gp4) P1, P11 guard the channel Capsid proteins integrated to membrane Assemble and package SSC genome Filamentous Phage M13

13. Don’t have to cross a cell wall Can enter through endocytosis (whole virus) Alternatively by fusion of viral envelope to plasma membrane (delivery of nucelocapsid) Must travel between organelles Host transcription machinery in nucleus Translation in cytoplasm Transport via ER, Golgi, endosomes Exit via cell lysis or budding Animal Viruses

14. Cause of major epidemics Capsid resistant to stomach acid Swallowing contaminated water Swimming pools, contaminated water supplies (+) Strand RNA Virus: Polio • Initially infects intestinal cells • Moves to neurons • Paralysis • Salk and Sabin vaccines • Inactivated virus • attenuated live virus

15. Picornavirus = pico (very small) +RNA virus (~30nm) Icosahedralcapsid Capsid: Vp1,Vp2, Vp3 external; Vp4 is internal Binds to receptor Endocytosed Uncoats inside endosome Releases ssRNA into cytoplasm (+) strand is translated directly Makes a single polyprotein (+) Strand RNA Virus: Polio

16. Single polyprotein processed Cuts itself into pieces by viral proteases (2A, 3AB, 3C) Capsid proteins Replicating RNA polymerase viral RNA has two roles in producing progeny virions (+) RNA in cytoplasm is translated; transcribed to (-)RNA (-)RNA in ER-derived vesicle is replicated => (+) RNA genome (+) Strand RNA Virus: Polio

17. Cell cannot replicate RNA RNA viruses encode own polymerase Since don’t use cell polymerase, can avoid nucleus Replicates in ER derived membranes (-) RNA is replicative intermediate Assembles in cytoplasm Exits cell (lysis?) (+) Strand RNA Virus: Polio

18. Pandemic of 1918 Greatest one-year loss of life in recorded history Especially deadly among college-aged Get your flu shot! (-) Strand RNA Virus: Influenza • Segmented genome: 8 separate RNAs • Each codes for one protein • lipid envelope • 2 major envelope proteins • Neuraminidase • Hemagglutinin

19. (-) strand RNA can’t be read by ribosome Must be transcribed to (+) RNA No replicating RNA polymerase in host All (-) RNA viruses must bring own polymerase protein into host cell Influenza binds to host sialic acids HA mediates membrane & envelope fusion in endosomeat low pH (-) Strand RNA Virus: Influenza

20. Animation: Influenza Virus Entry into a Cell

21. (-) strand RNA moves to nucleus (+) strand mRNA synthesized Move to cytoplasm Viral proteins made Envelope proteins placed in plasma membrane (+) strand used to make progeny (-) RNA Assembly at plasma membrane Budding to release progeny viruses (-) Strand RNA Virus: Influenza

22. Animation: Influenza Virus Replication

23. Rehearse your learning here.

24. Viral RNA polymerases are inaccurate Introduce many mutations Antigenic drift Rapid evolution New flu virus species every year New vaccine necessary Cell infected by 2 strains can recombine Assemble new combinations of RNAs Reassortment = antigenic shift (-) Strand RNA Virus: Influenza

25. HIV is a lentivirus, evolved from viruses infecting African monkeys HIV causes acquired immunodeficiency syndrome (AIDS) - transmitted through blood and through genital or oral-genital contact - There is no vaccine or cure - However, there are drugs that extend life expectancy Human Immunodeficiency Virus

26. Bullet-shaped capsid Encloses 2 identical copies of RNA Plus polymerase proteins Surrounded by envelope Envelope spike proteins (SU, TM) embedded Genome: 3 reading frames, gag, Pol, Env Accessory proteins: regulate latency and virion production Human Immunodeficiency Virus

27. Binding to CD4 and CCR5 on T cells Conformational changes Fusion peptide Membrane fusion Core released to cytoplasm HIV binding and entry

28. Reverse transcriptase has three different activities: 1) DNA synthesis from the RNA template - Primed by the host tRNA 2) RNA degradation: via RNase activity 3) DNA-dependent DNA synthesis: Generates a dsDNA, integrates into the host chromosome HIV Replication

29. RT replicates DNA from RNA tRNA priming RNA destroyed as DNA made Forms dsDNA Circularizes Moves to nucleus by the help of Vpr accessory protein integrase inserts it into host genome Latent form: provirus HIV replication

30. Cell RNA poltranscribes Full genome dimer Spliced mRNA for proteins Env (SU, TM) passgolgi-> CM gag and gag-pol Assembly in CM Active virions need viral protease Exit via budding HIV-1 life cycle

31. Animation: HIV Replication

32. Rehearse your learning here

33. Icosahedral capsid dsDNA genome Encodes over 70 proteins Surrounded by lipid envelope Multiple envelope proteins Tegument between capsid and envelope Includes many proteins Herpesviruses: DNA Viruses

34. Virus can bind several host receptors Envelope fuses with plasma membrane Capsid moves to nucleus Uses host polymerase to replicate Lytic infection mRNA moves to cytoplasm Proteins built, assembled Virus exocytosed from plasma membrane Herpes viruses: life cycle

35. Animation: Herpes Virus Replication

36. HSV-1 life cycle For your rehearsal

37. Unifying theme in DNA virus replication strategies • early genes • encode proteins involved in take over of host and in synthesis of viral DNA and RNA • viral DNA replication • usually occurs in nucleus (except for poxviridae) • early and late mRNA synthesis • usually by host RNA polymerase • Proteins used for packaging, capsid assembly and virion release

38. Unifying theme in RNA viruses replication strategies • very diverse reproductive strategies • four general modes of replication and transcription