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

2. Prokaryotic Viruses: Bacteriophages
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

3. Bacteriophage T4
T4 phage: virulent coliphage, order of Caudovirales (tailed viruses), family of Myoviridae
Host: enterobacteriacae eg E.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. Genes are functionally and temporally clustered
early
Genes
genes with
related
functions
are usually
found
clustered
late
Genes:
structural
proteins
Map of T4 genome. Genes of related function tend to be clustered together.

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
Genome replication
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 8

9. Phage Particles Self-Assemble
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
Holin creates holes in membrane
T4 lysozyme attacks the E. coli cell wall
~150 viral particles released

10. Filamentous Phage M13 Simple capsid structure
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

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

12. Filamentous Phage M13 Host enzymes replicate SSC Makes capsid proteins
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

13. Animal Viruses 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

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

15. (+) Strand RNA Virus: Polio
Picornavirus = pico (very small) +RNA virus (~30nm)
Icosahedral capsid
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

16. (+) Strand RNA Virus: Polio
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

17. (+) Strand RNA Virus: Polio
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?)

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

19. (-) Strand RNA Virus: Influenza
(-) 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 endosome at low pH

20. Animation: Influenza Virus Entry into a Cell

21. (-) Strand RNA Virus: Influenza
(-) 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

22. Animation: Influenza Virus Replication

23. Rehearse your learning here.23

24. (-) Strand RNA Virus: Influenza
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

25. Human Immunodeficiency Virus
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 25

26. Human Immunodeficiency Virus
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

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

28. HIV Replication 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 28

29. HIV replication 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

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

31. Animation: HIV Replication

32. Rehearse your learning here32

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

34. Herpes viruses: life cycle
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

35. Animation: Herpes Virus Replication

36. HSV-1 life cycle
For your rehearsal 36

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