Transcript The Viruses Part II: Viral Molecular Biology

Lecture #12
Bio3124
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
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
Genes are functionally and temporally clustered
early
Genes
• genes with
related
functions
are usually
found
clustered
late
Genes:
structural
proteins
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
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
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
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
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
Filamentous Phage M13
• 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
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
Filamentous Phage M13
• 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
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
(+) 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

(+) 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
(+) 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
(+) 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?)
(-) 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

(-) 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
Animation: Influenza Virus Entry into a Cell
(-) 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
Animation: Influenza Virus Replication
Rehearse your
learning here.
(-) 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
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
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
HIV binding and entry
• Binding to CD4
and CCR5 on T
cells
• Conformational
changes
• Fusion peptide
• Membrane fusion
• Core released to
cytoplasm
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
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
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
Animation: HIV Replication
Rehearse your
learning here
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
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
Animation: Herpes Virus Replication
HSV-1 life
cycle
For your
rehearsal
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
Unifying theme in RNA viruses replication strategies
• very diverse
reproductive
strategies
• four general
modes of
replication
and
transcription