Lecture Lecture #12#12

Bio3124Bio3124

Prokaryotic Viruses: BacteriophagesProkaryotic 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 T4Bacteriophage 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

earlyGenes

lateGenes: structural proteins

• genes withrelatedfunctions are usuallyfoundclustered

Genes are functionally and temporally clustered Genes are functionally and temporally clustered

• 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

Events during Binding and Entry of T4 phageEvents during Binding and Entry of T4 phage

• lytic cycle ~30 minutes

• Host chromosome is

degraded and nucleotides

are used

• Viral proteins synthesized

and, genome replicated

• Complete virions released

Brief life cycle of T4 phage Brief life cycle of T4 phage

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

• 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 replicationGenome replication

Phage Particles Self-AssemblePhage 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 M13Filamentous 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 InfectionPhage M13 Infection Binds to the pilus of

F+ cellsand to coreceptor

TolADNA released into

cytoplasm coat proteins

disassemble inserted to inner

membrane

Filamentous Phage M13Filamentous 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 VirusesAnimal 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(+) Strand RNA Virus: Polio• Cause of major epidemics

– Capsid resistant to stomach acid– Swallowing contaminated water

• Swimming pools, contaminated water supplies

Initially infects intestinal cellsMoves to neurons

Paralysis

Salk and Sabin vaccines Inactivated virus attenuated live virus

(+) Strand RNA Virus: Polio(+) 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(+) 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(+) 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 envelope2 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 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 shiftantigenic shift

• 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 VirusHuman Immunodeficiency Virus

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

• Reverse transcriptase has three different activities:1) DNA synthesis from the RNA template

- Primed by the host tRNA2) RNA degradation: via RNase activity3) DNA-dependent DNA synthesis: Generates a dsDNA, integrates into the host chromosome

HIV ReplicationHIV Replication

HIV replicationHIV 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 VirusesHerpesviruses: 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 cycleHerpes 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 strategiesUnifying 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 strategiesUnifying theme in RNA viruses replication strategies

• very diverse reproductive strategies

• four general modes of replication and transcription