Bacterial Viruses Flashcards
What is a virus?
Obligate intracellular parasite that can only multiply inside a host cell and must rely on it for energy, metabolic intermediates and protein synthesis.
What is the virion? What are its parts?
extracellular form of viruses that protect the viral genome when the virus is outside the host cell.
What is a capsid?
protein shell of a virion composed of a individual capsomere proteins that are often arranged in a precise and highly repetitive pattern around the nucleic acid
What is nucelocapsid?
Inner structure of ONLY nucleic acid and capsid protein. May be naked (no further layers) or enveloped in layers of phospholipids and glycoprotein to protect the genome and aid in attaching to host cell.
What are the two types of symmetry of a virion?
Rod shaped viruses: helical symmetry (e.g. human papillomavirus virion)
Spherical shaped viruses: icosahedral symmetry (ebola)
What is the difference in the way a virus enters a eukaryotic and prokaryotic cell?
Eukaryote: entire virion is taken up by the cell
prokaryote: only nucleic acid is taken up by the cell
Why do virus infections upset host bacteria’s regulatory systems?
there is a marked overproduction of viral nucleic acid and protein to make new virions
What are the two groups of proteins synthesized after infection?
early proteins: nucleic acid polymerases and other enzymes used to shut down host cell transcription and translation
late proteins: structural components of the virion and other components needed for assembly
what is burst size?
Average number of virions released from a host cell when it lyses.
What is the range in virus sizes?
viral genomes range almost a thousand fold from smallest to largest, and are grouped by genome structure
What is the baltimore classification of viral genomes?
7 class system (3 DNA, 4 RNA) of viral genome classification developed by virologist David Baltimore based on the relationship of viral genome to its mRNA.
What is the difference between plus and minus RNA and DNA strands?
(-) RNA: negative sense-RNA complementary to positive sense RNA. Cannot be directly translated into protein.
(+) RNA: positive sense-RNA. Can be directly translated into protein.
(-) DNA: strand is complementary to the (+) mRNA and serves as the template during transcription
(+) DNA: strand with the same sequence as the (+) mRNA (with T instead of U)
What is baltimore Class I form of genome, mRNA production, and replication?
Genome: dsDNA
mRNA Production: Host RNA polymerase transcribes viral DNA into mRNA.
Replication: Uses host or viral DNA polymerase.
What is baltimore Class VII form of genome, mRNA production, and replication?
Genome: dsDNA, but replicates via an RNA intermediate.
mRNA Production:
Host RNA polymerase transcribes viral dsDNA into mRNA. Reverse transcriptase converts mRNA back into DNA for new virions for infection.
Replication: Uses reverse transcriptase to return to DNA form.
What is baltimore Class II form of genome, mRNA production, and replication?
Genome: ssDNA
mRNA Production: by itself creates mRNA(-) so it is converted into dsDNA intermediate replicative form by host DNA polymerase, then transcribed into mRNA (+)
Replication: Uses host DNA polymerase on replicative dsDNA.
What is baltimore Class III form of genome, mRNA production, and replication?
Genome: dsRNA
mRNA Production:
Viral RNA-dependent RNA polymerase (replicase) transcribes the (-) strand of the two strands into mRNA(+).
Replication: Uses RNA replicase to copy RNA.
What is baltimore Class IV form of genome, mRNA production, and replication?
Genome: (+) ssRNA (acts as mRNA directly).
mRNA Production:
No transcription needed—ribosomes translate it immediately.
Replication: Uses RNA replicase to make a (-) RNA template, then more (+) RNA copies.
What is baltimore Class V form of genome, mRNA production, and replication?
Genome: (-) ssRNA (complementary to mRNA).
mRNA Production:
RNA replicase transcribes (-) RNA into (+) mRNA before translation.
Replication: Uses RNA Replicase to copy RNA.
What is baltimore Class VI form of genome, mRNA production, and replication?
Genome: (+) ssRNA but not used directly as mRNA.
mRNA Production:
Reverse transcriptase converts RNA to dsDNA. Host RNA polymerase transcribes viral DNA into mRNA.
Replication: Uses reverse transcriptase to turn to dsDNA and integrates into host genome.
What are bacteriophages?
Bacterial viruses intensively studied a model systems for the molecular biology and genetics of virus replication.
What is phage ɸX174? How does it work?
Parvovirus; Icosahedral Baltimore class II ssDNA (+) bacteriophage that binds specifically to the Lipopolysaccharides of E. coli and converted into double stranded replicative form by host enzymes.
What is the condition of overlapping genes?
When a phage genome has insufficient DNA to encode all viral specific proteins, parts of the genome are transcribed in more than one reading frame with different promoters.
How does phage ɸX174 undergo rolling circle replication?
- E. coli host’s primosome and DNA pol III synthesizes the (-) complementary strand to the (+)ssDNA, converting the genome into its double stranded replicative form.
- Viral A protein acts as endonuclease and “nicks” at the origin of replication in (+) strand of circular dsDNA, letting 3’ end act as a primer for rolling circle replication.
- (+) strand synthesis is initiated as dNTP’s are added to 3’ end using exposed (-) strand as template as 5’ peels away.
- Rotation of circle results in linear copy of (+) strand
- Viral A protein cuts out old (+) ssDNA and ligates ends of newly synthesized (+) ssDNA to make circular (+) genome.
- (+/-) dsDNA replicative form is ready for another round of replication, and there is an accumulation of (+) strand ssDNA genome copies to be packaged into capsid by single stranded DNA binding proteins.
How does E. coli host enzymes convert phage ɸX174 genome into the replicative form?
Secondary structure ssDNA folding allow recruiting primosome (helicase and primase) to lay down a primer.
What is a plaque?
a visible clearing or zone of bacterial lysis (cell death) on a bacterial lawn (a dense growth of bacteria) caused by the infection and replication of bacteriophages (viruses that infect bacteria)
What are the steps in a plaque assay?
- Mixture of top agar, bacterial cells and diluted phage suspension is poured onto a solidified nutrient agar plate
- Micture is left to solidify
- Incubation allows for lawn of bacterial growth and phage replication, creating plaques
What is Phage M13?
Filamentous ssDNA(+) virus with helical symmetry attaching to the pilus of its host cell. It replicates similarly to that of ɸX174 phage, but is released from host cell without cell undergoing lysis.
What are the stages in the Phage M13 life cycle?
- phage attaches to pilus of recipient cell allowing ssDNA(+) entry into the cell
- Host enzymes convert (+)ssDNA into double stranded replicative form to create the complementary (-)ssDNA for (+)mRNA transcription.
- Rolling replication occurs, creating new (+)ssDNA and peeling off the old (+)ssDNA. pV starts accumulating.
- Once enough pV proteins are present, they bind to displaced (+)ssDNA strands as they peel off, preventing it from being used as a template for (-) strand synthesis and marking it for phage assembly.
- pV coated (+)ssDNA is delivered to the phage assembly site at inner membrane in periplasm.
- pVIII replaces pV and coats the ssDNA forming the long filamentous structure
- pIII and pVI dock onto the assembling phage at the inner membrane.
- PIII releases particle from inner membrane and slides it out of outer membrane secretin protein (pIV) and out of the cell without killing the host.
What is steady state infection?
Also known as chronic infection: when a pathogen persists in the host cell for a significant period, continuously replicating and releasing progeny, without causing immediate death of the infected cells
What is the structure of the T4 Phage?
- Icosahedral head: capsid holding170 kbp dsDNA
- tail: helical tube surrounded by contractile sheath
- contractile sheath: contracts during infection, pushing the tail core through the bacterial membrane
- base plate and tail fibers/pins: recognize and attach to lipopolysaccharides
What is the T4 phage’s mode of reproduction?
Virulent: actively reproduce inside host cell
Lytic: reproduction leads to lysis of host cell
What are the stages in the lifecycle of the T4 phage?
- adsorption to cell surface
- T4’s genome injected at 0 minutes
- Transcription of T4 early genes begins <2min
- host chromosome is degraded at 4 min
- T4 genome replication begins at 5 min
- translation of late genes begins at 8 minutes
- Thousands of capsid proteins have been made by 13 min
- 15 minutes - viral parts are assembled, proheads filled
- 20 mins - first complete virions are formed
- 25 mins- T4 lysozyme has been produced, and cell lyses with 100-300 virions released
What are the stages of T4 infection?
- Adsorption: tail fibers contact LPS of E. coli cell on a phage receptor
- Attachment: base plate and tail pins contact surface of outer membrane
- Penetration: contraction of the tail sheath and injection of T4 dsDNA genome
What is the mechanism of injection of the genome?
- Tail enzyme (T4 lysozyme) degrades peptidoglycan layer signalling the sheath proteins to contract.
- 24 rings of 6 proteins have bound ATP hydrolyzed, turning into 12 rings of of 12 proteins.
- Contraction drives rigid tail through the bace plate and degraded cell wall, injecting genome.
What happens in the minute following T4’s ssDNA entering the cytoplasm?
Synthesis of host DNA and RNA ceases and transcription of early, middle and late phage genes begin.
Which early genes are transcribed/translated in the first 8 minutes of T4 genome injection?
- EARLY phage gene promoters: recognized by E.coli σ70/RNA polymerase
- T4 nuclease: Stops E. coli gene expression, provides nucleotides for synthesis of phage genome
- Enzymes for synthesis and glucosylation of unusual T4 base, 5-hydroxymethylcytosine (HMC)
- T4 replisome (DNA polymerase, primase, helicase, clamp, clamp loader, topoisomerase, ligase, etc.)
- Proteins that modify host RNA polymerase to recognize phage -30 box promoters of MIDDLE genes
What is the purpose of the synthesis and glucosylation of hydroxymethylcytosine?
Protects from T4 nuclease activity and host restrction endonucleases (degrade foreign DNA)
How long does T4 phage replication take?
Begins 5 minutes after infection and continues until halfway through late phase of gene expression
Which middle and late genes are transcribed/translated AFTER the first 8 minutes of T4 genome injection?
- RNA-polymerase modifying proteins for recognition of late gene promoters (including T4-encoded sigma factor)
- Structural proteins (capsid, tail, sheath)
- packaging motor proteins
- T4 lysozyme for degradation of peptidoglycan and eventual lysis of cell
What are the steps of the T4 phage replication?
- Helicase/primase complex is loaded at multiple origins
- Primers added by T4 primase
- Replication by T4 DNA polymerase
- Primer is degraded by T4 exonuclease, resulting in 5’ gap
- Redundant 3’ ends are complementary and line up to form BP, joined by T4 ligase to form concatemers.
- Contactemers are cut by T4 endonuclease at no specific sequence for packaging.
How are the linear segments of cut concatemers sized?
Headful packaging: sized to fit the phage head
Circularly permutated: Linear segment has full T4 genome (rearranged) plus a little extra redundant sequences at terminal ends
What are the steps in the assembly of T4 virions?
- Baseplate and tube assembly: base plate, tail pins, helical tube, sheath proteins
- Capsid assembly: proteins aggregate to form a prohead, end of concatemer is cut and begins to fill the prohead via host ATP-driven packaging motor
- Tail fibre assembly: tail proteins assemble fibres, and are added to mature tail as last step before maturation.
What is the structure of the T7 phage?
- small
- short tailed phage
- icosahedral head with 40kbp dsDNA genome
What are elements of the reproduction type of the T7 phage?
- virulent and lytic
- encodes its own T7 RNA polymerase that only recognizes T7 gene promoters
- replicates linear genome at one origin, bidirectionally
- packaging of genomes is achieved by specific cutting by T7 endonuclease
What are the steps of T7 phage’s bidirectional replication?
- replication starts at single origin near one end of DNA
- Two replilcation forks form and move in opposite directions, forming an “eye” form
- One side of the DNA will replicate until the short end, while the other will continue to replicate down the long end, forming the “Y” form.
- Replication will reach the end of the long end, resulting in completed strands with unreplicated terminal repeats.
- Unreplicated terminal repeats will pair, and its own DNA polymerase and DNA ligase will fill in the gaps, resulting a concatemer.
How is the T7 phage genome packaged?
T7 has a cutting enzme that makes single stranded cuts at exact positions on the concatemer, resulting in an overhang on each strand. DNA polymerase fills in the gap under the overhang, and T7 terminase enzyme feeds DNA into the capsid via ATP-powered translocation.
What is the difference between a virulent and a temperate phage?
Virulent: once infection begins, it will lyse and kill its host (e.g. T4)
Temperate: Can choose to take the lysogeny or lytic pathway (e.g. dsDNA ƛ phage)
What is the structure of the ƛ phage?
Linear dsDNA phage with an icosahedral head holding 47 kbp (~40 genes) and a tail tip that binds to LamB porin protein involved in maltose transport
What is the lytic pathway of a temperate phage?
- Pathway occurs immediately upon infection of the host or excision of the temperate phage from the host chromosome
- Viral genome is replicated, transcribed, and translated
- Synthesis of new viral particles
- Host cell lyses, releasing virions.
What is the lysogenic pathway of a temperate phage?
- Begins when temperate phage integrates into the host chromosome, becoming a prophage. The cell that harbours the virus is a lysogen.
- Integrated viral genome may cause lysogenic conversion.
- Very few genes are transcribed to maintain the dormant state
- Virus genome is replicated in synchrony with host chromosome and passed to daughter cells at cell division
How was lysogeny discovered?
Forms turbid plaques (fried egg appearance) on petri dishes with tiny colonies of bacteria growing, despite being surrounded by billions of bacteriophage.
What is lysogenic conversion?
Prophage induces phenotypic change in host cell, through changes to surface structure and toxin production to cause disease.
What is an example of changes to surface structure via lysogenic conversion?
ε prophage of Salmonella modifies LPS structure so that other ε phages can’t attach and infect the same cell again (superinfection immunity)
What are examples toxin production via lysogenic conversion?
Corynebacterium diphtheriae: diphtheria toxin is encoded on a prophage
Streptococcus pyogenes: reddening toxin (scarlet fever) is encoded on prophage
What are unusual properties of the lysogenic state?
- Immune to superinfection and cannot be infected by the same phage (but can be infected by other phages)
- spontaneous induction of prophage: during growth, some cells lyse and release normal burst size of phages
- Cell stress conditions can induce entire population to burst and release phages.
What are factors that promote induction of a prophage?
Decision for a temperate phage to go to lytic pathway is based on environmental conditions. Cell stress conditions can induce the excision of the prophage and proceeding to lytic pathway.
How is the linear genome of the ƛ phage circularized?
12 nucelotide segment (cos site) at 3’ end of each strand is single stranded and unpaired. Circularization occurs when there is base pairing between the cos sites, and DNA ligase seals nicks to form circular dsDNA replicative form. At this point, can choose to proceed with lytic or lysogenic pathway.
What are the three classes of genes on the ƛ phage genome.
Immediate early, immediate delayed, late.
How does the ƛ phage genome get transcribed?
Host RNA polymerase holoenzyme trnascribes ƛ genes bi-directionally from two immediate promoters: PR (rightward) and PL (leftward), and is controlled by two operators: OR (controls PR) and OL (controls PL)
What is the immediate mechanism following infection and circularization?
- PL and PR are immediately activated
- RNA polymerase moves rightward from PR to transcribe cro gene, and stops at an immediate early termination signal
- RNA polymerase moves leftward to transcribe N gene, and stops at early termination signal
- N mRNA is translated to anti-terminator N protein which modifies host RNA polymerase so that it can bypass immediate early termination signals
- Transcription proceeds. PR transcribes cro and subsequently cII (decision to lytic or lysogenic). PL transcribes N gene and subsequently CIII gene (complexes with CII to protect from proteolysis, mediated by E. coli proteases)
- Transcription continues until the delayed early terminator signals are encountered.
How is Lytic versus Lysogeny pathway chosen in temperate phages based on CII levels?
Decision is dependent on levels of cII protein. High CII = lysogenic, low CII = lytic.
What are conditions that promote lytic and lysogeny?
- nutrient conditions: high number of proteases = lytic, lower number of proteases = lysogenic
- Multiplicity of infection: high MOI promotes lysogeny (multiple copies of CII and CIII genes, sufficient
What does CII do?
- Activator of PRE, which transcribes LEFTWARD through region encoding cro and cI. Antisense cro transcript base pairs with cro mRNA to prevent translation (and thus lysis) and cI gene produces CI protein (prevents transcription of PR, and thus lysis)
- directs transcirption of integrase gene from PI
What does CI do?
Binds 3 sites each within OR and OL and
, stops transcription from Pr and PL, repressing promoters before delayed early phase is completed, outcompeting Cro. Translation of delayed early proteins is stunted and lytic cycle is aborted.
How is the lambda phage genome inserted in the host genome?
- Integrase enzyme (Int) binds to the attP site on the lambda genome (opposite cos sites) and the attB site on the E. coli genome between galactose and biotin operons
- attP and attB base pair due to sequence similarity (homology)
- Integrase cleaves phage and chromosomal strands via endonuclease function
- Phage genome is ligated and integrated as a prophage into nonessential region between galactose and biotin operons, permuting genes (changing order of lambda genes)
What are the two stages of the λ phage replication?
- After linear dsDNA is circularized upon entry into the cell, it is replicated by host machinery similar to bi-directional replication to produce faithful circularized copies.
- Once sufficient lambda DNA has been replicated, the replication process switches to rolling circle replication.
What are the two proteins used in the replication of the circular form of lambda?
DnaO: binds to the origin of replication on the circular lambda DNA similar to that of DnaA.
DnaC: recruits host DnaB (helicase) functionally similar to DnaC in E. coli.
What is the rolling circle replication of lambda following circularization?
- One strand of dsDNA is nicked. 3’ end acts as primer for continuous DNA synthesis with the intact strand as template.
- 5’ end is displaced and is unwound and host machinery makes okazaki fragments.
- Phage endonuclease cleaves the peeling 5’ end, generating cohesive ends m and m’. These concatemers are assembled into virions.
What occurs in the late phase of infection?
- High amounts of Cro protein accumulate
- Q protein has accumulated: activates PR and allows rightward transcription of late genes (proteins for capsid, tail, etc)
- Cell proceeds towards lysis and phage genome is synthesized and assembled with capsid proteins to form virions
What is the Cro protein’s role in the decision to go lytic or lysogenic?
Cro binds to the operator sites (OL and OR regions), blocking the transcription from PR and PL. This promotes lysis by preventing the transcription of the cI gene (which encodes the lambda repressor protein that maintains lysogenic state).
What is the Q protein’s role in the decision to go lytic or lysogenic?
Protein acts as transcriptional antiterminator, by bypassing termination of transcription in the region of the genome where the late genes are located (those involved in phage assembly and lysis of the host cell)
When is the decision made to go lytic or lysogenic?
During the delayed early phase when:
- some circular replicative forms are present
- Cro has not yet repressed early promoters PR and PL.
- Q protein has not yet enabled transcription of late genes.
What can CII and CIII activate upon accumulation?
Transcription from:
- PRE (CI synthesis - prevents expression of genes that trigger lytic cycle)
- PAQ (antisense to Q transcripts, blocking translation of virion parts)
- PI (integrase synthesis)
What happens when CI binds to OR?
Represses PR,
which inhibits transcription of lytic genes and shifts the expression of cI from PR to PRM, the promoter that produces cI at lower levels to maintain the lysogenic state.
What are the steps in decision making for lytic and lysogenic fates?
- Transcription begins at PR and PL: early proteins are made
- N protein extends trnascription from PR and PL: delayed early proteins are made
- A) If CII and CIII accumulate faster, PRE is activated, CI is produced, PRM is activated to continue CI production and phage remains in lysogeny
- B) If Cro and Q accumulate faster, Cro stops the early phase Q activates PR and starts the late phase, and phage undergoes virulent and lytic reproduction
What is MS2’s structure and function?
Structure: Very small ~25nm viruses, icosahderal, linear (+) ssRNA genome of 4 genes, 3.5kb.
Function: attach to lateral edge of F-pilus via maturation protein to inject ssRNA
What are the proteins that the MS2 phage translates to?
- Maturation protein
- Coat protein
- Lysis protein
- RNA replicase subunit
How does the small MS2 ssRNA regulate the synthesis of its viral proteins?
Genome folds into a complex secondary structure that controls access to the 4 AUG start codons. Early in infection, most accessible AUG codons are replicase and coat protein.
How is the MS2 replicase activated?
Non-functional, must combine with host ribosomal protein S1 and elongation factors EF-Tu and EF-Ts to be functional. When functional, acts as RNA-dependent RNA polymerase.
What does the activated MS2 replicase do? What is the function of the product?
Activated replicase synthesizes complementary ssRNA(-) from the ssRNA(+) genome. From there, it uses ssRNA(-) as a template to make more ssRNA(+). Resulting ssRNA replicates can be used as templates for synthesis of more replicative forms, mRNA for synthesis of viral proteins, or complete genomes to be incorporated into capsids.
What do the coat proteins of MS2 do?
When coat proteins accumulate, they bind to RNA and change secondary structure, hiding replicase start site, and revealing maturation and lysis start codons.
What does the maturation protein of MS2 do?
Bind to genomes, initiate assembly of coat proteins into capsids to form nucleocapsid. Nucleocapsid consists of 1 (+)ssRNA genome, 1 maturation protein, and many coat proteins.
What does the lysis protein of MS2 do?
Translated at the end of replication cycle, results in lysis of cell with burst size of 1000-2000 virions per cell.
