Data Loading...
Viruses VIRUSES Viruses are small packages of genes Flipbook PDF
Viruses Heyer 3 Many cell divisions produce a large population of bacteria infected with the prophage. The bacterium rep
127 Views
55 Downloads
FLIP PDF 1.44MB
Viruses
Viruses are small packages of genes
VIRUSES
Consist of protein coat around nucleic acids (DNA or RNA)
Viruses measured in nanometers (nm). Require electron microscopy.
Obligate intracellular parasites
Basic virus structure
• Cannot grow or reproduce by itself – Have no independent metabolic pathways for anabolic synthesis
• Reproduce (replicate) only by using host cell machinery • Non-cellular • (Is it alive?)
Figure 18.1
0.5 mm
Classification of Viruses
1. Nucleic Acid: DNA or RNA (not both) 2. Protein coat (capsid): usually helical-columnar or polyhedral.
Enveloped Viruses
Based upon • Morphology of capsid • Presence of an envelope around the capsid • Type of nucleic acid – DNA or RNA – Double-stranded (ds) or single-stranded (ss) – Circular or linear – (Most are dsDNA [in bacteria] or ssRNA [in plants & animals]) Envelope derived from host cell membrane + viral protein spikes.
Heyer
1
Viruses
Capsomere of capsid
Classification of Viruses RNA
Membranous Head envelope Tail Capsid sheath RNA Tail fiber
DNA
Capsomere
Glycoprotein 18 ¥ 250 nm 70–90 nm
Glycoprotein 80–200 nm
Classification of Viruses DNA *
80 ¥ 225 nm
*
20 nm
50 nm (c) Influenza viruses: enveloped helical RNA-virus
50 nm
(a) Tobacco mosaic (b) Adenoviruses: virus: helical polyhedral RNA-virus DNA-virus
50 nm (d) Bacteriophage: complex-polyhedral DNA-virus
* IV. =“ssRNA(+)” * V. =“ssRNA(–)”
Figure 18.4. Colorized TEMs
General viral reproductive cycle • Viruses use enzymes, ribosomes, nucleotides, amino acids, etc. of host cells to synthesize progeny viruses Entry into cell and uncoating of DNA
DNA Capsid
its tail fibers to bind to specific receptor sites on the outer surface of an E. coli cell.
5 Release. The phage directs production
Viral DNA
Transcription
2 Entry of phage DNA
and degradation of host DNA. The sheath of the tail contracts, injecting the phage DNA into the cell and leaving an empty capsid outside. The cell’s DNA is hydrolyzed.
of an enzyme that damages the bacterial cell wall, allowing fluid to enter. The cell swells and finally bursts, releasing 100 to 200 phage particles.
Translation
Phage assembly
mRNA Viral DNA
1 Attachment. The T4 phage uses
VIRUS
HOST CELL
Replication
The Lytic Cycle (virulent phage) • Bacteriophage T2 or T4
Capsid proteins
a DNA virus 4 Assembly. Three separate sets of
Figure 18.5
Self-assembly of new virus particles and their exit from cell
Head
Tails Tail fibers
proteins self-assemble to form phage heads, tails, and tail fibers. The phage genome is packaged inside the capsid as the head forms.
Figure 18.6
One-step Growth Curve
3 Synthesis of viral genomes
and proteins. The phage DNA directs production of phage proteins and copies of the phage genome by host enzymes, using components within the cell.
The Lytic Cycle • Bacteriophage
• Agar plate with “bacterial lawn” (solid white field of bacteria)
Attachment to lysis in 25 minutes!
Heyer
• Plaques: clear, bacteria-free region growing around one original viralinfected bacterium
2
Viruses
Lytic Cycles and the Transduction of Bacterial Hosts
Lytic / Lysogenic Cycles (temperate phage)
Phage DNA
• Bacteriophage l The phage attaches to a host cell and injects its DNA. Phage DNA circularizes
Phage
Bacterial chromosome (not degraded)
Lytic cycle The cell lyses, releasing phages.
Figure 18.7
Occasionally, a prophage exits the bacterial chromosome, initiating a lytic cycle.
Many cell divisions produce a large population of bacteria infected with the prophage.
Lysogenic cycle
The bacterium reproduces Certain factors normally, copying the determine whether prophage and transmitting it to Lysogenic cycle Lytic cycle is induced or is entered Prophage daughter cells.
New phage DNA and proteins are synthesized and assembled into phages.
Phage DNA integrates into the bacterial chromosome, becoming a prophage.
Figure 18.16
Enveloped RNA-virus structure •Influenza virus
Enveloped ssRNA(–) virus cycle
• Coronavirus (SARS) Capsid
1 Glycoproteins on the viral envelope bind to specific receptor
molecules(not shown) on the host cell, promoting viral entry into the cell.
RNA
• Envelope membrane
Envelope (with glycoproteins)
2 Capsid and viral genome enter cell
• 8 RNA segments • 10 proteins
HOST CELL
Template
strands also function as mRNA, which is translated into both capsid proteins (in the cytosol) and glycoproteins for the viral envelope (in the ER).
mRNA ER
6 Vesicles transport envelope glycoproteins to the plasma membrane.
Figure 18.8
Enveloped RNA-virus cycle
Capsid proteins Copy of genome (RNA)
Glycoproteins
A capsid assembles around each viral genome molecule.
8 New virus
Budding off new viruses without cell lysis.
RNA-virus cycles ssRNA (+) viruses
ssRNA (–) viruses
•
•
The (–) strand cannot act as an mRNA
•
The virus must provide some RNA-dependent RNA polymerase (RDRP)
•
RDRP replicates (–) strand to produce (+) strands
•
the (+) strands are templates to translate viral proteins and for RDRP to replicate (–) strands
•
Genomic (–) strands & viral proteins assembled into new viruses
•
E.g., rabies virus & influenza virus
•
The (+) strand acts as an mRNA
Including RNA-dependent RNA polymerase (RDRP) – Uses RNA as a template to replicate more RNA
•
Heyer
4 New copies of viral genome RNA are made using complementary RNA strands as templates.
7
– Can be translated by host cell ribosomes to make viral proteins
Budding off new viruses without cell lysis.
3 The viral genome (red) functions as a template for synthesis of complementary RNA strands (pink) by a viral enzyme (RNA-dependent RNA polymerase).
Viral genome (RNA)
5 Complementary RNA
RDRP replicates (+) strand to produce complementary (–) strands, and then the (–) strands to make more (+) strands
•
↑↑ (+) strands Æ more viral protein & genome for newly assembled viruses
•
E.g., poliovirus & cold virus
– Cannot be translated
3
Viruses
HIV
Membrane of white blood cell
The Retrovirus cycle
1 The virus fuses with the cell’s plasma membrane. The capsid proteins are removed, releasing the viral proteins and RNA.
• HIV
2 Reverse transcriptase catalyzes the synthesis of a DNA strand complementary to the viral RNA.
Reverse transcriptase
HOST CELL Viral RNA
0.25 µm
HIV entering a cell
RNA-DNA hybrid
HIV integrates vDNA into host chromosome
3 Reverse transcriptase catalyzes the synthesis of a second DNA strand complementary to the first. 4 The double-stranded DNA is incorporated as a provirus into the cell’s DNA.
DNA NUCLEUS
Provirus
Chromosomal DNA RNA genome for the next viral generation
mRNA
5 Proviral genes are transcribed into RNA molecules, which serve as genomes for the next viral generation and as mRNAs for translation into viral proteins. 6 The viral proteins include capsid proteins and reverse transcriptase (made in the cytosol) and envelope glycoproteins (made in the ER).
New HIV leaving a cell
Figure 18.10
8 Capsids are assembled around viral genomes and reverse transcriptase molecules.
9 New viruses bud off from the host cell.
7
Vesicles transport the glycoproteins from the ER to the cell’s plasma membrane.
Viroids
Human Immunodeficiency Virus (HIV)
Addenda to the Central Dogma!
• Small loop of RNA with no capsid. – Infect plant cells.
• 1950s: All known life forms use the same mechanism to produce protein • 1970s: RNA viruses use RNA to make RNA! • 1990s: Retroviruses use RNA to make DNA!!
Viral-caused pathology
Nucleoside Analogs as therapeutic inhibitors of viral polymerases
• Cell lysis & death • Release of intracellular degradation enzymes into surrounding tissues. • Toxic viral proteins • Disruption of cellular function – Diminished vital functions – Production of toxic metabolites •E.g., Acyclovir, AZT, 3TC, DDI, DDC
Heyer
4
Bacterial Genetics
Escherichia coli
~ 4.6 million base pairs; ~4300 genes
Escherichia coli
• Extra-chromosomal DNA • Plasmid genes are not necessary for the usual survival of the bacterium
Gene Expression
Escherichia coli
RIBOSOMES • Make up major part of cytoplasm • 15,000+ per cell • Smaller and with different protein/ rRNA composition than in eukaryotes.
Heyer
Binary fission (asexual reproduction)
Gene Expression
1
Bacterial Genetics
Replication of the circular bacterial chromosome or plasmid
Binary Fission
Replication fork
Origin of replication
Termination of replication
Figure 18.14
Exponential Growth
• Population multiplies by a constant factor when growth rate not limited by resources. • In vitro doubling time = 20 minutes • In situ doubling time = 12 hours
HOW BACTERIA GAIN NEW GENETIC INFORMATION
Recombinant reproduction
Binary fission (asexual reproduction)
Gene Expression
Mutation Rates • # of mutations per cell generation
• MUTATIONS • RECOMBINATION – TRANSDUCTION – TRANSFORMATION – CONJUGATION
• TRANSPOSITION
Heyer
– Higher in bacteria than in animal cells • Lack level of repair mechanisms
– Yet only 1 mutation out of 107 to 108 replicated genes
• But most cell cultures contain about 109 cells per ml – and the average bacterial chromosome is about 4000 genes
∴ Each ml contains ~40,000 mutations that were not there one generation before ∴ Each ml contains ~10 cells with a mutation in any specific gene
2
Bacterial Genetics
HOW BACTERIA GAIN NEW GENETIC INFORMATION
HOW BACTERIA GAIN NEW GENETIC INFORMATION
Mutations vs. Recombination
Mutations vs. Recombination
EXPERIMENT Researchers had two mutant strains, one that could make arginine
but not tryptophan (arg+ trp–) and one that could make tryptophan but not arginine (arg- trp+). Each mutant strain and a mixture of both strains were grown in a liquid medium containing all the required amino acids. Samples from each liquid culture were spread on plates containing a solution of glucose and inorganic salts (minimal medium), solidified with agar. Only bacteria that can make both amino acids should survive on this minimal medium.
Mutant strain arg+ trp–
Mutant strain arg- trp+
Only the samples from the mixed culture, contained cells RESULTS that gave rise to colonies on minimal medium, which lacks amino acids. Mixture
Mutant strain arg+ trp– No colonies (control) CONCLUSIONS
Mutant strain arg- trp+ No colonies (control)
Colonies grew
Figure 18.15
1. Despite
all that spontaneous mutation, neither mutant strain re-acquired the ability to synthesize amino acids.
2. Thus,
Mixture
Figure 18.15
each cell from the mixture that formed a colony on the minimal medium must have acquired one or more genes from a cell of the other strain by genetic recombination.
Genetic Recombination
HOW BACTERIA GAIN NEW GENETIC INFORMATION • MUTATIONS • RECOMBINATION
Partial diploid
– TRANSDUCTION – TRANSFORMATION – CONJUGATION
• TRANSPOSITION
Genetic Recombination
Donor DNA fragment 5’ 5’
Recipient chromosomal DNA
Bacterial Recombination • Donor DNA is nicked and digested to form single strand end
Crossing over
• Exchange of genes between two DNA molecules
• Strand invasion: Recombination protein complex • binds donor ssDNA to recipient dsDNA • unzips recipient dsDNA • complementary recipient strand is displaced [D-loop] • homologous donor and recipient DNA complementary pair • Branch migration: additional donor DNA displaces more D-loop • Termination: • D-loop excised or degraded • DNA-polymerase fills in gaps • Ligase joins ends
Heyer
3
Bacterial Genetics
Bacterial Recombination
Non-homologous DNA may be recombined if it is flanked by homologous regions of DNA
HOW BACTERIA GAIN NEW GENETIC INFORMATION • MUTATIONS • RECOMBINATION – TRANSDUCTION – TRANSFORMATION – CONJUGATION
• TRANSPOSITION
TRANSDUCTION
Phage DNA
TRANSFORMATION
A+ B+
— GENERALIZED 1 Phage infects bacterial cell that has alleles A+ and B+
A+ B+ 2 Host DNA (brown) is fragmented, and phage DNA
Donor cell
and proteins are made. This is the donor cell.
3 A bacterial DNA fragment (in this case a fragment with
the A+ allele) may be packaged in a phage capsid. 4 Phage with the
A+
A+
allele from the donor cell infects a recipient A–B– cell, and crossing over (recombination) between donor DNA (brown) and recipient DNA (green) occurs at two places (dotted lines).
Crossing over A+ A– B– Recipient cell
+ – 5 The genotype of the resulting recombinant cell (A B )
differs from the genotypes of both the donor the recipient (A–B–).
(A+B+)
and
A+ B– Recombinant cell
Figure 18.16
TRANSFORMATION •
Cells after death, release DNA –
•
– –
• •
Heyer
Only in certain stage of cell cycle Near completion of cell wall synthesis
Competent cells-receptor sites on cell wall and membrane Release competence factor that helps in uptake – – –
• •
Naked DNA in solution
Cells may take up DNA
Alters cell wall Makes it more permeable to DNA E. coli needs to be treated to be competent
Transduction & Transformation • In vivo, restricted to recombination from closely related strains of bacteria – Transducing virus must bind and infect both cells. – Transforming DNA must be recognized by cellsurface receptors. – Donor cell DNA must be homologous to recipient cell DNA for crossing-over.
Endonucleases cut DNA into small pieces Recombination occurs between donor & recipientcomplementary base pairing
4
Bacterial Genetics
Plasmids Extra chromosomal DNA • High copy number: many copies per cell • Low copy number: expression inhibits its own replication
Plasmids grouped by transmissibility:
• Non-transmissible
•
Conjugative — can cause donor cell to initiate contact with recipient cells –
•
F plasmid Conjugation
Carries genes for sex pili and for rolling replication
Mobilizable — can prepare plasmid DNA for transfer in concert with conjugative plasmid
“Rolling Replication” of plasmid DNA
Single-stranded-DNA-binding proteins displace 5’ end.
• Donors with F plasmids (F+ cells) transfer plasmid to recipient cells (F-) which become F+
F plasmid Conjugation
• Plasmid codes for proteins allowing insertion into chromosomal DNA
• If plasmid integrates into the chromosome, it converts cell to Hfr cell (high frequency of recombination)
Hfr cell Conjugation
• F factor inserted into Hfr chromosome can cause plasmid-like “rolling replication” of large portions of donor chromosomal DNA into recipient cell. • More DNA that may be recombined into recipient genome. • Hence “High frequency of recombination”!
Heyer
5
Bacterial Genetics
Extra chromosomal DNA
•
Carries genes for sex pili (tra genes), rolling replica