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Evolu&onLecture 22
Biology W3310/4310Virology
Spring 2013Anything produced by evolu3on is bound to be a bit of a mess.SYDNEY BRENNER
Around here, it takes all the running you can do just to stay in the same place.LEWIS CARROLLAlice in Wonderland
1
• Where did viruses come from?
• Where are they going?
• How are they geLng there?
• How would we know?
Today’s ques&ons
2
Modern virology has provided a window on the mechanisms of evolu&on
• As host populaNons grow and adapt, virus populaNons are selected that can infect them-‐ New viral popula-ons emerge every day
• It also works the other way-‐ Viral popula-ons can be significant selec-ve forces in the
evolu-on of host popula-ons
• If a host populaNon cannot adapt to a lethal virus infecNon, the populaNon may be exterminated
3
The public is constantly confronted with the reality of viral evolu&on (even if they don’t
believe in evolu&on)
• New viral diseases: AIDS, West Nile virus in the US, hepaNNs C, Ebola, Marburg...
• Regular bouts every year with influenza and common cold viruses
• Drug resistant HIV
Simple fact: viruses evolve faster than many can comprehend
4
Viral evolu&on: The constant change of a viral popula&on in the face of selec&on pressures
• If the populaNon can’t change or adapt, it disappears
• Viral populaNons display spectacular diversity -‐ why they are successful
• Sources of diversity:-‐ Muta-on
-‐ Recombina-on
5
• Virus evoluNon is defined in terms of a populaNon, not an individual virus parNcle
• Viral populaNons comprise diverse arrays of mutants that are produced in prodigious quanNNes
• It is misleading to think of an individual parNcle as represenNng an ‘average’ virion for that populaNon -‐ virologists are populaNon biologists whether they know it or not
Viral evolu&on
6
When it comes to viral evolu&on, the median is not the message (Stephen Jay Gould)
• Viral populaNon diversity provides surprising avenues for survival, yet every individual is a potenNal winner
• SomeNmes even the most rare genotype in a populaNon may be the most common a]er a single selecNve event
7
Four main drivers of virus evolu&on
• Large numbers of progeny
• Large numbers of mutants
• Quasi-‐species effects
• SelecNon
8
Virus-‐infected cells produce large numbers of progeny
The interface of host defense and virus replicaNon is ferNle ground for selecNon and evoluNon
Principles of Virology, ASM Press
9
Replica&ng viruses produce large numbers of mutant genomes
• EvoluNon is possible only when mutaNons occur in a populaNon
• MutaNons are produced during copying of any nucleic acid molecule
10
• The masters of error-‐prone replicaNon: RNA polymerases cannot correct errors
• Average error frequency: 1 in 104 or 105 nucleoNdes polymerized
• In a 10 kb RNA virus genome, a mutaNon frequency of 1 in 104 results in about 1 mutaNon per genome
RNA viruses
11
DNA viruses
• Different lifestyle from most RNA viruses
-‐ Narrow host range
-‐ Persistent infecNons common
• Genome replicaNon not as error prone as RNA viruses
• Proofreading
• Most DNA viruses generate less diversity, evolve slower than RNA viruses
12
The quasispecies concept
• Analysis of an RNA bacteriophage populaNon (Qβ):“A Qβ phage populaNon is in a dynamic equilibrium with viral mutants arising at a high rate on the one hand, and being strongly selected against on the other. The genome of Qβ cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences.” E. Domingo, D. Sabo, T. Taniguchi, C. Weissmann. 1978. NucleoNde sequence heterogenity of an RNA phage populaNon. Cell 13:735-‐744.
• This discovery was far ahead of its Nme, not appreciated by most virologists
• Virus populaNons exist as dynamic distribuNons of nonidenNcal but related replicons, called quasispecies
13
Viral quasispecies
Principles of Virology, ASM Press
14
• Most viral infecNons are iniNated not by a single virion, but rather by a populaNon of parNcles
• The large number of progeny produced are complex products of intense selecNve forces inside the host
• The survivors that can re-‐infect a new host reflect the intense selecNve forces outside the host
• When small populaNons of virus parNcles replicate (as in laboratories), extreme fluctuaNons in genotype and phenotype are possible
Quasispecies effects
15
• For a given RNA virus populaNon, the genome sequences cluster around a consensus or average sequence, but virtually every genome can be different from every other
• It is unlikely that a genome with the consensus sequence is actually replicaNng in the populaNon
The myth of consensus genome sequences
16
Selec&on
• SelecNon favors both the creaNon of diversity and single dominant mutaNons-‐ Counterintui-ve -‐ the drive for survival must result in diverse
popula-ons as well as selected muta-ons
17
• Survival of the fi?est: A rare genome with a parNcular mutaNon may survive a selecNon event, and this mutaNon will be found in all progeny genomes
• Survival of the survivors: However, the linked, but unselected mutaNons, get a free ride
• Consequently, the product of selecNon a]er replicaNon is a new, diverse populaNon that shares only the selected mutaNons
Selec&on
18
Selec&on for survival inside a host
• At the end stage of AIDS, the billions of virions that survive are T-‐cell tropic, engage CXCr4 receptor
• When infecNon is passed to a new host, the iniNal replicaNng genomes are M-‐tropic, engage CCr5 receptor
• Progeny genomes have passed through a booleneck, only a few pass on
• Virions that ulNmately devastate the immune system are not the most fit for infecNon of new hosts
19
• Increased mutaNon rates are selected during virus evoluNon: mutaNon is good for viral populaNons
• It is possible to isolate mutaNons in viral polymerases that reduce the frequency of incorporaNon errors
• What was learned from the study of these mutants
-‐ They do not have a selecNve advantage when wild type and anN-‐mutators are propagated together
-‐ Lower rates are neither advantageous nor selected in nature
-‐ The mutants are o]en less pathogenic
• High mutaNon rates are a posiNve force in virus evoluNonhop://www.virology.ws/2009/05/15/increased-‐fidelity-‐reduces-‐viral-‐fitness/
Diversity is selected
20
Error threshold
• The capacity to sustain prodigious numbers of mutaNons is a powerful advantage
• There must be a point at which selecNon and survival balance geneNc fidelity and mutaNon rate
• This limit is called the error threshold. If you exceed it, you are dead. If you live too far below it, you cannot produce enough mutaNons to survive selecNon
• RNA viruses: evolve close to their error threshold
• DNA viruses: evolve far below their error threshold21
Error threshold
• Expose a cell culture infected with a DNA virus to a base analog such as 5-‐azacyNdine
• 5-‐azacyNdine is incorporated as a C, but templates as a T (G to A transiNons)
• MutaNon rate among viral progeny increases several orders of magnitude
• When a similar experiment is done with an RNA virus, the error frequency per genome increases only two-‐ to threefold at best -‐ cannot make any more mutaNons
22
Error threshold
anNviral ribavirin and poliovirus
23
Gene&c boNlenecks
• Extreme selecNve pressures on small populaNons that result in loss of diversity, accumulaNon of non-‐selected mutaNons, or both
• A single RNA virus plaque is picked and expanded
• Next, a single plaque is picked from the expanded stock
• The process is repeated over and over
Principles of Virology, ASM Press
24
Gene&c boNlenecks
• The booleneck arises by restricNng further viral replicaNon to the progeny found in a single plaque
-‐ A few thousand progeny viruses derived from a single founder virus
25
• A]er about 20-‐30 cycles of single-‐plaque amplificaNon, many virus populaNons are barely able to grow
• They are markedly less fit than the original populaNon
• The environment is constant, and the only apparent selecNon is that imposed by the ability of the populaNon of viruses from a single plaque to replicate
• Why does fitness plummet?
Gene&c boNlenecks
26
• The answer lies in a phenomenon dubbed Muller’s ratchet: Small, asexual populaNons decline in fitness over Nme if the mutaNon rate is high
• ReplicaNng RNA viruses produce many mutaNons; they survive close to their error threshold
• By restricNng populaNon growth to serial single founders (the booleneck) under otherwise nonselecNve condiNons, so many mutaNons accumulate (exceed the threshold) that fitness decreases
Gene&c boNlenecks
27
The ratchet metaphor: each of the new mutaNons works like a ratchet, allowing the gear to move forward, but not backward
Each round of error-‐prone replicaNon works like a ratchet, “clicking” relentlessly as mutaNons accumulate at every replicaNon cycle
28
Principles of Virology, ASM Press
29
Fitness decline compared to ini&al virus clone aPer passage through a boNleneck
Principles of Virology, ASM Press
30
• InfecNon by a limited virus populaNon and subsequent amplificaNon are o]en found in nature-‐ Small droplets of suspended virus during aerosol
transmission
-‐ Ac-va-on of a latent virus from a limited popula-on of cells
-‐ Small volume of inoculum introduced in infec-on by insect bites
• How do infecNons that spread by these routes escape Muller’s ratchet?
BoNlenecks in the real world?
31
• Subject a more diverse viral populaNon to serial passage-‐ Don’t pick a single plaque, pool several plaques
• More diversity in the replicaNng populaNon facilitates construcNon of a mutaNon-‐free genome by recombinaNon or reassortment, removing or compensaNng for mutaNons that affect growth adversely
Avoiding the ‘ratchet’
Principles of Virology, ASM Press
32
• Even if such a recombinant is a rare species, it has a powerful selecNve advantage for growth-‐ Its progeny will ul-mately take over the popula-on
• The message is simple: Diversity of a viral populaNon is important for the survival of individual members
-‐ Remove diversity, and the popula-on suffers
Avoiding the ‘ratchet’
33
By exchange of gene&c informa&on
Sick virus 1
Sick virus 2
Healthy viral recombinant
34
• Allows the construcNon of viable genomes from debilitated ones and avoids Muller’s ratchet
• A similar mechanism is reassortment among genomic segments when cells are co-‐infected with segmented RNA viruses
• It is an important source of variaNon, as exemplified by orthomyxoviruses and reoviruses
Exchange of gene&c informa&on
35
Selec&on: Gene&c shiP & driP
• SelecNon of viral mutants resistant to eliminaNon by anNbodies or cytotoxic T cells is inevitable when sufficient virus replicaNon occurs in an immunocompetent individual
• Dri] -‐ diversity arising from copying errors and immune selecNon -‐ may occur each Nme a genome replicates
• Shi] -‐ diversity arising a]er recombinaNon or reassortment -‐ is relaNvely rare
36
Influenza viruses
• Influenza A viruses are classified by anNgenic composiNon, by serologic tesNng of HA and NA
• CombinaNons of H and N are called HxNy
• x = 1-‐17; y = 1-‐9
• H1-‐17 can infect birds; H1, H2, H3 can infect and transmit between humans
37
Principles of Virology, ASM Press
38
39
An&genic driP: Influenza virus
Principles of Virology, ASM Press
40
Exchange of gene&c informa&on
Principles of Virology, ASM Press
41
Selec&on: Is virulence a posi&ve or nega&ve trait?
• Idea: increased virulence reduces transmissibility because hosts die faster, reducing exposure to uninfected hosts
• ExpectaNon: all viruses evolve to be maximally infecNous and avirulent
• But this is not what is observed
• Persistent infecNons lie dormant for years, then kill host at end stage
• Virulent viruses for one species may be maintained as asymptomaNc infecNons in another
• For some diseases, increased virulence increases transmissibility and is selected for in natural infecNons
42
An experiment in virus evolu&on
• In 1859, the European rabbit was introduced to Australia for hunNng purposes
• Lacking natural predators, it reproduced to plague proporNons in a short Nme
43
An experiment in virus evolu&on
• The myxoma leporipox virus was released in Australia in the 1950s in an aoempt to rid the conNnent of the rabbits
• The natural host of myxoma virus is the cooontail rabbit, the brush rabbit of California, and the tropical forest rabbit of Central and South America
• The virus is spread by mosquitoes; infected rabbits develop superficial warts on their ears
• European rabbits are a different species, infecNon is 90-‐99% fatal
44
• In the first year, the released virus was efficient in killing rabbits with a 99.8% mortality rate
• By the second year the mortality dropped to 25%
• In subsequent years, the rate of killing was lower than the reproducNve rate of the rabbits, and hope for 100% eradicaNon was dashed
An experiment in virus evolu&on
45
An experiment in virus evolu&on
• Both rabbits and viruses produce large numbers of offspring
• The virus evolved to kill fewer rabbits and to extend the life of lethally infected rabbits so that the virus could overwinter and spread in spring mosquitoes
• The rabbits evolved to become more resistant or tolerant of the virus
• As predicted for an evolving host coming to an equilibrium with the pathogen
46
What was learned?
• You always get what you select, but you o]en don’t get what you want
• EliminaNon of rabbits with a virus was flawed idea-‐ Selec-ve forces that could not be controlled were at work
• In Australia, apparently nothing: experiments in biological control of rabbits using a lethal calicivirus are under way
47
The origin of viruses
• Regressive theory: viruses are derived from intracellular parasites that have lost all but essenNal genes
• Cellular origin theory: viruses arose from cells by reducNve evoluNon
• Independent-‐en&ty theory: viruses coevolved with cells from the origin of life (‘pickpockets’)
• But there is no fossil record, and few viral stocks more than 80 years old
• BioinformaNcs has provided insight48
Origin of RNA viruses from RNA cells
Different cell lineages
Biochimie 87 (2005) 793-‐803
49
Very large viral DNA genomes
• Mimivirus (1.2 Mbp), Phycodnavirus (330 kbp) genomes have sequence coherence: are not mosaics
• Homogeneity of genomes within family, lack of homology among families, difficult to explain using model that they arose by the acquisiNon of exogenous genes by a primordial precursor viral genome
• Megavirus
Principles of Virology, ASM Press
50
Origins of DNA viruses
• By rela*ng *mescale of herpesviral genome evolu*on with that of hosts, believe that three major groups of herpesviruses (alpha, beta, gamma) arose ~180-‐220 million years ago
51
Human viruses
• All known types of viruses likely evolved long before humans appeared on Earth
• All human viruses have therefore evolved from animal viruses
52
Origins of smallpox virus
• Sequence analysis of 45 isolates: genomes can be organized into 3 clades, which cluster geographically (West Africa, South America, Asia)
• Gene content is constant; lack of diversity indicates recent introducNon into humans
• Only member of poxvirus family with credible homology to smallpox is a gerbil poxvirus
• Perhaps human smallpox virus arose a]er a zoonoNc infecNon from infected gerbils
53
Origins of measles virus
• Measles virus is closely related to rinderpest virus, a bovine pathogen
• Probably evolved from an ancestral rinderpest virus when humans first began to domesNcate caole
• Established in the Middle East ~5,000 years ago, when human populaNons began to congregate in ciNes
• Spread around the world by colonizaNon and migraNon, reaching Americas in 16th century and destroying naNve Americans
54
The fundamental proper&es of viruses constrain and drive evolu&on
• Despite many rounds of replicaNon, mutaNon, selecNon, we can recognize a herpesvirus or influenza virus genome by sequence analysis
• Viral populaNons o]en maintain master or consensus sequences, despite opportuniNes for extreme variaNon
• How is stability maintained?
• All viruses share fundamental characterisNcs that define and constrain them
• Viruses that can funcNon within the constraints survive
55
Constraining viral evolu&on
• Extreme alteraEons in viral consensus genome do not survive selecEon
• The viral genome is one constraint
-‐ DNA cannot become RNA, or vice versa
-‐ Replica3on strategy -‐ cannot change; consider interac3on with host proteins
• Physical nature of capsid
-‐ Icosahedral capsids: defined internal space, fixes genome size
• SelecEon during host infecEon
-‐ A mutant too efficient in bypassing host defenses will kill host, suffer the same fate as one that does not replicate efficiently enough
56
Evolu&on of new viruses
• AssumpNon: new viruses can only arise from viruses that are now in existence, not de novo
• What is the number of all possible mutaNons of a viral genome?
• It is so large in fact that all the possibiliNes can never be tested in nature
• Sequence comparisons of several RNA virus genomes have demonstrated that well over half of all nucleoNdes can accommodate mutaNons
57
Evolu&on of new viruses
• For a 10 kb viral genome, there are more than 45000 sequence permutaNons that define all possible mutants
• If one considers deleNons, recombinaNon, and reassortment, the numbers become even larger
• Considering that there are roughly 4135 atoms in the visible universe, this is a large number indeed
58
• New mutants will always arise, and the possibiliNes literally are unimaginable
• Future viruses arise from extant viruses: mutants, recombinants, reassortants of what is out there
• We only know about 1% of the viruses that exist
• As mutaNon rates are probabilisNc and viral quasispecies are indeterminate, the very concept of predicNng the direcNons of viral evoluNon has liole meaning
Virus evolu&on is relentless
59
Food for thought
• It took 8 million years or more for simian primates to change 2% of their genomes to become human
• By contrast, poliovirus can change 2% of its geneNc structure in five days!
• This is about the Nme it takes for the virus to pass from the human mouth to the gut
• Imagine what a virus can do with 8 million years
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