Principles of Molecular Virology

300 Pages·2013·9.02 MB·English

Principles of Molecular
Virology
Fifth Edition
Alan J. Cann
University of Leicester, UK
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Preface to the Fifth Edition
ThisisthebookInearlydidn'twrite.Quitealothaschangedformesincethe
fourth edition was published, and when discussions began as to whether to
produceanewedition,Ihadmydoubts.Frommyperspective,oneofthemain
thingsthathaschangedisMicrobiologyBytes.comdthere,andinrelatedspaces
such as the MicrobiologyBytes page on Facebook, in the words of Elvis
Costello,“EverydayIwritethebook.”Sowhyareyoureadingthis?Although
theInternetisfarbetterthanprintedbooksformanythings,thereisclearlystill
a strong demand for this book because sometimes it’s good to have a core of
knowledgeinoneplace(e.g.,whenitcomestorevisionforexams).Addtothat
the translations into other languages(including Chinese) that the last edition
received, plus the Amazon Kindle version, and I can reach a wide and appre-
ciative audience inthis format.
OnceIhadagreedtowriteanewedition,Iwantedtodotwothings.Iwantedto
update the technical knowledge throughout, but I also needed to completely
rewritethewholebooktomakeitfarmoreaccessible,basedontheexperienceI
havegainedwritingonlineoverthepastfewyears.HopefullyIhaveachievedthat.
For the fifth edition, readers can now access student and instructor materials
online.Pleasevisitstore.elsevier.comandsearchforISBN978-0-12-384939-7.
Student materials are available via the companion site link and instructor
materials are available via the manuals text link on the Resources tab at the
bottom of the page.
I would like to thank all the staff of Elsevier, without whose hard work and
persuasion this edition would never have appeared. Although we have never
metfacetoface,theyresideinmyinbox,constantlyremindingmethatchapters
are overdue.
Alan J. Cann
UniversityofLeicester,UK
[email protected]
January2011
ix
CHAPTER 1
Introduction
WHAT’S IN THIS CHAPTER? CONTENTS
What Are
n Westartbyaskingwhatavirusis,andwelookathowvirusesaredifferent
Viruses?................2
fromallotherorganisms.Arevirusesalive?Itdoesn’tmattertoavirus,but
Arevirusesalive?.....3
it is afrequently asked question, so wewill considerpossible answers.
The History of
n Thenwespendashorttimelookingatthehistoryofvirology,becausethe
Virology ...............4
wayourpresentknowledgewasacquiredexplainshowwecurrentlythink
about viruses. LivingHost
n We finishby describingsome techniquesusedto study viruses, ending Systems................6
rightuptodatewiththemostrecentmethods,whichwouldhavesounded Cell Culture
like science fiction when the first edition of this book was published. Methods...............8
Serological/
Immunological
Methods.............10
Thisbookisabout“molecularvirology,”thatis,virologyatamolecularlevel.It Ultrastructural
looks at protein-protein, protein-nucleic acid, and protein-lipid interactions, Studies................11
whichcontrol the structureofvirus particles;the waysvirusesinfect cells;and Molecular
how viruses manage to replicate themselves. Later we will also examine the Biology................18
consequences of virus infection for host organisms, but it is important to
Summary............23
considerthebasicnatureofvirusesfirst.Beforegoingintodetail,itisusefulto
Further
knowalittle about thehistoryofvirology, andinparticular,howour present
Reading..............23
knowledge of viruses was achieved. Understanding this helps to explain how
wethinkaboutvirusesandwhatthecurrentandfutureconcernsofvirologists
are. That is the reason for this chapter.
There is more biological diversity between viruses than in all the rest of the
bacterial, plant, and animal kingdoms put together. This is the result of the
success of viruses in parasitizing all known groups of living organisms;
understanding this diversity is the key to comprehending the interactions of
viruses with their hosts. The principles behind some of the experimental
techniquesmentionedinthischaptermaynotbewellknowntoallreaders,so
it may be helpful to explore the further reading at the end of this chapter to 1
PrinciplesofMolecularVirology.DOI:10.1016/B978-0-12-384939-7.10001-8
Copyright(cid:1)2012,ElsevierLtd.Allrightsreserved.
2 CHAPTER 1: Introduction
become more familiar with these methods or you will not be able to under-
standthecurrentresearchliterature.Inthisandsubsequentchapters,termsin
the text in bold red print are defined in the glossary found in Appendix 1.
(the WEB icon) tells you that you can find an interactive learning
resource on the web site.
BOX 1.1. DON’T SAY VIRALTO ME
Ifyoureadaboutviruses,you’llprobablycomeacrosstheword“viral”quitequickly.Most
virologistsuseitthesedays,butIhateit.That’sbecausethewordvirusisanoun,but“viral”
isanadjectivedescribingsomethingrelatingto,orcausedbyavirus.AsfarasI’mconcerned,
using nouns as adjectives is wrong, but most people don’t seem to mind this one. If you
wantedtodescribechickensoup,wouldyousayitwaschickenalflavored?Ifyouwantto
usethewordviral,goodlucktoyou,justdon’tsayittomeunlessyouuseit properly,for
example,“antiviraldrug.”
WHAT ARE VIRUSES?
Viruses are submicroscopic, obligate intracellular parasites. They are too
small to be seen by optical microscopes, and they have no choice but to
replicate inside host cells. This simple but useful definition goes a long way
toward describing viruses and differentiating them from all other types of
organisms. However, this short definition is not completely adequate. It is
not a problem to differentiate viruses from multicellular organisms such as
plants and animals. Even within the broad scope of microbiology covering
prokaryotic organisms as well as microscopic eukaryotes such as algae,
protozoa, and fungi, in most cases this simple definition is enough.
However, a few groups of prokaryotic organisms also have specialized
intracellular parasitic life cycles and overlap with this description. These are
the Rickettsiae and Chlamydiaedobligate intracellular parasitic bacteria that
have evolved to be so cell-associated that they can exist outside the cells of
theirhostsforonlyashortperiodoftimebeforelosingviability.Acommon
mistakeistosaythatvirusesaresmallerthanbacteria.Though thisistruein
most cases, size alone does not distinguish them. The largest virus known
(Mimivirus) is 400 nm in diameter, while the smallest bacteria (e.g., Myco-
plasma) are only 200 to 300 nm long. Nor does genetic complexity separate
virusesfromotherorganisms.Thelargestvirusgenome(Mimivirus,1.2Mbp
(million base pairs)) is twice as big as the smallest bacterial genome
(Mycoplasma genitalium, 0.58 Mbp), although it is still shorter than the
smallest eukaryotic genome (the parasitic protozoan Encephalitozoon, 2.3
Mbp). For these reasons, it is necessary to go further to produce a definition
of how viruses are unique:
What Are Viruses? 3
1. Virusparticlesareproducedfromtheassemblyofpreformedcomponents,
whileother biological agents grow from anincreasein the integrated sum
of their components andreproduce by division.
2. Virusparticles(virions) donot grow or undergo division.
3. Viruseslackthegeneticinformationthatencodesthetoolsnecessaryforthe
generation of metabolic energy or for protein synthesis (ribosomes).
No known virus has the biochemical or genetic means to generate the energy
necessary to drive all biological processes. They are absolutely dependent on
theirhostcells forthisfunction.Lackingtheabilitytomakeribosomesisone
factor that clearly distinguishes viruses from all other organisms. Although
therewillalwaysbesomeexceptionsanduncertaintiesinthecaseoforganisms
that are too small to see easily and in many cases difficult to study, these
guidelines are sufficientto define what avirus is.
A number of virus-like agents possess properties that confuse the previous
definition, yet are clearly more similar to viruses than other organisms. These
arethesubviralelementsknownasviroids,virusoids,andprions.Viroidsare
small(200e400nucleotide),circularRNAmoleculeswitharod-likesecondary
structure. They have no capsid or envelope and are associated with certain
plantdiseases.Theirreplicationstrategyislikethatofvirusesdtheyareobligate
intracellularparasites.Virusoidsaresatellite,viroid-likemolecules,abitlarger
than viroids (approximately 1000 nucleotides), which are dependent on the
presenceofvirusreplicationfortheirmultiplication(thereasontheyarecalled
satellites). They are packaged into virus capsids as passengers. Prions are
infectiousproteinmoleculeswithnonucleicacidcomponent.Confusionarises
fromthefactthattheprionproteinandthegenethatencodesitarealsofound
in normal uninfected cells. These agents are associated with diseases such as
CreutzfeldteJakob disease in humans, scrapie in sheep, and bovine spongi-
form encephalopathy (BSE) in cattle. Chapter 8 deals with these subviral
infectious agents in more detail.
Genomeanalysishasshownthatmorethan10%oftheeukaryoticcellgenome
is composed of mobile retrovirus-like elements (retrotransposons), which
mayhavehadaconsiderableroleinshapingthesecomplexgenomes(Chapter
3). Furthermore, certain bacteriophage genomes closely resemble bacterial
plasmids in their structure and in the way they are replicated. Research has
revealed that the evolutionary relationship between viruses and other living
organismsis perhaps more complex than was previously thought.
Are viruses alive?
As discussed earlier, viruses do not reproduce by division but are assembled
from preformed components, and they cannot make their own energy or
proteins.Avirus-infectedcellismorelikeafactorythanawomb.Oneviewis
4 CHAPTER 1: Introduction
that inside their host cell viruses are alive, whereas outside it they are only
complexarrangementsofmetabolicallyinertchemicals.Chemicalchangesdo
occurinextracellularvirusparticles,asexplainedinChapter4,butthesearein
not the growth of a living organism. This is a bit problematicdalive at some
timesbutnotatothers.Virusesdonotfitintomostofthecommondefinitions
oflifedgrowth,respiration,andsuch.Ultimately,whethervirusesarealiveor
notisamatterofpersonalopinion,butitisusefultomakeyourdecisionafter
considering the facts. Some of the reading at the end of this chapter will help
youconsiderthe evidence.
BOX 1.2. ARE VIRUSES ALIVE? WHO CARES?
Virusesdon’tcareifwethinktheyarelivingornot.AndIdon’tcaremucheither,becauseasfar
asI’mconcerneditismuchmoreimportanttounderstandhowvirusesreplicatethemselvesand
interactwiththeirhosts.Butyoumightcare,eitherbecauseyouareaphilosophicalpersonwho
likesthinkingaboutthesethings,orbecauseyouhavetowriteanessayoransweranexam
questiononthesubject.Inthatcase,itisimportanttoconsiderhowyoudefinewhataliving
organismisandhowvirusesaresimilarordifferenttomicroorganismsweconsidertobealive
(you’regoingtomakelifehardforyourselfifyoustartcomparingthemtohumans).Thisisnot
asimplequestion,andanysimpleansweris,quitesimply,wrong.
THE HISTORY OF VIROLOGY
Humanknowledge ofvirusdiseases goes back alongway, although it is only
much more recently that we have become aware of viruses as distinct from
other causes of disease. The first written record of a virus infection is a hiero-
glyph from Memphis, the capital of ancient Egypt, drawn in approximately
3700 BCE, which depicts a temple priest showing typical clinical signs of
paralyticpoliomyelitis.PharaohRamsesV,whodiedin1196BCEandwhose
well-preservedmummifiedbodyisnowinaCairomuseum,isbelievedtohave
diedfromsmallpoxdthecomparisonbetweenthepustularlesionsontheface
of this mummy and those ofmorerecent patients isstartling.
Smallpox was endemic in China by 1000 BCE. In response, the practice of
variolation was developed. Recognizing that survivors of smallpox outbreaks
wereprotectedfromsubsequentinfection,peopleinhaledthedriedcrustsfrom
smallpox lesionslikesnuffor,inlatermodifications,inoculated thepus from
a lesion into a scratch on the forearm. Variolation was practiced for centuries
andwasshowntobeaneffectivemethodofdiseaseprevention,althoughrisky
becausetheoutcomeoftheinoculationwasnevercertain.EdwardJennerwas
nearlykilledbyvariolationattheageofseven!Notsurprisingly,thisexperience
spurredhimontofindasaferalternativetreatment.OnMay14,1796,heused
cowpox-infectedmaterialobtainedfromthehandofSarahNemes,amilkmaid
The History of Virology 5
from his home village of Berkeley in Gloucestershire, England, to successfully
vaccinate 8-year-old James Phipps. Although initially controversial, vaccina-
tion against smallpox was almost universally adopted worldwide during the
nineteenth century.
Thisearlysuccess,althoughatriumphofscientificobservationandreasoning,
wasnotbasedonanyrealunderstandingofthenatureofinfectiousagents.This
arose separately from another line of reasoning. Antony van Leeuwenhoek
(1632e1723),aDutchmerchant,constructedthefirstsimplemicroscopesand
with these identified bacteria as the “animalcules” he saw in his specimens.
However, it was not until Robert Koch and Louis Pasteur in the 1880s jointly
proposedthe“germtheory”ofdiseasethatthesignificanceoftheseorganisms
becameapparent.Kochdefinedfourfamouscriteria,whicharenowknownas
Koch’s postulates and still generally regarded as the proof that an infectious
agent isresponsible for aspecificdisease:
(cid:1) Theagent must be present ineverycase ofthe disease.
(cid:1) Theagent must be isolated from the host and grown in vitro.
(cid:1) The disease must be reproduced when a pure culture of the agent is
inoculated intoahealthysusceptible host.
(cid:1) The same agent must be recovered once again from the experimentally
infected host.
Subsequently, Pasteur worked extensively on rabies, which he identified as
beingcausedbyavirus(fromtheLatinfor“poison”),butdespitethishedid
not discriminate between bacteria and other agents of disease. In 1892,
Dimitri Iwanowski, a Russian botanist, showed that extracts from diseased
tobacco plants could transmit disease to other plants after being passed
through ceramic filters fine enough to retain the smallest known bacteria.
Unfortunately, he did not realize the full significance of these results. A few
yearslater(1898), Martinus Beijerinickconfirmed and extended Iwanowski’s
resultsontobaccomosaicvirus(TMV)andwasthefirsttodevelopthemodern
idea of the virus, which he referred to as contagium vivum fluidum (soluble
livinggerm).FreidrichLoefflerandPaulFrosch(1898)showedthatasimilar
agent was responsible for foot-and-mouth disease in cattle, but, despite the
realization that these new found agents caused disease in animals as well as
plants,peoplewouldnotaccepttheideathattheymighthaveanythingtodo
with human diseases. This resistance was finally dispelled in 1909 by Karl
LandsteinerandErwinPopper,whoshowedthatpoliomyelitiswascausedby
a“filterableagent”dthefirsthumandiseasetoberecognizedasbeingcaused
by a virus.
Frederick Twort (1915) and Felix d’Herelle (1917) were the first to recognize
viruses that infect bacteria, which d’Herelle called bacteriophages (eaters of
bacteria).Inthe1930sandsubsequentdecades,pioneeringvirologistssuchas
6 CHAPTER 1: Introduction
SalvadorLuria,MaxDelbruck,andothersusedthesevirusesasmodelsystems
to investigate many aspects of virology, including virus structure (Chapter 2),
genetics (Chapter 3), and replication (Chapter 4). These relatively simple
agentshavesinceprovedtobeveryimportanttoourunderstandingofalltypes
of viruses, including those of humans, which can be much more difficult to
propagate and study. The further history of virology is the story of the devel-
opment of experimental tools and systems with which viruses could be
examinedandthatopenedupwholenewareasofbiology,includingnotonly
thebiologyofthevirusesthemselvesbutinevitablyalsothebiologyofthehost
cells on whichtheyare dependent.
LIVING HOST SYSTEMS
In1881,LouisPasteurbegantostudyrabiesinanimals.Overseveralyears,he
developed methods of producing attenuated virus preparations by progres-
sively drying the spinal cords of rabbits experimentally infected with rabies,
which,wheninoculatedintootheranimals,wouldprotectfromdiseasecaused
by virulent rabies virus. In 1885, he inoculated a child, Joseph Meister, with
this, the first artificially produced virus vaccine (since the ancient practice of
variolation and Jenner’s use of cowpox virus for vaccination had relied on
naturallyoccurringviruses).Wholeplantshavebeenusedtostudytheeffects
of plant viruses after infection ever since tobacco mosaic virus was first
discovered by Iwanowski in 1892. Usually such studies involve rubbing
preparations containing virus particles into the leaves or stem of the plant to
cause infection.
During the SpanisheAmerican War of the late nineteenth century and the
subsequent building of the Panama Canal, the number of American deaths
due to yellow fever was colossal. The disease also appeared to be spreading
slowly northward into the continental United States. In 1900, through
experimental transmission of the disease to mice, Walter Reed demonstrated
that yellow fever was caused by a virus spread by mosquitoes. This discovery
eventually enabled Max Theiler in 1937 to propagate the virus in chick
embryosandtoproduceanattenuatedvaccinedthe17Dstraindwhichisstill
in use today. The success of this approach led many other investigators from
the 1930s to the 1950s to develop animal systems to identify and propagate
pathogenic viruses.
Culturesofeukaryoticcellscanbegrowninthelaboratory(thisisknownas
invitroortissueculture)andvirusescanbepropagatedinthesecultures,but
these techniques are expensive and technically demanding. Some viruses
such as influenza virus will replicate in the living tissues of developing
embryonated hen eggs. Egg-adapted strains of influenza virus replicate well
in eggs and very high virus titres can be obtained. Embryonated hen eggs
Living Host Systems 7
were first used to propagate viruses in the early decades of the twentieth
century. This method has proved to be highly effective for the isolation and
culture of many viruses, particularly strains of influenza virus and various
poxviruses (e.g., vaccinia virus). Counting the pocks on the chorioallantoic
membrane of eggs produced by the replication of vaccinia virus was the first
quantitative assay for any virus. Animal host systems still have their uses in
virology:
(cid:1) Toproducevirusesthatcannotbeeffectivelystudiedinvitro(e.g.,hepatitis
B virus)
(cid:1) Tostudythepathogenesisofvirusinfections(e.g.,HIVanditsnearrelative,
SIV)
(cid:1) To test vaccine safety (e.g., oral poliovirus vaccine)
Nevertheless, they are increasinglybeingdiscardedfor the following reasons:
(cid:1) Breeding and maintenance of animals infected with pathogenic viruses is
expensive.
(cid:1) Thesearecomplexsystemsinwhichitissometimesdifficulttoisolatethe
effects ofvirusinfection.
(cid:1) Results obtained are not alwaysreproducibledue to host variation.
(cid:1) Unnecessary or wasteful use of experimental animals is morally
unacceptable.
With the exception of studying pathogenesis, use of animals is generally
being overtaken by faster and cheaper molecular biology methods. In the
1980s the first transgenic animals were produced that carried the genes of
other organisms. Inserting all or part of a virus genome into the DNA of an
embryo (typically of a mouse) results in expression of virus mRNA and
proteins in the animal. This allows the pathogenic effects of virus proteins,
individually and in various combinations, to be studied in living hosts.
SCID-hu mice have been constructed from immunodeficient animals
transplanted with human tissue. These mice form an intriguing model to
study the pathogenesis of human immunodeficiency virus (HIV) because
there is no real alternative to study the properties of HIV in vivo. Similarly,
transgenic mice have proved to be vitally important in understanding the
biology of prion genes. Although these techniques raise the same moral
objections as old-fashioned experimental infection of animals by viruses,
they are immensely powerful new tools for the study of virus pathogenicity.
A growing number of plant and animal virus genes have been analyzed in
thisway,buttheresultshavenotalwaysbeenasexpected,andinsomecases
it has proved difficult to equate the observations obtained with those
gathered from experimental infections. Nevertheless, this method has
become quite widely used in the study of important diseases where few
alternative models exist.