380 PART IV host cell.

Microbial Repkcation ond Growth

of the
integrity
the glycoproteins

disrupt viral
cell
of the to the
bud through internal membranes such as the endo immediately
insertion
impart
However, by
viruses

plasmic reticulum or Golgi apparatus (rotaviruses). surface,

these
and
g l y c o p r o t e i n s ,

cell target
or inner nuclear membrane into the proteins a
(herpesviruses), form specificity
surface
and
does
become

ing vesicles. These viruses obtain their envelopes new infected

cell can
host.
of the virus,
from the membranes of the vesicles rather than and the inmune
defenses
such as
Sendai

from the cytoplasmic membrane surrounding the for the virions,

facilitate
the fusion
enveloped that
host cell. The vesicles the assembled
Some cell-surface

containing cell-surface
proteins
with the
host
Viruses migrate to the inner surface of the have inserted

mic membrane and fuse with it,

cytoplas of the
virion
envelope that are
be-
effectively releas Viral
glycoproteins

promote
fusion

ing intact virions to the outside. membrane. membrane

surface m u l t i n u c l e a t e d

the host to
Viruses that are released by budding vary con- into
cells,
leading
neighboring
cells
neighboring
siderably in their effects on host cell metabolism tween of the
The fusion the virus.
and integrity. Some, such as herpesviruses, cause cell
formation.
cell-to-cell
spread of
efficient
host cell lysis. Others, such as retroviruses, do not allows

Situational Problem 81

Debating Whether Viruses are Alive of life

of the meaning
understanding that dis-
Microbiologists often argue about whether or not
acellular microorganisms should be considered
damental

and the ability

to examine the
processes
entities.
nonliving
as living entities. Geneticists may consider that tinguish living from of the univer-
member
are a
Consider that you the next
information flow is the principal life function
team and that
the topic for
debate
and accordingly view viruses as alive because sity alive?" As in any
Are They
they store and use genetic information to specify debate is "Viruses: to argue ei-
must be prepared
their own replication. Physiologists, on the other formal debate, you
notes for debate,
issue. Prepare
hand, may consider viruses as nonliving entities ther side of this
both sides. Think
that you consider
because of their inability to carry out physiolog- making s u r e of viruses can
be
ical functions such as ATP generation. Thus the about how the properties whether
viewed as evidence for
determining
perspective of an individual can bias the opin-
Also, decide what experi-
ion on whether viruses should be viewed as liv- they are or not alive.
ing or not. A philosophical or scientific discus- ments could be designed
to help resolve this
sion of whether viruses are alive requires a fun- issue.

REPLICATION OF The replication of lytic phage can be observed as

BACTERIOPHAGE plaques (zones of clearing) when permissive bacte
ria are grown on a solid medium (see Box 8-2). In
Bacteriophage replicate within host bacterial cells. the absence of phage infection, the bacterial growth
Bacteriophage are differentiated based on their is confluent, forming a "lawn" of bacterial cells.
morphologies, genomic nucleic acids, and host cell Several kinds of mutations can occur during the
ranges (Table 8-3). Some replicate only
within a replication cycle of a bacteriophage that alter its
Others replicate various
in host cell range or the
specific host cell strain. appearance of the plaques it
Gram-negative or Gram-positive bacterial cells. forms (Table 8-4).
Within the host bacterial cell the phage uses the Some bacteriophage
nucleotides) and alternate
are
capable of lysogeny, an
cell's molecules (e.g., ATP and replication strategy in which the phage
structures (including ribosomes) to make new genome is incorporated into the bacterial chromo-
phage. In most cases the complete replication cycle some. Such
phage are called temperate bacterio-
results in death of the host cell. Phage phage because only the integrated
of the phage phage genome is
released
that kill host bacterial cells when they are replicated along with the replication of host cell
are called lytic phage. DNA. Phage capsids are not
made and
complete
 8 Viral Replication 381
Chapter

1Characteristics
Table 8-3 Cha of Some of
the Groups of
Bacteriophage

Tailed bacteriophage
Cubic b a c t e r i o p h a g e
Filamentous bacterio.
phage (Inoviridae)
Description Bacteriophage
Genome: DNA,
double-stranded. Virion:
capsomers. The tails of the complex shape, Di
number of
phage are 1on8 ridae), tractile in group
myoviridae), long and and very hort
in
group C (Pedoviridae).noncontractile in group B (Styloviridae). 2
Example: T-even coliphages.
TOup 1 (Microviridae)-Genome: DNA. single:stranded. Virion: icosahedral, sy"
metry, 12
capsomers. Example: 6X174.
TOup 2
(Corticoviridae)-Genome: DNA. double-stranded.
symmetry,
Virion: icosan dral
enveloped. Example: PM-2. CuDIC
eviviridae)-Genome: RNA, single-stranded. Virion: icosahedra.
symmetry, 32
capsomers. Example: 1,
GrOup 4 (Cystoviridae)-Genome: RNA, double-stranded. Virion: icosaneara 7*
metry, enveloped. Example: d6.
Genome: DNA, single-stranded. Virion: rod-shaped, helical symmetry. Example: M13.

Table 8-4 Types of Mutations in Bacteriophage

Type of Mutation Change Produced

Host range Change in the range of host cells the phage can infect is due to a change in receptor
Sites of the host cell, a change in the phage or host enzymes involved in replica

tion, or in the restriction enzymes or modification of the phage genome.

host cell
nange in the characteristics of the plaques (zones of clearing on a solid surfacelysis
Plaque morphology due to
that
Ormed by the phage growing on host bacterial cells spread
is clear
may be seen as a change in size of the plaque or whether
the plaque or
and
turbid; plaque morphology is characteristic of the rate of phage replication
the efficiency of host cell lysis.

such that the

Temperature sensitive
Change in range of temperatures over which the phage can replicate
but not at a higher temperature
phage remains able to replicate at one temperature
at the higher
due to a change in a phage protein that makes the protein unstable
mutation because the
temperature; this is an example of a conditionally "lethal"
phage is unable to replicate at the higher temperature, but replicates at the lower
temperature.
forms a stop codon,
Change in the nucleotide sequence of the phage genome that
Nonsense protein; in some host cells
causing termination of the synthesis of a phage-coded
the nonsense codon is suppressed and the phage will replicate, whereas in other
is unable to replicate; this limits the
host cells lacking a suppressor the phage
host cell range of the phage.

bacterial chromosome, the phage genome (referred

The host cell is
phage progeny are not produced. to as a prophage) is replicated with the
bacterial
not killed by lysis in this mode
of phage replica-
DNA during normal host cell DNA replication. At a
tion. later time, the prophage can be excised from thhe
is usually incor-
In lysogeny, the phage genome bacterial chromosome or plasmid DNA, reestab-
porated into the bacterial
chromosome by nonreci- lishing a lytic replicative cycle.
into the
procal recombination. Once
incorporated
382 PART IV MKrobiol Replkcotion ond Growth

BOX82
METHODOLOGIES
Assaying for Lytic Bacteriophage nutrient medlum
and dllutions of the phgpe
confluent lawn of
sutoble solid bocteria forma
To astoy for prepared on a the
infectve bocteria is
bocteriophoge
f o w m of
of obsence
bocteriophoge,
lytic
ension ore then spreod over the some surfoce. In the phoge. Lyss by acteriophoge
boctere
growth release ofthe to
due to the lytikc coepn
ploqueof
in regions bocterio ore kiled
wherephoge replication occurs, the bocterio kiled due
ore terig (see fgure). Eoch infectious
indicated by the formation of a ycle. Thespreod
zone of clearing or ploque,
infectious unit and
ntlated lytic replicatve
ts ytic
initiated its repmthe bacte ria in the wicinity of the initiol
nesite where single bocteriophoge octed
a as an
esults in the sis
of riate dilution foctors can be
phoge from the initialy infected bocteriol cell to the surrounding cels that develop ond the oppropriate low percentoge of
relatively lowpe
phoge ond hence this zone of clearing The number of plaques used sov has
oe in these assoys hos a relatively
r d e the phoges
but does not permit
used to The medum
calculate the number of bacteriopha nasomple. uninfected cells
oPar and therefore is called soft agar; it permits diffusion ofphoge to nearby
that are produced to
move to remote ports of the plate. because when cell s hedvil a
size is limited
the ploque inhibition,
ne to spreod indefinitely
With T4 phoge,
phenomenon known as ysis
reinfected with b the hsisof the cellis inhibited, a in size because the host
which is octual on e he time of normal lyss,synthesis. In other phage, ploques
are limited

are no
cells are
bocterial cells
period ofphope
longer in ea growth phose in which phages con be produdeu
no lon
00cterial

Plaques from Bacteriophage Replication. Replication of

yic bacteriophage causes the formation of plaques in a lawn of
bacterial cells growing on an agar surface.

GROWTH CURVE OF LYTIC PHAGE 80

lysis of the bacterial cell releases large num-
The of a
60 Latent
ber phage simultaneously. Consequently, the
lytic replication cycle exhibits a one-step growth
curve with high numbers of phage released period-
ically (Fig. 8-16). The growth curve for lytic phage
40
begins with an eclipse period during which there
are no complete infective phage particles and the 20-
naked DNA within the host cell is unable to infect
other cells to initiate a new replicative cycle. The Eclipse
end of the eclipse period is the time at which an
average of one infectious unit is produced for each 0 10 20 30 40 50
productive cell. The eclipse period, thus, is the Time (min)
time between entry of the phage DNA and forma- Fig. 8-16 One-step Growth Curve for
tion of the first complete phage within the host Because many viruses are released
Bacteriophage.
cell. cell lyses, lytic viruses exhibit a
simultaneously when a host
one-step growth curve
The eclipse period is part of a longer period, the
latent period, which, like the eclipse period, be-
gins when the phage injects DNA into a host cell. end of the eclipse period and the end of
the latent
but which does not end until the first assembled
period, assembled phage accumulate within the
phage from the infected cells appear extracellu- bacterial cell. Completely assembled
mulate within the bacterial cell until phage
larly. The latent period for a T-even phage typically accu-
the cell lyses,
is about 15 minutes. During the time between the releasing the phages into the extracellular fluid.
 Chapter 8 Viral Replication 383

The burst size, which varies

from cell to cell, LYTIC T-EVEN PHAGE
presents he average number of
units produc per cell; a typical infectious
burst
phage The developmental sequence of T-even phage is
T-even phage may be high as 200. As size for a
as Oxemplified by the replication of phage T4, a bac-
release of infective result of
a
the simultaneor teriophage with an icosahedral head capsid that
number of phages that initiate a phage, the
can
Contains the genome and a tail portion of the cap
tion cycle increases lytic replica- sid that is involved in attachment to the host cell.
greatly in a
single step. The en-
tire lytic growth cycle for some The genome of phage T4 is a large double-stranded
ocur in less than 20
T-even phage can linear DNA macromolecule that is highly folded
minutes under
ditions. optimal con within the head of the phage (Fig. 8-17). The repli
cation of T4 results in lysis of the host cell.

nis

&

10

dex A

DNA

ONA
repl.

Fig. 8-17 Genome of T-even Bacteriophage. The genome of T-even phage encodes the in.
formation for phage DNA replication and synthesis of numerous proteins. The genome is tran-
scribed in difering directions (orrows) for the production of the phage structures. Numbers refer
to specific genes. Direction of transcription (orrow).
384 PART Iv Marobal Repkoton ond Growth

ATTACHMENT AND PENETRATION and sheath, and the tail sheat

baseplate
in the
cur
contract because
it is reorganized fro
Replication of bacteriophage T4 begins with the a appears to sheath contains
rings. The deoxv.
Tachment of phage tail fibers to the outer surtace ot 24 rings to 12
the energy tor the powerful con.
a host cell, such E. coli (Fig ATP that provides
T,like other T-even phages of E. coli,Bacteriophag
as
8-18). tail structureis
traction. The central
core
of the
use
cel w forced through the
bacterial wall and the phaoo
popolysaccharides proteins
or as
receptors. Viirld-
tion in oceptors on ditferent strains of E. coli re- the periplasmic space by the
DNA is pushed into
contraction of the sheath. The phage tail penetrates
sults in
difterent host cells for T4 phage. The entire
phage does
after the
not
penetrate the bacterial cell. Rather. the cell wall but not the cytoplasmic membrane.
baseplate of the phage is attached to the Subsequently, the phage DNA migrates across the
Surtace of the host membrane and into the cell.
cell, conformational changes cytoplasmic
oC

Head

Collar
Contracted
Extended sheath
sheath

Cytoplasmic membrane
Attochment Penetration

A18
Teven bacteriophage Bacteriophage-Penetration
Fi
Teven into
is involved
Host Cell. The
in adsorption and penetration. Contraction tail structure of a
forces portion of the phage tail
a
through the cytopiasmic membrane of the hostof cell
the phage DNA (right of illustration and
the tail sheath
right colorized micrograph). and iniects
 Chacar feg ejiogon
r deg

degai.g 1t piage
getre. T4 ynage
lertide. The
ziaylaticn ef the T4 phage
s o
hoet cell tha dres
a

dne duposptingacre the fnr prriu rerdanans

osylated. and it phage DiA will ne
iesf the phagr IA Va terminaily
ONt Ces erause its DNA
b y strictior endonurleases
EARLY GENE EXPRESiON
LATE GENE EXPRESSION
T4 Cdes for cver 2h
new Aher there tarly events in the phage 1sat St
sized early ater prrneina
infertiom. The that at
Cie, thene is a further medtinainn od the k pdy
eatly perdottir
teins represen tthe od mTR 1esuiting in the esatin f unber vy
ophage replicat deveicrgnaesta thenis ed the eatiy phage paneins KiA piymerae
mesTOVE in these sters(ilortively
at
the en
1elerte
ary protenns (proteins that ate hade
tr a
Ag peTetrati
Thee a:y srni anes shit in the 1ersapinisn oite d ttn 2SA piymetzse
aCut the
stoppageohest (el
prsits ing
SUnrtess An eriy prrMe f T4
marzreneierzulat late prrnina
that degades the host ( phae is a tueie The late phage qe ute bn tte vatinrus p
D the
dornyr
rapThe tail, tail ftea, and head sttud3es d h

i n i s o d in tha ean.zeisn d tha ta tra

apha 14 mkÀ
nAe iicientky. The enaie

ASAEMBAL
 Chapter 8 Viral Replicotion 389

of the phage genome, but the mechanism of repli-
cating the double-stranded RNA genome of this
phage has not been elucidated.
BACTERIOPHAGE LAMBDAA
Bacteriophage lambda (A) is a double-stranded
DNA phage that is capable of alternating between
the lytic and lysogenic replication cycles. The
amino groups of adenine and cytosine are
lated in lambda phage DNA and this protects the
phage genome against destruction by the restric-
tion endonucleases of host E. coli cells. If
methyla-
tion of the DNA does not occur, the host cell
range
of lambda phage is changed. For
if example,
lambda phage replicates in E. coli K12 host cells
thatare methionine deficient,
methylation of the
phage genome does not occur, and the lambda
phage produced a r e unable to replicate in other E.
coli K12 host cells. However, such lambda phage
still replicate in
lacking rmethylated genomes could
E. coli C host cells because that bacterial strain
does not produce restriction endonucleases that
degrade the phage DNA. Lambda phage that repli-
cate in E. coli C host cells do not produce modified
(methylated) genomes and therefore, although they
can replicate in E. coli C, they are unable to repli-
methy- cate in E. coli K12 host cells.
The regulation of lambda phage replication,
which determines whether the replication cycle is
lytic or lysogenic, is an interesting example of
molecular-level control of gene expression (Fig
8-22). During the lytic replication cycle of lambda,
transcription begins at two promoter sites during
the early phase of replication. One of the promoter
sites initiates rightward transcridtion: the other ini

Immunity
operon

Immunity repressor prm repressor

Inhibits growh of Tfi1 -
Activates cre
Antiterminator -
Alleviates B, Krestricion
Activities cre Co Origin of replicarion
Kills induced lysogens
K DNA replicati#on
Inhibits exo V
gom
Exonuclease PL Antiterminator (late)
PO
Inhibits growh exo pm
in Pa lysogens
del PR RI - lysis
Excision XIS Let OR PR

Integration int operon

Atochment IR3 Endolysin
Several proteins End join (cos
Right
operon
Endonuclease

Phoge
heod

**** ** D

M
GV

Phoge
ail

Fig. 8-22 Genome of Bacteriophage

Lambda. The
codes the functions needed for the replication of this phage.
genome of bacteriophage lambda en-
transcribed in opposite directions. This figure indicates Replication
involves two operons that
are the genes,
operons, and direction of
transcription.
 390 PART IV Microbial Replication and Growth

rex d cro cY
N ex ccro
With p

gam
gam Without pQ
ber
@xo
del del

Cin Phage
head

Phage
tail
Immediate Delayed Late
early stoge early stoge stage
P P

NOO2c3rc
Left transcript Right transcript
Detail of promoter sites
Fig. 8-23 Genome Expression of Bacteriophage Lambda. Replication of bacteriophage
lambda involves two operons (left operon with promoter P and operator O and right operon with
promoterP and operator O) that are transcribed in opposite directions. Expression of the genes
in each operon different times for production of early and late proteins. The nes
occurs at
the N protein transcribed in the immediate early stage. This in turn activates the
are
delayed early
stage. In turn, a Q protein is made that activates the late stage of protein synthesis. The cl gene
codes for an inhibitor.

tiates leftward transcription. The completion of the circular DNA then is integrated into
rightward transcription of the phage genome re- of the E. coli bacterial
a
specific site
quires the expression of a Q gene, which codes for
chromosome. Integration re-
quires a site-specific topoisomerase that is coded
a Q protein required for late gene expression
(Fig. for by the int gene. During cell growth, the lambda
8-23). The complete transcription of the lambda
genome requires expression of the N gene that
repression system prevents the expression of the
codes for an N protein. Both the N and Q genes
integrated lambda genes except for the gene cl,
which codes for the lambda
must be expressed for the transcription of the com-
grated lambda repressor. The inte
plete genome. cell DNA during
phage DNA is replicated with host
The expressionreproduction
The lambda phage genome contains a cl gene, of the host cell.
of the cl is itself
which codes for a repressor protein that binds to
the operator that controls the expression of the N
regulation. Transcription ofgene subject to
cl increases when
the
lambda repressor is
protein. In the absence of N protein synthesis, the and present in low concentrations,
high concentrations of
replication of lambda phage DNA cannot proceed. hibit further repressor protein in-
The repressor protein also binds to another opera- lambda repressor transcription.
If the
concentration of
tor region, blocking the rightward transcription of protein declines
the lambda phage DNA and, thus, the production
permit further transcription of the sufficiently to
of the Q protein. This leads to a conversion to lyso protein, coded for by the cro gene, isphage genome, a
cro
protein represses further produced. The
stopping synthesistranscription
genic replication. of
For integration of the lambda phage genome, ho
gene, thus the cl
tein of the
mologous overlapping ends of the linear lambda responsible for
preventing repressor pro-
sion of the
phage complete expres-
molecule. This
phage can carry outgenome. When this occurs,
to form a circular DNA
genome join the
lytic replication
a
cycle. The
 392 PART IV
Microbial Replieotion and Growth

Table a-s
Plant Virus
Groups of Plant Viruses
Description
Description Plant Virus

Bromovirus (brone Isometric RNA virunen

mosaic virus) Small, icORahodrnl RNA Tobaceo necrosis
viruss virus
Cauliflower mosnic Rod-shaped, helical sym-
virus (DNA Double-strandod DNA: Tobamovirun (1o
metry, single-stranded
viru
higher plants)
of
replicates in cytoplasm bacco mosalc
RNA
virus)
Cucumovirus (cu Naked, icosahedral, RNA Tobravirus (tobacco
Rod-shaped, nematode

cumber mosaic transmittod, plus-

virus)
Luteovirus (barloy
yellow dwarf virus)
Nepovirus (tobacco
ringspot virus)
Potexvirus (potato
virus X)
Potyvirus (potato
virus Y)
viruses rattle virus)
stranded RNA viruses,
segmented genom
Small isometric virus, Small RNA viruses, cubic
RNA gonome Tombusvirus (tomato
symmetry, resistant to
Polyhedral, nematode
bush stunt virus)
elevated temperatures
transmittod, RNA and organic solvents
viruses Icosahedral virus, RNA
Flexous rods, 480-580 Tymovirus (turnip genome, transmitted by
yellow mosaic
nm, RNA genom flea beetles
virus)
Flexous, rod-shaped, he Watermelon mosaic
Flexous rods, 700-950
lical symmetry, single- nm, RNA genome
virus
strandod RNA

acids to ini-
from the host plant cell. Most plant viruses exhibit RNA is capable of binding with amino
The RNA molecule
great host cell specificity and cause various symp- tiate the assembly of the virus.
toms in the plants they infect. Plant viruses are typ- forms a loop, and the protein disk subunits are
ically named on this basis (Table 8-5). added continuously to the looped end of the RNA.
the forces that
electrostatic
The RNA overcomes

of the
protein subunits, and with-
TOBACCO MOSAIC VIRUS prevent binding
the subunits will not bind at
out the RNA protein
in
Tobacco mosaic virus (TMV) is a plant virus that physiological pH and low ionic strength. Thus,
RNA the protein units will
infects tobacco and other plants. It has a single- the absence of the core,

within a helical not assemble, whereas with the RNA, self-assembly

stranded RNA genome contained
subunits that comprise the viral occurs.
array of protein the cyto-
Within infected plant cells, the replicated TMV
TMV o c c u r s within
capsid. Replication of cell. The RNA genome of particles form cytoplasmic inclusions. These viral
plasm of the infected inclusions are crystalline in nature. The chloro-
TMV codes for RNA-dependent RNA replicase
an
of a complementary plast of a TMV-infected leaf becomes chlorotic (yel-
that is used for the synthesis for the syn-
low due to loss of chlorophyll), leading to the death
s e r v e a s a template
RNA ((-) strand) to of the cell. The death of the plant cell releases com
thesis of the RNA genome
([+] strand) of TMV. The
strand RNA also acts as a tem- pletely assembled TMV particles and viral nucleic
complenentary(-) subse- acid that has not been packaged with the protein
of mRNA, which is
plate for the synthesis cell ribosomes for subunits. Within plants, completely assembled vi
translated at the plant ral particles and viral RNA can move from one cell
quently coat subunits. After
of the protein to another, establishing new sites of infection. As a
the production components of
TMV a r e syn
the RNA and protein the protein coat around consequence of the viral replication within the
of
thesized, the assembly
c a n proceed
spontaneously; plant cells, the plant develops characteristic dis-
RNA c o r e ease symptoms, including the appearance of a
the central self-assembled. mo-
saic pattern of chlorotic spots on the leaves that
is
that is, TMV TMV
involves the at-
assembly
The initiation of RNA to a protein
disc sub- gives both the disease and the virus their names
tachment of the viral 8-25). The TMV (Fig. 8-26).
s t r u c t u r e (Fig.

assembly of the c o r e
 Chapter 8 Viral Replhcotion 393

RNA

Helical capsid

Protein disk
Atachment of disk

Fig. 8-25 Assembly of Tobacco Mosaic Virus. The assembly of tobacco mosaic virus in-
volves sequential addition of protein discs to surround the viral RNA genome.

Fig.8-26 Symptoms of Tobacco Mosaic Viral Infection. Leaf infected with tobacco mosaic virus.

VIROIDS
are synthesized, there is no evidence that they have
Viroids are infectious agents that are small highly mRNA activity. Viroid RNA does not appear to be
structured RNA genomes lacking protein capsids. translated into viroid-specified polypeptides.
In essence, a viroid is simply an RNA macromole The presence of viroids sometimes manifests as
cule that can be preserved and transmitted to cells, disease symptoms in the host organism, and cer-
where it is replicated. Viroids have between 246 tain plant diseases have been identified as caused
and 375 ribonucleotides. The RNA is circular and by viroids. Diseases caused by viroids include,
RNase. The circular among others, potato spindle tuber, citrus exocor
very resistant to digestion with
RNA forms a rod-like structure as a result of exten- tis, chrysanthemum stunt, cucumber pale fruit, av-
sive base pairing within the molecule. Replication ocado sunblotch, and coconut cadang-cadang
circle mechanism that pro- Compared to viruses, viroids introduce far less
may occur by a rolling
duces a complementary RNA template. Viroid RNA genetic information into host cells. It is not yet
is within host cells, using host cell en- clear how viroids survive outside of host cells or
replicated
zymes and the viroid RNA as a template. Evidence
how they are transmitted to
compatible host cells.
DNA-dependent While the origin of viroids is unclear, some have
suggests the involvement of a

RNA polymerase in the production of new viroids.

been found to have nucleotide sequences in com
RNA strands mon, suggesting a common
ancestry.
Although both positive and negative
 402 PART Iv
Microbial Replication and Growth

Adsorpthion
Penetration
Virus
nr Uncoating

Transeription

Replication
Viral coat
Translation

Envelope proteins inserted

into host membrane

Virus released by budding

Fig. 8-33 Influenza Viruses. Influenza viruses replicate within the cell nucleus.

Influenza viruses are

only RNA-containing virusesunique in that they are the REPLICATION OF REOVIRUSES
cell nucleus that replicate in the Reoviruses contain segmented double-stranded
(Fig. 8-33). Influenza mRNA
tion from the
genomic RNA transcrip- RNA genomes. Reoviruses initially attach to
cells
host cell nucleus.
Host cell
segments occurs in the via a hemagglutininand a surface protein that in-
cleaved by nascent transcripts teracts
a
virus-encoded endonuclease,
and the
are
mainly with cells of the immune
system.
5-P end of the host Only portions of the capsid are removed, and the
for synthesis of viral transcript is
used as a primer viral genome expresses all its
mRNA from the viral functions even
Thus influenza virus genome. though it is never fully released from the
transcription complexes in-
tervene in the host mRNA The reovirus
genome is
capsid.
obtain primer molecules formaturation pathway to different double-stranded RNA
segnmented,
containing 10
the viral genome molecules.
process. transcription The double-stranded genome cannot be
Replication of the viral in cells until it is
transcribed expressed
involves the production of(-) strand RNA genome that is translated.
to (+) strand RNA
Reovirus carries in its core an
strand RNA that then serves ascomplementary (+)
a
RNA polymerase that is used for the
synthesis of new viral (-) strand template synthesis of
a
for the new viral
RNA genomes. The (+) RNA molecules are
The
viral-specified RNA polymerase alsogenomes.
sizes viral mRNAs but synthe-
placed into capsids and integral
case uses this as a capsid-RNA repli-
and requires
the host cell's own primer molecules of the template to form the (-) strand
uses
mRNAs to initiate double-stranded RNA genome.
transcription of (+) strand mRNA. The
REPLICATION OF RETROVIRUSES
thesized (-) strand RNAs are assembled newly syn-
assortment of the eight different RNA by random Retroviruses, such as the human
into capsid segments ciency viruses (HIVs) that immunodefi-
proteins. Not all influenza viral cause
eny contain the correct arrangement of prog-
genome seg-
nodeficiency syndrome (AIDS), areacquired
that use reverse
immu-
RNA viruses
ments and therefore many are a
noninfectious
their release. Mature particles exit the host after molecule within thetranscriptase
host cell
to
produce a DNA
cell by duction of the DNA (Fig. 8-34). The
budding. which slowly releasesencapsulated molecule requires pro-
(lipid-enveloped) influenza viruses from infected dependent DNA polymerase an RNA-
host cells. tocarry out reverse (reverse transcriptase)
The DNA molecule transcription of the viral RNA.
"transcribed" from the viral
 Chapter 8VnalRepleoton 403

RNA
Retrovirus
Rarver se Irenwripese

Rerverse trancription

Host cell

Nucleus

Transcription
RNAreplicatiof

Synthesis of Viral
Virol protein assembly

New virus released

by budding

Fig. 8-34 Retroviruses. Retroviruses replicate using reverse

is used to transcriptase to form DNA that
produce viral proteins and RNA genomes for viral progeny.

RNA genome by reverse

transcriptase codes for vi self coded for by pro gene. Retroviruses also con
a
ral replication wi in the host cell. Reverse tran- tain two
scriptase is a DNA polymerase that has additional
proteins coded
for by the pol gene. Like
the gag gene product, the
enzymatic activities. It can synthesize DNA with pol gene product is pro-
an RNA template (reverse transcription). It also can
teolytically cleaved into two proteins: (1) the re
synthesize DNA with a DNA template (normal
verse
transcriptase, which can transcribe RNA or
DNA into DNA and also contains RNase
DNA poly1nerase activity). It also has ribonuclease and (2) an integrase activity
H activity, which removes RNA from RNA/DNA protein, which is probably re-
sponsible for the integration of the viral-specified
hybrid molecules. DNA into the host DNA.
Retroviruses contain a central capsid core sur Some retroviruses carry a
rounded by an inner protein coat and an outer gene that codes for a
protein that causes the host cell to be transformed
lipid-containing envelope to which protein spikes from a normal cell into a cancer cell.
These retro
are atlached. Two proteins that form the envelope viruses are said to be v-onc, that
is, oncogenic.
spikes are coded for by the env gene. Several other Oncoviruses are retroviruses that cause animal cell
structural proteins are coded for by the gag gene, transformations and its members were
which is translated into a primary protein product called RNA tumor viruses; formerly
and then proteolytically cleaved into individual
they are known to be
oncogenic, that is, to cause malignancies in birds
proteins. The enzyme that cleaves this protein is it- and mammals,
including humans. The specific onc
 404
PART IV
Mikrobiol Replication and Growth

BOX8-4
A CLOSER LOOK

Replication of HIV
The human
immunodefidiencyomposed
viruses contain inus that couses AIDS cormblex retrovirus that has
g unique propertes (Fig A. These
some
of tvo identicol sngle strands of RNA (n a sense they ore diploid) and two associated
molecules of
p9 Surroun reverses
transcriptase
e that
that copy the RNA into DNA The genome is also associoted with two small proteins, p7 and
envelope thot s oc outer core proteins p17 and Inner core proteins p24. Surrounding the core proteins s an
coproteins, gp4l and 9 s as t buds out of the host cel. This outer envelope layer contoins two HIspeciic

Pyproteins of the irus oin genes common to all retroviruses, gag pol, and env, that code for the major
e s of the virus (F. B). These poyprotelns are cleared by viral protease to yield respectively the
Pp,
nD20).
py, pl7 and p24), replication enzymes (reverse
The HIV tronscribtase, protease, and integrose) andnucieocopstd
genome codes for six additional genes that are envelope
core
gYcoprotelns
c 0 n to regulate the expression of the HIV genome. Two of unique the inus. hese Benes
to

wnich codes for a regulator of mRNA these genes are tat, which codes for a
nsOctvation transcription. The Tat protein binds to an RNA sequence on trans-acuvator
response element-which increases the
protein,
and vpu
genes that assist in the amount of RNA genome-IAR
transcripts formed. HIV also contains VIit, pr, net,
epicaton begins withregulating transcription.
adsorption of the viral particles to CD4 receptors on cells of the immune
pnogytes and mocrophoges) and to system (
atochment focilitates the interaction ofglial cells of the brain (Fig.
C). The viruses attoch via their envelope proteins,helper20.
gpI20
nterocbon eposes a site on the gp4 viral envelope with another protein on the surface of the host cel, CD26. The gpI This
yoplasmic membrane leading to gycoprotein, which induces gpI20-CD26
penetration of the virus into the cel. The viral fusion ofis the viral envelope with host cell
Benome s transcribed by reverse transcriptose particle uncoated and the single-stranded RNA
required for entry into the cell This DNA, calledinto double-stranded DNA. Additional
ntegrated with the host genome at specific sites on proviral DNA, is transported into thesurface cofactor interactions are also
host cell nucleus where it
integration of the viral genome is the chromosome. The integrated viral becomes
in a
lotent state for a long performed by a viral integrase enzyme. The HIV
genome is called a provirus.
period of time. can remain integrated in the host
Events that ead to activation
HIV genome of the host cell may couse the
chromosome
begins when RNA
polymerase binds to latent HIV genome to also
become
long terminal repeat (LTR) activated.
on, tat, nef, and rev
weak and RNA genes are expressed, as well aspromoters in the
the structural
Transcription of the
region of the HIV genome.
polymerose tends to dissociate
resulting in short RNA transcripts. However, from the viral DNA easily, polyproteins gag-pol and env. The LTR Eorly
promoter is initially
protein and NF-xB to the LTR converts the binding of the Tat
strong one. The rev gene HIV promoter to a
HIV genome. The product also controls
emphasis of viral transcriptiontranscription of the
produce major structural and
the then shifs to p17 9p41 gP120
Chronic HIVinfection enzymatic proteins of the HIV.
constitutive NF-RB DNA of monocytes and macrophages leads to
regulation of NF-xB leodsbinding activity. This change in the
to enhancement
also production of of HIV LTR activity and p24
chemical mediators (ytokines)
immune defense system. There of the body's
Capsid
is even
greater
response when monocytes and mocrophages arepotentiation
encounters with viral and bacterial
of this
activated by
important in the pathogenesis of HIVpathogens.
and lead
This may be
replication. to increased
viral
During replication of HIV, the gag p9
translated into a large gene is transcribed and
cleaved by an HIiVcodedpolyprotein (p53). This
into the corepolyprotein
is
protease
pl7, and p24. The pol gene is transcribed proteins, p7, p9,
large and translated into a
polyprotein, which is also proteolytically cleaved into RNA
tronscriptase, protease, and integrose reverse genome
gene is similary
transcribed, translated polypeptides.
into a
Finally, the en
and processed into polyprotein (gpl60) Reverse
The g 20 and gbl20gp4l
and gp4l envelope gycoproteins. transcriptase
into the host envelope gycoproteins are incorporated
cytoplasmic
from the viral structural andmembrane. Intact virions are
enzymatic ossembled
RNA tronscripts are also inc proteins and some of the Human
Mature viral particles are
released
into maturing viral
slowly and particles.
continuously from
infected host cells by budding (see FiIG,8.11contnuously
tion of HIV,
tains
Immunodeficlency
A, Virus-Structure and Replica
various surfaceHIV is an enveloped
1). penetracion of HIV proteins, such as retrovirus. This virus
RNA genome and into host cells. It gpl20, that are co
reverse also contains involvedof 1in
transcriptase needed for its copies
two
replication
 Chapter 8 Viral Replikation 405

Nucleocopsid
core proleins
Envelope
gycoproteins
3-OH
-O H
5-P 9o9 pol pr pu) env c
LTR
LTR
Reverse transcriptase,
prolease, integrose, and tat or rev
ribonuclease
h eH genome has long terminal repests (LTRs) at is ends, which enables it to enter intoa
ProU tate. t also has three genes common to all retroviruses, gog pol, and env. These three
genes code for polyproteins that are subsequenty cleaved by a viral protease to lorm in
teins. Addidonally it has several
regulator protelns.

Viral
budding
O 0000

CD4receptor
OOoVrcl asenbly
Cytoplasmic
Membrane fusion Translation
membrane
and internalization

Viral RNA v Viral mRNA

Reverse transcription
Viral DNA www

Nuclear
membrane

U3RUS U3RUS
HIV Vwwwwww.sLTR- Vwwww tat
rev
Transcriptional
regulation Induction of transcription
NF-KB, SP1,
antigens, oytokines, Nucleus
other viral proteins

C, HIV replicates within T cells that have CD4 receptors on their surfaces. The CD4 acts as a re-
ceptor for viral adsorption. Wichin an infected cell, reverse transcription produces double-stranded
DNA that can be incorporated into a chromOsome of the host cell. Transcription and translation
produce the copies of the RNA viral genome and proteins for the viral capsid. Maturation of the
virus and release by budding yields mature viruses.
 400
PART IV Microbiol Replicotion and Growth
Situational Problem 8-2 production of capsid
proteins :and
coding for the RNA polymerase. Other
vin
Should RNA-dependent
iruse
the
Smallpox Virus be Destroyed? such as the
amyxoviruses,
r h a b d o v i r u s e s , param

c o n t a i n a n RNA genome
that
or nearly two centuries, scientists tried to
elim-
orthomyxoviruses
as
mRNA. In these viruses, the first

n
inate the smallpox virus (which occurs naturauy cannot serve

the replication cycle must be the transe

human hosts only) as a human disease. Suc- step in
strand RNA to (+) strand RNA, TH se
cess was achieved in 1977 tion from (-) RNADol
through global vacci their o w n RNA-dependent
nation programs carried out by the World
Health
viruses carry
which enter the
i
host cell with the iral
Organization. However, vials containing the merases,
initiate viral replication.
Smallpox virus are presently maintained in genome to
Moscow and Atlanta. Now that the REPLICATION OF PICORNAVIRUSES
disease
smallpox has been eliminated, scientists face
dilemma of whether to also the The picornaviruses
are small, single-stranded RNA
totally eliminatethe has icosahedral symme.
Smallpox virus that is being maintained in cul- viruses. The nucleocapsid
Maturation of the pi.
ture collections. Should the last and is nonencapsulated.
smallpox viruses be eliminated? What remaining
are the
try
cornaviruses occurs in the cytoplasm of the host
benefits and losses of rhinovirus have members that
doing so? How should this cell. Enterovirus and
decision be made? infect humans. Species of rhinovirus cause 25% of

all c o m m o n colds in adults. The enteroviruses in-

clude poliovirus, echovirus, hepatitis A virus, and
coxsackievirus. Diseases caused by members of
this group include poliomyelitis, infectious hepati-
REPLICATION OF RNA tis, and foot and mouth disease. Picornaviruses
ANIMAL VIRUSES also cause mild infections of the gastrointestinal
RNA animal viruses exhibit
many diverse and respiratory tracts.
for replication. n some cases, the RNA strategies The most studied picornavirus, poliovirus, is
the virus acts as an mRNA on
genome of
the host cell,
entering very specific in its adsorption to cells (Fig. 8-31). It

5-P 3-OH
Poliovirus
mRNA

Translation
RNA dependent
polymerase
3-OH 5-P

Replicative
torm

Polyprotein RNA dependent

Cleavage by polymerase
proteasees 5-P 3-OH

New poliovirus RNA

Viron proteins and

RNA polymerase
Fig. 8-31 Poliovirus. In poliovirus (colorized micrograph, blue), RNA serves as
a
for production of a polyprotein that is then cleaved to form capsid proteins and RNA messenger
The RNA also serves as the template for producing a replicative form that in turn is polymerase.
for new poliovirus genomic RNA. the template
 Viral Replication 401
Chapter 8

is mediated by the viral protein, VP1. The po- The genome of the influenza
viruses is a seg
of eight different RNA
liovirus virions are internalized by endocytosis mented genome composed
different monocistronic
nd RNA is released into the cytoplasm. Interest- molecules that code for a
and 8 are spliced to-
ingly, the poliovirus single-stranded RNA codes for mRNA molecule (segments 7
One of the
two proteins).
avery large polypeptide chain, a polyprotein gether and each codes for codes for the
which is cleaved by proteases to form many differ- RNA genome segments specifically for
required
ent proteins. The proteases are encoded by both the RNA-dependent RNA polymerase
virus and the host cell. The proteins formed by pro- transcription of the viral genome.
There are two types of protein
spikes on the in-
tease cleavage include an RNA-dependent RNAA H and N (Fig.
polymerase and four proteins of the viral capsid. fluenza virus envelope, designated
different types of H pro-
The RNA polymerase is used to produce a comple- 8-32). There are at least 13
found on different
mentary replicative RNA strand that can act asa teins and 9 types of N proteins
influenza virus strains.
The hemagglutinin (H)
template for the synthesis of new viral genomes. of the vi-
for the attachment
The capsomer proteins assemble into pentamers spikes are responsible brings about
that condense into capsids. The assembly of the ral particle to the cell. Hemagglutinin
with the cytoplas-
the fusion of the viral envelope
capsid and insertion of the RNA genome is fol- The cell receptor is
lowed by the release of numerous viral particles. mic membrane of the host cell.
on cell surface gly-
Release of the poliovirus occurs because blockage neuraminic acid residues found
of protein spike is a
of cellular protein synthesis by the poliovirus leads coproteins. The second type
neuraminic acid
to breakdown of lysosomes. The digestive enzymes neuraminidase (N) that cleaves
facilitate the
released from the lysosomes cause cell lysis. residues from the cell surface and may
the host cell.
release of newly formed virus from
REPLICATION OF ORTHOMYXOVIRUSES inside the host
Influenza virus is transported
of the endosomal
influenza viruses, cell by endocytosis. The low pH
The orthomyxoviruses, such as
that results
RNA viruses vesicle causes a change in the H spikes
are single-stranded, enveloped (-) the vesicle mem-
in the viral envelope fusing with
that exhibit helical symmetry. Many influenza the cytoplasm.
indi- brane. This releases viral cores into
viruses are referred to by common names that to the
cate their geographic origins, such as Hong Kong The RNA-protein complex then migrates
host cell nucleus.
flu virus.

Hemogglutinin (H)
Segmented RNA
Lipid Protein
membrane capsid
Polymerase

N tetramer
A
Stalk

Peptide Head
binding sites
T E Neuraminidase (N)
H trimer

Lipid membrane

A, Influenza viruses
8-32 Influenza Viruses.
Fig. that pro-
have envelope proteins
called H and N spikes
Colorized micrograph
of in-
trude from the surface. B,
fluenza viruses. (81,000x).