Transcript Viroids are small (~300 nt) circular RNA molecules that are

Defective Interfering RNAs
DI RNAs are formed by deletion and recombination during viral RNA
replication
They require helper virus to replicate and to be packaged into DI particles
They must retain all cis-acting signals required for replication and
packaging
Most DI RNAs are not translated
They interfere with the replication of helper virus by competing with it for
resources
Because they interfere, they may serve to ameliorate the symptoms of viral
infection
Amelioration has been shown in model systems, but no convincing evidence
for amelioration of any natural infection by DIs has been produced
A Rhabdovirus (VSV) and DIs derived from it
Viral RNA 3’
5’
le
N
P
M
G
L
tr
Types of VSV DI’s
tr
c
Class II - Hairpin
L
tr
L
Class I - Panhandle
L
tr
tr
c
Class III - Simple internal deletion
le
N
P
M
G
L
tr
N
P
M
G
L
tr
Class IV - Mosaic
tr
c
Minus strand RNA sequences
Plus strand sequences
le Leader
tr Trailer
tr
c
le
Complement of trailer
A Coronavirus (MHV) and DIs derived from it
le
ORF1a
ORF1b
MHV viral RNA
An
(~30 kb)
MHV DIs
DIssA
(~25 kb)
An
kilobases
0
2
10
22
24
26
28
30
An
DI-a
(5.5kb)
An
DIssF
An
DIssE
An
(3.6 kb)
(2.2 kb)
B36
(2.2kb)
kilobases
0
2
4
6
The Effect of Defective Interfering Particles on Viral Evolution
Yield of infectious virus or DI RNA
A.
1
Concentration
of DI RNA
Concentration
of helper virus
2
1) Early in an undiluted passage series,
standard virus replicates well, but DI’s are
beginning to accumulate.
2) DIs replicate at high efficiency, and
interfere with standard virus.
3) So little standard virus is produced that
there is little helper function, and DI
replication drops.
3
4
4) With little DI replication, interference is
reduced and standard virus titers rise again.
Time in Days (Tens of Virus Passages)
Yield of infectious virus or DI RNA
B.
ST 1
ST 2
DI 1
DI 2
1’
4’
2’
3’
Time in Months (Hundreds of Virus Pas sages)
1’) DI 1 interferes strongly with replication
of standard virus (ST 1).
2’) A new variant of standard virus (ST
emerges that is resistant to interference by
DI 1 and does not serve as a helper for DI
2)
1.
3’) Without helper assistance, DI 1
disappears, and ST 2 replicates vigorously.
4’) New DIs of ST 2 (DI 2) appear and begin
to depress ST 2 replication.
SATELLITES AND SATELLITE VIRUSES
GROUP
dsDNA Satellite
Bacteriophage P4
GENOME
SIZE
11.5 kb (10-15 genes)
ssDNA Satellite Viruses
Dependovirus (AAV)
dsRNA Satellites
M satellites of yeast
ssRNA Satellite Viruses
Chronic bee-paralysis
virus associated satellite
Tobacco necrosis virus
satellite
HELPER
VIRUS
HOST(S)
P2 bacteriophage
Bacteria
-
COMMENTS
All structural proteins
from P2
4.7 kb
Adenovirus
H erpesvirus
Vertebrates
See Table 6.15
1 to 1.8 kb
Totiviridae
Yeast
Encode “killer” proteins;
encapsidated in helper
coat protein
1239 nt
Chronic bee-paralysis Bees
virus
Tobacco necrosis virus Plants
1.7 kb
H epatitis B virus
Humans
Encode two forms of 
antigen, encapsidated by
helper proteins
0.8 to 1.5 kb
Various plant viruses
Plants
Encode nonstructural
proteins, rarely modify
disease syndrome
C-type linear RNA satellites
<0.7 kb
Various plant viruses
Plants
D-type circular RNA satellites
“virusoids”
~350 nt
Various plant viruses
Plants
Commonly modify disease
caused by helper
Self-cleaving molecules
ssRNA Satellites
H epatitis delta virus
B-type mRNA satellites
3 RNAs, each 1kb
Viroids
Viroids are small (~300 nt) circular RNA molecules that are
infectious and commonly cause disease in plants
They are not translated and are not packaged into proteincontaining particles
Transmission is commonly inadvertent, occurring during
horticulture
Replication of the RNA is effected by host polymerases,
usually RNA Polymerase II
Some viroids are capable of self-cleavage and self-ligation to
form the circular RNA “genome”. Others use cellular
enzymes
VIROIDS
GROUP
GENOME
SIZE
HOST(S)
COMMENTS
Group A:
ASBVd group
246 to 339 nt Plants
Replicate by a symmetric strategy
in chloroplasts of infected plants;
can form self-cleaving hammerhead
ribozymes in both plus and minus strands
246 to 375 nt Plants
Noncleaving, r eplicate by an asymmetric
strategy in nucleus of infected cells
Group B
PSTVd, HSVd,
CCCVd, ASSVd,
and CbVd-1 groups
Viroids do not require helper viruses
Vd = viroid; ASBVd = avocado sunblotch viroid; PSTVd = potato spindle tuber viroid;
HSVd = hop stunt viroid; CCCVd = coconut cadang-cadang viroid; ASSVd = apple scar skin viroid;
CbVd-1 = Coleus blumei viroid-1.
Structures of Different Subgroups of Non-Self-Cleaving Viroids
A.
PSTVd
CNNGNGGUUCCUGUGGG
HSVd
AA
UGGGG
C
UCCCC
CCCVd
UGGGGAA
C
UCCCC
B.
GGAUCC CCGGG
CGCUUCAG
GAAACCUGGAGCG
AACAAAGGUGGCCC
AUCA
UCGAAGUC
C G
C
G
C G
C G
U A
A
A
G
A
G C
G C
A U
C G
U•G
U A
C G
AGG A GCC CCGG
G AAC
UC
GC
CCA ACGGU GGCC
AG CGC AG
CU
AAA
GAGGG AUCC GCCGGG
CUC
AACA UAGG
U GGCCC
AUCA
PSTVd
ASSVd
CbVd1
CNNGNGGUUCCUGUGGG
CNNGNGGUUCCUGUGGG
UCGUCG UCGAC
GAAGG
CAGG GAGCUG
AUCGCC
C
UUC GCCU GCAGAAC
AAU
CU GGG
CGCUGC
GGAAACGUU
A GCG
Conserved Sequences
Domains
Terminal Conserved Region (TCR)
Terminal Left (T
Terminal Conserved Hairpin (TCH)
Terminal Right (T
Central Conserved Region (CCR)
GG
L)
R)
Pathogenesis (P
Variable (V)
)
Secondary Structure of Peach Latent Mosaic Viroid and its
Hammerhead Ribozymes
A.
1
339
3’
B.
Minus strand
20
AU
G
A
G
A
G
A
G
20
40
CA A
55 U
U
A
A
C G
C
C
G
C
C
A G U U U C G C U A U U C A A GG CU CA U C A G U G G C U U A G C C A G A C U U
A
285
C
U CA A A G C G G U A A G U U CU G A G U A G U C A C C G A A U C G G U CU G A GA
A A
A
C
A
U
U
C
A
UUU
300
G
320
GU
1
A C G G C GA
U G C CG A
G
G
A
G
U
U
U
U
C
A
A
U A
5’
C
U
C
55
A
U
A
A
G
U
C
U GGG CU
GU
CA C C CGA
40
C.
Plus strand
320
AU
A
A
A
G A G A C GA
CU C U G A
GU
G
3’
U
U
U
C
U
C
AA
C AG
5’
G
A
285
A
G
A
G
U
C
UGUG CU
U
CA CA CGA
300
A
Hepatitis Delta
Hepatitis  is commonly called a virus and abbreviated HDV, although
technically it is a satellite of hepatitis B virus that is related to viroids
HDV requires HBV as a helper and exacerbates the symptoms of HBV
infection
It can establish a chronic infection if HBV becomes chronic
The HDV genome of 1.7 kb is a covalently closed circular RNA molecule
The genome is effectively a viroid into which has been inserted a single gene
encoding the hepatitis  antigen
Replication of the genome is carried out in the nucleus by host RNA
Polymerase II, and  antigen is required
The HDV core is composed of two forms of  antigen and the RNA; budding
to produce virions uses helper HBV surface glycoproteins
Global Distribution of Hepatitis Delta Infection as measured by the incidence of
Hepatitis  antigen in the serum of Hepatitis B patients with Hepatitis
K
u
w
ai
t
Percent of Hepatitis B Patients
with Hepatitis  Antigen
No Data
0-5%
6-20%
21-60%
>60%
Equator
Patterns of Anti-Hepatitis Antibodies, Hepatitis Antigens, and
Elevated Liver Enzymes in Patients Coinfected with Hepatitis B and
Hepatitis 
A. Simultaneous coinfection with hepatitis B and hepatitis

Convalencence and
Recovery
Acute Dis ease
ALT
HBs Ag
HDV RNA
HD Ag
0
2
4
6
8
10
12
24
32
B. Acute hepatitis  after superinfection of a chronic hepatitis B patient
Acute Dis ease
HD Ag
0
2
4
6
8
10
12
24
32
C. Chronic hepatitis after superinfection of a chronic hepatitis B patient
Severe chronic hepatitis B and chronic hepatitis

HD Ag
0
2
4
6
8
10
12
24
32
Weeks after Exposure (coinfection or superinfection)
Serology
Antigens, RNA, and liver function markers
ALT level (alanine aminotransferase)
HD RNA (hepatitis
 RNA)
HD Ag (hepatitis  antigen)
HBsAg (hepatitis B surface antigen)
Antibody levels
IgM
Anti HBc (anti-hepatitis B core)
IgG
IgM Anti HD (anti-hepatitis
IgG
 antigen)
A.
Viroid Domain
HD-Ag Coding Domain
1015
Genomic RNA ~1683 nt
795
0/1683
(Ori)
1631
1638
688/689
950 1017
Antigenomic RNAs
3’
An
0.8 kb mRNA
1601
L-HD Ag
S-HD Ag
5’
903/904
Antigenome
template RNA
795
5’
1638
An
0/1683
3’
1015
688/689
RNA editting site
Ribozymes
Ribozyme cleavage sites
ORF for HD antigen
903/904
A n Polyadenylation site
Initiation codon
Termination codon
RNA-binding
domain
B.
S-HDAg (195aa)
136-146
195-214
97-107
66-88
31-52
2-27
L-HDAg (214aa)
Amino acids
200
160
120
80
40
0
Coiled-coiled sequence (dimerization signal)
Packaging signal
Nuclear localization signal (NLS)
S-HD Ag-specific epitope
Arginine-rich motif (ARM)
Cryptic RNA-binding domain
Editing of Hepatitis d Antigen
S-HDAg is 195 aa in length and terminates at a UAG codon
S-HDAg is required for RNA replication
Editing occurs to change this codon to UGG during infection
Editing occurs by deamination of A
The edited mRNA is translated into L-HDAg of 214 aa
L-HDAg suppresses RNA replication
Both L-HDAg and S-HDAg are present in the core of HDV
The envelope proteins of HBV are used to assemble HDV virions
Prion Diseases
Prion diseases, also called transmissible spongiform
encephalopathies, are slowly progressing but uniformly
fatal neurological diseases
They are characterized by alterations in the metabolism of a
brain protein called the prion protein
The normal function of the prion protein is not known
The nature of the infectious agent remains controversial but
a favored hypothesis is that an altered form of the prion
protein is itself the infectious agent
Human TSEs
Human TSEs may occur sporadically, may be inherited , or
may be acquired by infection (the infectious agent is often
called the scrapie agent)
Sporadic TSE, usually a form of CJD, occurs at a frequency of
about one per million
Inherited or familial TSEs are always associated with
mutations in the prion protein, and the probability of
developing TSE may approach 100% in the case of some
mutations
Sporadic or familial TSEs are usually transmissible once they arise
HUMAN PRION DISEASES
Disease (Abbreviation)
Experimental
Hosts
Kuru
Primates, mice
Creutzfeldt-Jakob disease (CJD)
Primates, mice
iCJD (iatrogenic)
nvCJD(new variant)
fCJD (familial)
Gers tmann-Strauss ler-Scheinker
syndrome (GSS)
Fatal s poradic ins omnia (FSI)
Infection through ritual cannibalism
Infection from prion-contaminated human
growth hormone, dura mater grafts, etc.
Somatic mutation or spontaneous conversion
of PrP c to PrP Sc
Ingestion of bovine prions?
Germline mutation in PrP gene
sCJD (sporadic)
Fatal familia ins omnia (FFI)
Cause of Disease
Germline mutation in PrP gene
Primates, mice
Germline mutation in PrP gene (D178N, M129)
Somatic mutation or spontaneous conversion
of PrP c to PrP Sc
Human Prion Diseases
Although all human TSEs are characterized by changes in the
metabolism of the prion protein, the symptoms differ, in part because
different areas of the brain are affected.
Kuru is characterized by progressive ataxia leading to total
incapacitation. It was spread by canabilism.
CJD is characterized by dementia and ataxia. It may occur sporadically,
may be contracted iatrogenically, or may be familial.
nvCJD is characterized by psychiartric symptoms, usually depression.
Onset of symptoms occurs much earlier in life than CJD. It is thought to
be contracted by consumption of beef from cattle infected with BSE.
FFI is characterized by intractable insomnia. It is usually an inherited
disease but sporadic cases have been reported.
GSS is characterized by cerebellar disorders and a decline in cognitive
ability. It is an inherited disease.
Two Victims of Kuru Among the Fore People
Spongiform Encephalophy
In
Creutzfeld-Jacob Disease
A and B illustrate two
different forms of vacuolar
degeneration of the gray
matter
C illustrates astrocytic gliosis
PRION DISEASES of Other Vertebrates
Disease (Abbreviation)
Natural Host
Scrapie
Sheep and goats
Mice, hamsters, rats
Infection in genetically susceptible
sheep
Transmissible mink encephalopathy (TME)
Mink
Hamsters, ferrets
Infection with prions from
sheep or cattle
Chronic wasting disease
Mule deer, white tail
deer and elk
Ferrets, mice
Cattle
Mice
Bovine spongiform encephalopathy (BSE)
Experimental
Hosts
Cause of Disease
Unknown
Infection with prion-contaminated
meat and bone meal
Feline spongiform encephalopathy (FSE)
Cats
Mice
Infection with prion-contaminated
beef
Exotic ungulate encephalopathy (EUE)
N yala, oryx and
greater kudu
Mice
Infection with prion-contaminated
meat and bone meal
Isoforms of the Human Prion Protein
PrP c
Precursor human prion protein
N
S
22aa
N N
181 197
H1
H2
S
231
H3
209aa
S C
23aa
Maturation
Mature cellular prion protein
CHO CHO
PrP c
CHO
H1
H2
H3
Proteinase K
CHO
Conversion
Modified prion protein
Truncated prion protein
PrP Sc
CHO CHO
CHO CHO
Proteinase K
209aa
CHO
+
~142 aa
N-linked carbohydrate chains
H1
GPI (glycosyl phosphatidylinositol)
Repeats of 8 amino acids,
PrP27-30
PQ
HGGGWGQ
H elical regions of PrP
-sheets in PrP c
c
Mutations in the Human Prion Protein Gene
Polymorphisms associated with prion disease
E200K
Ins ertion of
2-9 octarepeats
Pre HPrP c
P105L
A117V
P102L
S
Polymorphisms that are
phenotypically wild type
F198S R208H
T183A
V210I
V180I
D178N
H1
Deletion of an
octarepeat
Beta sheets
Q217R
M232R
H2
M129V N171S
H1
H3
S
E219K
Alpha helices
D178N - Point mutation associated with FFI
P102L - Point mutations associated with GSS
E200K- Point mutations and insertions associated with familial CJD
M129V - homozygosity at this locus increases susceptibility to sporadic CJD
Structure of the Prion Protein in Solution
aa121
S1
H2
S2
H3
aa231
H1
Conversion of PrPc to PrPsc
The conversion of PrPc to PrPsc involves a transition from
helices to beta-sheet and the acquisition of partial resistance
to protease.
Such a conversion will occur in vitro when PrPc is exposed to
PrPsc, which appears to act as a seed to induce the conversion
of PrPc.
Mouse studies have shown that a neuron must express PrPc
before is is susceptible to being killed by exposure to the
scrapie agent (PrPsc?).
Why neurons die upon exposure to the scrapie agent is not
known, nor is the nature of the toxic substance that leads to
neuronal death understood (is it PrPsc?).
The Importance of the Prion Protein for TSE Disease
Mice that do not express the prion protein do not develop TSE
upon infection with the scrapie agent
Mice that overexpress the prion protein are more sensitive to
the development of TSE and may even develop TSE
spontaneously
There is a species barrier to infection by scrapie derived from
another animal because of differences in the sequence of the
prion protein in different animals
Mice that express, for example, the hamster prion protein are
more easily infected by scrapie from hamsters than scrapie
from mice, and vice versa
The Protein Only Hypothesis
The protein only hypothesis proposes that the infectious agent that
transmits TSE is PrPsc.
In this model, PrPsc is a seed that induces the formation of more of itself.
The seed may arise spontaneously or by infection with PrPsc. Mutations in
the prion protein make formation of the seed more probable.
Transmission of ingested PrPsc to the brain might require “replication” of
the agent in lymph nodes followed by invasion of peripheral nerves.
Biochemical studies of the scrapie agent have found nothing other than PrPsc
in purified preparations, but because of the very low specific infectivity of
such preparations, contamination by a virus or other infectious agent cannot
be rigorously excluded.
The Protein Only Hypothesis (con)
One of the major criticisms of this hypothesis is that
multiple strains of scrapie exist that cause different
symptoms, but there is only one prion protein. How can
one protein assume multiple conformations that “breed
true” and why should different symptoms be produced?
There have been shown to be at least two different strains
of scrapie whose PrPsc can be distinguished on the basis
of their structure, and that “breed true”. The conversion
to the two different structural forms can be demonstrated
in vitro. Thus, it is possible that multiple conformational
states do in fact exist that can act as a seed to induce the
formation of more of themselves.
Spread of the BSE epidemic in the British Isles
1987
Avo
n
1989
Avo
n
1991
Avo
n
ð
1993
1995
Incidence of BSE
Cases per 1000 head of cattle
Avo
n
Avo
n
None
<1
1 to 2
2 to 3
3 to 4
4 to 5
>5
Confirmed Cases of BSE in British Cattle (1986-1996)
20
Reported BSE cases per one/half year in thousands
1
2
3
4
15
10
5
0
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
% of Total nvCJD Onsets
A.
40
2.0
30
1.5
20
1.0
10
0.5
10
20
30
40
50
Age in Years
60
70
80
Annual Sporadic CJD Deaths/
Million Population
Age Distribution of vCJD and sCJD in Britain
Chronic Wasting Disease
A TSE of deer and elk
Found in the western U.S. and Canada
Has been known for at least 35 years
In areas of Wisconsin 3% of the white tailed deer are infected
There have occurred 5 unusual cases of CJD in the U.S.
Victims were age 30 or younger
Two were hunters and one was the daughter of a
hunter and regularly ate deer or elk
The disease was different from BSE
Postulated Topology of PrP Proteins in Cellular Membranes
A. Conformations of the human prion protein translated
N N
181 197
STE TMI
S
N
S
E1
in vitro
S
231
C
E2
C N
N
C
Lumen
Microsomal
membrane
Cytosol
N
C
CtmPrP
secPrP
N tmPrP
B. Maturation of secPrP in cells
s
N
N
Lumen
ER
C
Lumen
ER
Cytosol
CHOCHO
Maturation
s
Transport through
post-ER compartments
Plasma
membrane
Cytosol
secPrP
STE - stop transfer effector
CHOCHO
N
Cytosol
PrPc
PrPc
CHO
N-linked carbohydrate chains
TMI - transmembrane domain
GPI- glycosyl phosphatidylinositol
E1 (epitope for MAb 3F4)
Repeats of 8 amino acids
E2 (epitope for MAb 13A5)
Prions in Fungi
If prions are defined as
Proteins that have two or more conformational forms
One form of which is soluble
Other forms aggregate and can induce conversion of
the soluble form to the aggregated form
Then prions have been found in yeast
The prion form of protein represents loss of function
It occurs spontaneously at low frequency
Once it appears the prion form is dominant but
reversible by treating with denaturants
Comparison of two yeast prion proteins
Yeast Prion Protein Ure2p
Prion-inducing
domain
1
N-repression domain
Glutathione-S-transferase
65 80
151 158
221 227
348 354
Yeast Prion Protein Sup35p
Prion-propagating
domain
1
Translation-termination
domain
114
254
Prion-promoting sequences
Prion-inhibiting sequences
Domains with known non-prion functions
685