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

Sequences in DIa expanded 4x

Sequences in DIb expanded 4x

DIblength = 2286; complexity= 1169

CAP

5' 3'

A n

DIa

length = 1652;complexity = 680

Structural ProteinsNonstructural Proteins

Virus Genome

Subgenomic Promoter

Genome Organization of Defective Interfering Particles of Semliki Forest Virus

A Rhabdovirus (VSV) and DIs derived from it

N P M G LViral RNA

Types of VSV DI’s

Class I - Panhandle tr

le tr

trc

L trtrc

Class II - Hairpin

Class III - Simple internal deletionN G

Class IV - Mosaic

Minus strand RNA sequencesPlus strand sequences

le Leader

trTrailer

trc

Complement of trailer

3’ 5’

P M L trle

trc

N P M G Lle tr

L

L

A Coronavirus (MHV) and DIs derived from it

MHV viral RNA

DIssA (~25 kb)

0 2 4 6kilobases

An

ORF1a ORF1ble(~30 kb)

A n

A n

A nDI-a (5.5kb)

DIssF (3.6 kb)

DIssE (2.2 kb)

A nB36

(2.2kb)

kilobases30282624220 2 10

An

MHV DIs

Concentration of DI RNAConcentration of helper virus

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.

4) With little DI replication, interference is reduced and standard virus titers rise again.

Time in Days (Tens of Virus Passages)

1 2

3 4

Yie

ld o

f in

fect

iou

s vi

rus

or

DI

RN

A

1’) DI 1 interferes strongly with replication of standard virus (ST 1).

2’) A new variant of standard virus (ST 2) emerges that is resistant to interference by DI 1 and does not serve as a helper for DI 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.

ST 1ST 2

Time in Months (Hundreds of Virus Passages)

Yie

ld o

f in

fect

iou

s vi

rus

or

DI

RN

A

1’

2’ 3’

4’

DI 1DI 2

A.

B.

The Effect of Defective Interfering Particles on Viral Evolution

SATELLITES AND SATELLITE VIRUSES

HOST(S) COMMENTSGROUP GENOME SIZE

HELPER VIRUS

dsDNA SatelliteBacteriophage P4 11.5 kb (10-15 genes)P2 bacteriophage Bacteria All structural proteins

from P2ssDNA Satellite Viruses

Dependovirus (AAV) 4.7 kb AdenovirusHerpesvirus

VertebratesSee Table 6.15

dsRNA SatellitesM satellites of yeast 1 to 1.8 kb Totiviridae Yeast Encode “killer” proteins;

encapsidated in helper coat proteinssRNA Satellite Viruses

Chronic bee-paralysis virus associated satellite

3 RNAs, each 1kb Chronic bee-paralysis virus

Bees

Tobacco necrosis virus satellite

1239 nt Tobacco necrosis virus

Plants

ssRNA SatellitesHepatitis delta virus 1.7 kb Hepatitis B virus Humans Encode two forms of

antigen, encapsidated by helper proteins

B-type mRNA satellites 0.8 to 1.5 kb Various plant virusesPlants Encode nonstructural proteins, rarely modify disease syndrome

C-type linear RNA satellites<0.7 kb Various plant virusesPlants Commonly modify disease caused by helper

D-type circular RNA satellites “virusoids”

~350 nt Various plant virusesPlants Self-cleaving molecules

-

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 protein-containing 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

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.

ASBVd group Plants246 to 339 nt Replicate by a symmetric strategy in chloroplasts of infected plants; can form self-cleaving hammerhead ribozymes in both plus and minus strands

Group A:

Plants246 to 375 nt Noncleaving, replicate by an asymmetric strategy in nucleus of infected cells

PSTVd, HSVd, CCCVd, ASSVd, and CbVd-1 groups

HOST(S) COMMENTSGROUP GENOME SIZE

VIROIDS

Viroids do not require helper viruses

Group B

Variable (V)

Terminal Conserved Region (TCR) Terminal Left (TL) Pathogenesis (P)

Terminal Conserved Hairpin (TCH) Terminal Right (TR)

Central Conserved Region (CCR)

DomainsConserved Sequences

CNNGNGGUUCCUGUGGG CGCUUCAG GG A UCC CCGGGG

AAACCUGGAGCG

CGAAGUCU AACAAGGUGGCCCAUCAA

A GG A GCC CCGG GCAACUC

CCA CUCGG GGCC CG

CAGAGA

G

U

GGGG

CCCC

AAU

UC

GAG AUCC CCGGGGAAAGG

CUC UAGG GGCCCAACA AUCAU

GGGG

CCCC

AAU

UC

UCGUC G UCGACGA AGG

CAGGCGAGCUGAUCG

CC

CNNGNGGUUCCUGUGGG

AA AGGA CGUU GCG

C GGG CCU GCAG CGCUGC GG U UUC G AAC AAUCNNGNGGUUCCUGUGGG

PSTVd

HSVd

CCCVd

ASSVd

CbVd1

A. B.

C GC GC G

C GU AA AG AG CG CA UC GU G•U AC G

PSTVd

Structures of Different Subgroups of Non-Self-Cleaving Viroids

GGG

GGG

G

G

GA

G

G

G

CC

CCCC

C

CC

C

UUA

A UG

AA

AA

A AA

U U

UU 40

13’ 5’

20

55

G CUA

G CU A

U AU A

GCA U

U U

GUG

G

GG

G

G

GG

G

A

U

CC

CACC

C

UC

A

CU

A

A UA

AA

AA

A A

AU U

UC300

3’ 5’

320

U GU A

C GU A

GCA U

285U A

UU

U

G

G

G

GA

A

A55

AAAAA

A A AA

AA A

AA

AA

AAA

AAA

AA

AA

AA

C

C

CC

C

C

C

CC

CC

C

CC

C C C

C C C

C

C

CCC C

CUUU

U

UUU

U

U

U

U

UUU

UU

U

UUUU

U

UUUU

UG

G

GGG G

GG

G G

GG

G

G

G

G G

G

G G

G

AA

1

339

320 300

20 40

285G

G

A.

B. C.Minus strand Plus strand

Secondary Structure of Peach Latent Mosaic Viroid and its Hammerhead Ribozymes

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

Kuwait

No Data0-5%

6-20%21-60%>60%

Percent of Hepatitis B Patients with Hepatitis Antigen

Global Distribution of Hepatitis Delta Infection as measured by the incidence of Hepatitis antigen in the serum of Hepatitis B patients with Hepatitis

Equator

Anti HD (anti-hepatitis antigen)

ALT level (alanine aminotransferase)

HD Ag (hepatitis antigen)

HBsAg (hepatitis B surface antigen)

HD RNA (hepatitis RNA)

Antigens, RNA, and liver function markers Antibody levels

IgMIgG

IgMIgG

Anti HBc (anti-hepatitis B core)

HD Ag

Severe chronic hepatitis B and chronic hepatitis

C. Chronic hepatitis after superinfection of a chronic hepatitis B patient

Weeks after Exposure (coinfection or superinfection)

0 2 4 6 8 10 12 24 32

HD Ag

B. Acute hepatitis after superinfection of a chronic hepatitis B patient

Acute Disease

0 2 4 6 8 10 12 24 32

HBsAg

HDV RNA

HD Ag

A. Simultaneous coinfection with hepatitis B and hepatitis

Acute DiseaseConvalencence and

Recovery

ALT

0 2 4 6 8 10 12 24 32

Serology

Patterns of Anti-Hepatitis Antibodies, Hepatitis Antigens, and Elevated Liver Enzymes in Patients Coinfected with Hepatitis B and

Hepatitis

688/689

903/904

Ribozyme cleavage sitesRibozymes

Initiation codon Termination codon

ORF for HD antigen

1015RNA editting site

An Polyadenylation site

An

L-HD AgS-HD Ag

Antigenomic RNAs

0.8 kb mRNA

688/689

903/904

0/16831638

Genomic RNA ~1683 nt 1015

795 1638

795

An 0/16835’

3’

Viroid Domain HD-Ag Coding Domain

Antigenome template RNA

5’

3’

A.

1638

(Ori)1631

950 16011017

B.

04080120160200

S-HDAg (195aa)

L-HDAg (214aa)

Amino acids

RNA-binding domain

97-107136-146 66-88 31-52195-214

Coiled-coiled sequence (dimerization signal) Packaging signal

Nuclear localization signal (NLS) S-HD Ag-specific epitope

2-27

Arginine-rich motif (ARM) Cryptic RNA-binding domain

0/1683Genomic RNA

An

An5’

5’903

by host enzymes

Translation

HDAg

Ribozyme cleavage

HDAg mRNA

1)

2)

0/1683An

0/1683An

903/904 Ribozyme cleavage at 903/904

Self-ligation

Antigenomic RNA 795 16310/1683

3)

4)

5)

6)

The antigenome is replicated by a similar mechanism using steps 1), 4), 5), and 6), with ribozyme cleavage at 688/689

7)

688/689

0/16831631795Genomic RNA

Processing and polyadenylation

5’

16315’

0/1683An 5’

903/904

1631

An

Replication of Hepatitis- RNA

Editing of Hepatitis d Antigen

S-HDAg is 195 aa in length and terminates at a UAG codon

Editing occurs to change this codon to UGG during infection

Editing occurs by deamination of A

S-HDAg is required for RNA replication

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) Cause of Disease

Fatal sporadic insomnia (FSI) Somatic mutation or spontaneous conversion

of PrPc to PrPSc

Gerstmann-Straussler-Scheinker syndrome (GSS)

Germline mutation in PrP gene

Experimental Hosts

Kuru Infection through ritual cannibalismPrimates, mice

Fatal familia insomnia (FFI) Germline mutation in PrP gene (D178N, M129)Primates, mice

iCJD (iatrogenic) Infection from prion-contaminated human growth hormone, dura mater grafts, etc.

nvCJD(new variant) Ingestion of bovine prions?fCJD (familial) Germline mutation in PrP gene

Creutzfeldt-Jakob disease (CJD) Primates, mice

sCJD (sporadic) Somatic mutation or spontaneous conversion

of PrPc to PrPSc

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.

CJD is characterized by dementia and ataxia. It may occur sporadically, may be contracted iatrogenically, or may be familial.

Kuru is characterized by progressive ataxia leading to total incapacitation. It was spread by canabilism.

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 Cause of DiseaseExperimental Hosts

Scrapie Sheep and goats Infection in genetically susceptible sheep

Mice, hamsters, rats

Transmissible mink encephalopathy (TME)Mink Infection with prions from sheep or cattle

Hamsters, ferrets

Chronic wasting disease Mule deer, white tail deer and elk

Unknown

Ferrets, mice

Bovine spongiform encephalopathy (BSE)CattleInfection with prion-contaminated meat and bone meal

Mice

Feline spongiform encephalopathy (FSE)CatsInfection with prion-contaminated beef

Mice

Exotic ungulate encephalopathy (EUE)Nyala, oryx and greater kudu

Infection with prion-contaminated meat and bone meal

Mice

22aaN C

209aa 23aa

N181

N S197 231

Precursor human prion protein PrPc

Mature cellular prion protein PrPc

Proteinase K

Modified prion protein PrPSc

209aa ~142aa

Truncated prion protein PrP27-30

Proteinase K

Maturation

Conversion

CHON-linked carbohydrate chains

GPI (glycosyl phosphatidylinositol)

H1 Helical regions of PrPc

-sheets in PrPc

Repeats of 8 amino acids,

+

P GGGWGQQH

SS H1 H2 H3

H1 H2 H3

CHOCHO

CHO

CHO

CHOCHO CHO CHO

Isoforms of the Human Prion Protein

Insertion of 2-9 octarepeats

M232RA117V V180I Q217R

P102L D178N

P105L T183A V210IF198SR208H

E200K

SS H1 H2 H3Pre HPrPc

Polymorphisms that are phenotypically wild type

Deletion of an octarepeat

M129V N171S E219K

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

Polymorphisms associated with prion disease

Beta sheets H1 Alpha helices

Mutations in the Human Prion Protein Gene

aa121

aa231

S1

S2

H1

H2

H3

Structure of the Prion Protein in Solution

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.

Mouse studies have shown that a neuron must express PrPc before is is susceptible to being killed by exposure to the

scrapie agent (PrPsc?).

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.

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.

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.

Transmission of ingested PrPsc to the brain might require “replication” of the agent in lymph nodes followed by invasion of peripheral nerves.

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.

Avon

1987

Avon

1989

Avon

1991

Avon

1993

Avon

1995

Incidence of BSECases per 1000 head of cattle

None<11 to 22 to 33 to 44 to 5>5

ð

Spread of the BSE epidemic in the British Isles

1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 19960

5

10

15

20R

eport

ed B

SE

case

s per

on

e/h

alf

year

in t

hou

san

ds

1 2 43

Confirmed Cases of BSE in British Cattle (1986-1996)

3010 20 40 50 60 70 80

% o

f Tota

l n

vCJD

On

sets

An

nu

al

Sp

ora

dic

CJD

Death

s/

Mil

lion

Pop

ula

tion

Age in Years

10

20

30

40 2.0

1.5

1.0

0.5

A.Age Distribution of vCJD and sCJD in Britain

Year1999199419891984

Sporadic CJDIatrogenic CJDFamilial CJDGSSnvCJD

Case

s p

er

Year

10

20

30

40

50

60

Creutzfeldt-Jakob Disease (CJD) in Britain

Chronic Wasting Disease

A TSE of deer and elk

Found in the western U.S. and Canada

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

Has been known for at least 35 years

N C

N181

N S197 231

SS

A. Conformations of the human prion protein translated in vitro

STE TMI

E1 E2

N

C

C

N

CtmPrP NtmPrP

C

N

secPrP

Microsomal membrane

Cytosol

Lumen

B. Maturation of secPrP in cells

ER

secPrP

Maturation Transport throughpost-ER compartments

ER

PrPc

Lumen LumenPlasma membrane

Cytosol Cytosol Cytosol

C

N s

s

N N

PrPc

E2 (epitope for MAb 13A5)

TMI - transmembrane domain GPI- glycosyl phosphatidylinositol

E1 (epitope for MAb 3F4) Repeats of 8 amino acids

STE - stop transfer effector N-linked carbohydrate chainsCHO

CH

OC

HO C

HO

CH

O

Postulated Topology of PrP Proteins in Cellular Membranes

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

Once it appears the prion form is dominant but reversible by treating with denaturants

It occurs spontaneously at low frequency

1 65 80 151158 221 227 348 354

Glutathione-S-transferaseN-repression domain

Prion-promoting sequences

Prion-inhibiting sequences

Yeast Prion Protein Ure2p

Prion-inducingdomain

domainPrion-propagating

1 114 254 685

Domains with known non-prion functions

domainTranslation-termination

Yeast Prion Protein Sup35p

Comparison of two yeast prion proteins