Transcript Chestnut - Rutgers

Reverse transcribing viruses other
than retroviruses
Transposons
• Class I - retrotransposons
– Very common in most organisms, especially plants
– Similar to retroviruses (e.g., HIV), but most particles don’t
have envelopes and most are not infectious
– Stress induced, are likely a major contributor to variation
and evolution in all organisms
– Two families classified with viruses - Metaviridae and
Pseudoviridae, differ in gene order and relationships
– Most do not contain env gene, but some do
– Most have not been shown to be transmissible, some have
• Class II - DNA mediated transposons
– Well-known in bacteria, plants, and animals; more recently
identified in fungi
– Not classified with viruses
Transposition of Class II transposons
(not classified with viruses;
e.g., TN5 used for mutagenesis)
PstI
PstI
IR
IR
DR
1
Transposase
DR
1
Short ITRs; encode transposase
PstI
PstI
2
Transposition of Class II transposons
PstI
PstI
IR
IR
DR
1
DR
1
Transposition is by cut-and-paste mechanism
PstI
PstI
2
Transposition of Class II transposons
PstI
PstI
DR DR
1 1
Circular DNA intermediate migrates to new
position on same or different chromosome
PstI
PstI
2
Transposition of Class II transposons
PstI
PstI
DR DR
1 1
“Footprint” is evident because direct
repeat sequences are left behind
PstI
PstI
2
Transposition of Class II transposons
PstI
PstI
DR DR
PstI
PstI
2
Transposition of Class II transposons
PstI
PstI
DR DR
1 1
Different size restriction fragments
are used to identify new insertions
quickly, confirmed by sequence
PstI
PstI
IR
DR
2
IR
DR
2
Transposition of Class II transposons
PstI
PstI
IR
IR
DR
1
DR
1
When transposition occurs during
meiosis, new and original copy
may still be evident in progeny
PstI
PstI
IR
DR
2
IR
DR
2
Transposition of Class I retrotransposons
(classified with viruses)
PstI
PstI
LTR
LTR
gag
pol (RT)
DR
1
DR
1
Have LTRs; encode gag
(CP), pol (RT), RNaseH,
protease, integrase; some
have env
PstI
PstI
2
Transposition of Class I retrotransposons
PstI
PstI
LTR
LTR
gag
pol (RT)
DR
1
DR
1
Transposition is by
transcription, reverse
transcription, integration
RNA
PstI
PstI
2
Transposition of Class I retrotransposons
PstI
PstI
LTR
LTR
DR
1
DR
1
Transposition is by
transcription, reverse
transcription, integration
RNA
DNA
PstI
PstI
2
Transposition of Class I retrotransposons
PstI
PstI
LTR
LTR
DR
1
DR
1
Transposition is by
transcription, reverse
transcription, integration
RNA
DNA
PstI
PstI
LTR
DR
2
LTR
DR
2
Properties of retrotransposons
• Useful as genetic markers for population studies
• May represent the majority of nuclear genome
• They contribute to genetic variability and
plasticity of organism
• Transposition may be induced by environmental
or developmental stresses
• Knowing what induces transposition may allow
one to predict behavior of organism in given
conditions
Retrotransposon replication
• Most of the general features of replication
similar to retrovirus replication (see
retrovirus lecture for details of replication)
• For most, virion (without envelope) has
been shown to be an essential part of
replication cycle
• Non-enveloped particle is not infectious
under most circumstances - can’t leave cell
Retrotransposon properties
• Two virus families: Metaviridae and Pseudoviridae
differ in genome organizations and phylogenies
• Genome sizes 4-13 kb
• Some LTR have envelope gene (env), most do not
• Infectivity has been demonstrated for those
containing env protein
• Horizontal transfer has been demonstrated for many
transposons without env gene
• No disease has been associated with infection and
replication
• Genetic disease or mutation may be associated with
transposition
• Transposition hot-spots may be found in host DNA
Phylogenetic relationships among reverse-transcribing elements
Retrotransposons
Bacterial msassociated DNA
Retrotransposons
from two families are
not each other’s
closest relatives
Mauriceville
plasmid
In the figure, the length of the box indicates the relative diversity
of reverse transcriptase genes within the family (longer is more
diverse); the width of the box indicates the relative number of
sequences used for the comparison (wider is more sequences).
From Boeke et al.,
2001 ICTV, 7th Report
Phylogenetic relationships among reverse-transcribing elements
Phylogenetic relationships among reverse-transcribing elements
Color code: orange, DD site of RT; purple, RNase domain (RH); yellow, integrase domain (INT);
blue, cysteine-histidine motif (C-H); green protease domain (PR); pink, envelope/movement protein
domain (ENV/MP). 25
From Hansen and Heslop-Harrison. 2004. Adv.Bot.Res.
PARARETROVIRUSES:
CAULIMOVIRIDAE
Pararetroviruses vs. Retroviruses
Characteristics
Pararetroviruses
Retroviruses
Enveloped particles
No/Yes
Yes
Genome nucleic acids
DNA
RNA
Encapsidation of reverse transcriptase
No
Yes
Replication intermediates
RNA
DNA
Integration into host chromosomes
No (usually)
Yes
Pregenomic RNA as polycistronic RNA
Transcription,
splicing
Transcription,
splicing
Gag-pol fusion protein
Yes
Yes
Full length transcripts with terminal
repeats
Yes
Yes
Caulimoviridae
• Isometric 50 nm T=7 particles or bacilliform
particles; no envelope
• Nicked dsDNA genomes ~ 8 kb
• First plant virus shown to have DNA genome
• Replication is by reverse transcription
• Transcription is in nucleus; DNA replication in
cytoplasm
• Most do not integrate into host genome
Caulimoviridae
• Most have narrow host ranges
• Most are relatively unimportant as pathogens;
exception is Rice tungro bacilliform virus, part of the
most important rice virus complex
• Most are transmitted by invertebrate vectors
• Viruses do not replicate in vector; use virus-coded
helper protein to aid transmission
• Promoter elements commonly used in genetic
engineering of plants
• Caulimoviruses are not very versatile as plant gene
expression vectors because of packaging
constraints/instability
Visualization of Viral Particles
• Electron micrographs of thin sections
of caulimovirus-infected tissue showing
42-46 nm virus particles and inclusion
bodies.
(Photos courtesy of Dr. T.A. Chen)
Caulimovirus Life Cycle
• Virus enters plant cell, capsid
protein is removed
Helper
factor
Mature
particle
with DNA
Inclusion
• dsDNA enters nucleus; gaps
closed; transcription to 35S and
19S RNAs
• In the cytoplasm, the 19S RNA is
translated to produce protein that
forms inclusion bodies
•Five ORFs are translated from 35S
RNA by complex combination of
strategies
•Other copies of 35S RNA are
reverse transcribed and packaged
into virions
•Particles exit cells through
plasmodesmata or by aphids
(Shaw, Fund. Virology, 3rd Ed., 1996)
Cauliflower mosaic virus genome structure
•Seven ORFs on CaMV
genome
Translation regulator
Movement protein
Helper component
Inclusion,
transactivator
Replication
factor
Coat
protein
Reverse
transcriptase
•Translation of seven
proteins from two
transcripts
•ORF 2 is the only
dispensable ORF
•ORFs 6 and 7 are
involved in translation
regulation
•Packaged genomic
DNA has discontinuities
on both strands
•Replication is from
tRNAmet primer
Genome Organization of Caulimoviridae
Petuvirus
I
PVCV
Caulimoviruses
VII
CaMV
I
II
II
II
I
IV
V
VI
FMV
CERV
Soymoviruses
VII
SbCMV
I
a
b
c
IV
V
VI
BRRV
Cavemoviruses
I
II
III
IV
CsVMV
Tungroviruses
I
II
RTBV
Badnaviruses
ComYMV
I
II
II
I
IV
III
V
Hepadnaviruses
Hepatitis B
Hepatitis Viruses
The Liver is a target for
six viruses that cause
acute hepatitis. These are
Hepatitis Virus A, a
picornavirus passed in a
fecal-oral pattern, the
pararetrovirus Hepatitis
B, that is transmitted via
blood products, IV drug
use, from mother to child
& by unprotected sex,
Hepatitis C, a flavivirus
transmitted via IV drug
use & earlier by blood
products, Hepatitis Delta,
a viroid-like satellite
associated with HBV and
Hepatitis E, a calicivirus
passed via feces contaminated water.
Hepatitis G is a relatively rare flavivirus
transmitted via blood and possibly during sex.
Fig. 24.01
& Liver Disease
Characteristics & Control of Hepatitis Viruses
Acute Viral Hepatitis by Type, United States, 1982-1993
34%
47%
16%
3%
Source: CDC Sentinel Counties Study on Viral Hepatitis
Hepatitis A
Hepatitis B
Hepatitis C
Hepatitis
Non-ABC
Chronic Liver Disease: USA 1999
NASH
10%
Alcohol
25%
Hepatitis B
accounted for only
4.4% of newlydiagnosed chronic
liver disease
Hepatitis C
57%
Hepadnavirus properties
• Enveloped icosahedra 42-50 nm
• Partially double-stranded, non-covalently closed
circular DNA 3-3.5 kb
• Viral polymerase is reverse transcriptase, and is
covalently linked to 5’ end of – sense DNA
• Capped 19 nt RNA primer is linked to 5’ end of +
strand DNA
• Encode 7-8 proteins, 6-7 are structural
• Every nucleotide of viral genome is used in coding
sequences
• Coding capacity = 1.5 X genome size
Hepadnavirus properties
• Replication is by reverse transcription in
particles
• Virus assembly in nucleus
• Specificity for liver cells
• Cause liver disease, hepatocarcinoma
• Genome and genomic fragments may integrate,
but not necessary to replication
Hepadnavirus associated disease
• Hepadnaviruses show narrow host specificity
– Experimental infection with particles is difficult
– Infection from cloned DNA has been demonstrated
for several hepadnaviruses
– Because of compact genome with many overlapping
ORFs, genome manipulation is difficult
– Viruses markedly hepatotrophic, but can be found in
other cells e.g, pancreas, spleen, blood cells
– Infection may be transient or persistent
Hepatitis B transmission
•
•
•
•
Through blood exchange, e.g., drug use
Sexual contact
Perinatal transmission from infected mother
Other exposure to open skin breaks and mucous
membranes, especially in low socio-economic
Hepatitis B prevention
• Vaccination
• Personal hygiene
• Precautions with drugs & sex
Hepatitis B virus structure and
genome organization.
A. Mature, enveloped virions
and incomplete particles
B. Genome organization.Light
blue minus strand DNA has
one nick and polymerase
bound to 5’ end; dark blue +
strand DNA has a large gap.
Transcription begins at
different sites, but
polyadenylation is at one site
Hepadnavirus infection cycle
1-4.Receptor-mediated virus entry
into hepatocyte; core entry
into nucleus and DNA repair
5-6.Synthesis of pregenome and
subgenomic transcripts; export
to cytoplasm
7-9. Translation of surface and X
proteins from short RNAs,
capsid and polymerase (RT)
proteins from pregenome
10-11. Packaging of pregenome
with RT, followed by DNA
synthesis
12. Early in infection, recycling of
DNA to nucleus
13-15. Particle assembly,
membrane acquisition, and
egress
Hepadnavirus & Caulimovirus vs. Retrovirus RT Replication
(final product boxed in grey)
genome
template RNA
dsDNA (incomplete circle)
Synthesized by host pol II
pregenome RNA, mRNA
redundant ends for
template switch
dsDNA (complete, relaxed circle)
Synthesized by host pol II
pregenome RNA, mRNA
redundant ends for
template switch
RNA (diploid)
Synthesized by host pol II
genome RNA, mRNA
redundant ends for
template switch
viral enzyme
P (RT/RNase H,
no IN function)
POL (RT/RNase H,
no IN function)
RT/RNase H (with IN function)
DNA in nucleus
Nonintegrated episome
Nonintegrated episome
Integrated into host genome, provirus
Primer: strand 1 (–)
strand 2 (+)
viral P
derived from template RNA,
terminal RNase H product (cap)
cytoplasm, subviral core
in virion assemply
host tRNA
derived from template RNA,
internal RNase H product (ppt)
cytoplasm, assembled
viral capsid
host tRNA
derived from template RNA,
internal RNase H product (ppt)
cytoplasm, subviral core
in uncoating upon entry
RT reaction
Hepadnaviruses and Caulimoviruses: DNA Genomes
Reverse Transcribed from a +RNA Template
Hepatitis B
Virus (HBV)
Cauliflower Mosaic
Virus (CaMV)
Relaxed dsDNA circle:
• HBV: 1 complete (–) and 1 incomplete (+) strand
• CaMV: 2 complete strands with discontinuities
(2 in + strand, 1 in – strand)
Circle maintained by overlapping (redundant) 5’ ends
Polymerase (RT/RNase H) in virus particle
• HBV: Covalently attached to 5’ end of –DNA strand
RNA sequence(s) at 5’-end(s) of + strand
Structural anomalies are ‘hallmarks’ of replication strategy
dsDNA
mRNA
dsDNA
From Flint et al. Principles of Virology (2000), ASM Press