Viruses with multi-partite genomes have the information required for the virus infection

1. Plant Viruses
Bradley Hillman
Dept. of Plant Biology and Pathology
339 Foran Hall, Cook
932-9375 X 334
hillman@aesop.rutgers.edu
Comparative Virology course website:
http://www.rci.rutgers.edu/~bhillman/comparative_virology/Index.htm

2. Plant Viruses
• Introduction and history
– Why study plant viruses?
– How do they relate to animal viruses?
– How has their study impacted virology?
• Symptoms
• Composition and structure
• Taxonomy and nomenclature
– Four families contain both plant and animal viruses
– Seven families contain only plant viruses
– Many plant viruses belong to genera without family affiliation

3. Plant Viruses (cont’d)
• Survey of some major plant viruses
– Positive strand RNA
• RdRp supergroup 3 (Sindbis-related)
– Tobacco mosaic virus (Tobamovirus)
– Brome mosaic virus (Bromoviridae)
• RdRp supergroup 2 (Flavivirus-related)
– Turnip crinkle virus (Tombusviridae)
– Red clover necrotic mosaic virus (Dianthovirus)
• RdRp supergroup 1(Picornavirus related)
– Tobacco etch virus (Potyviridae)
– Cowpea mosaic virus (Comovirus)
– Negative strand RNA
• Sonchus yellow net virus (Rhabdoviridae)
– Ambisense RNA
• Tomato spotted wilt virus (Bunyaviridae)

4. Plant Viruses (cont’d)
• Survey of some major plant viruses (cont’d)
– Double-stranded RNA
• Wound tumor virus (Reoviridae)
– Single-stranded DNA
• Bean golden mosaic virus (Geminiviridae)
– Double-stranded DNA (pararetroviruses)
• Cauliflower mosaic virus (Caulimoviridae)
• Expression strategies of + strand RNA viruses
• Plant virus infection cycle
• Cell-to-cell movement and movement within plants
• Plant-to-plant transmission
• Brome mosaic virus, a well-studied plant virus
• Satellites, defective-interfering RNAs, viroids
• Plant defense response to virus infection
• Plant viruses and biotechnology

5. Host Systems: Plants 1
• Eukaryotic, but fundamentally different from
animals
• Plants don’t move, so vectors are very important
for moving viruses from one plant to another
• Plants are autotrophic and easy to grow in
quantity– great bioreactors
• Plants have rigid cell walls and very small cell-to-
cell connections (plasmodesmata)
• Synchronous infection of many cells can be
achieved using plant protoplasts (primary cell
cultures with cell walls removed)

6. Host Systems: Plants 2
• Developed plant cells are totipotent
• Virus in one part of a plant moves to another
slowly by cell-to-cell connections; more rapidly
through vascular system, mostly phloem
• Plant defense response system exists, but is
less specific than vertebrate or invertebrate
systems
• Plants are developmentally complex; viruses
may be excluded from some tissues

7. CHARACTERISTICS OF PLANT
PATHOGENIC VIRUSES
• Loss due to plant viruses is often difficult to quantify, but
they are often of great importance as plant pathogens
(fungi are most economically important )
• Viruses do not usually kill plants, and symptoms on
plants are often subtle
• Virus diseases of plants are not subject to chemical
control – no effective cure in individual plants
• Symptoms, serological, electron microscopic, or
molecular methods are used to identify plant viruses
• Plant virus disease cycles often are dependent on
vectors or alternate hosts

8. Selected highlights of plant
virology research

9. Tulipomania – late 16th century
Traded for 1 Viceroy
tulip bulb:
4 tons of wheat
8 tons of rye
4 fat oxen
8 fat pigs
12 fat sheep
2 hogsheads of wine
4 barrels of beer
2 barrels of butter
1000 lbs of cheese
1 bed with
accessories
1 full dress suit
1 silver goblet
Before it was known to be caused by a virus, tulips with color
breaking symptoms were prized and traded for large sums of
goods – this led to “tulipomania” in the late 1500’s

10. • Adolf Mayer –1886 – showed that Tobacco
mosaic virus was transmissible, could not
find bacteria or fungi associated with
disease
TMV

11. • Dmitri Ivanowski - 1892– showed that
Tobacco mosaic virus was not retained by
filters that retained all bacteria known at
that time

12. • Martinus Beijerinck - 1898– repeated
demonstration that Tobacco mosaic
virus was not retained by filters that
retained all bacteria known at that time
Believed results
Did extensive dilution experiments
Showed diffusion of infectious agent
through agar
Named “contagium vivum fluidum”, later
virus

13. Wendell Stanley – 1935
• At Rockefeller Foundation in Princeton
• Crystallized TMV, thought it was only
protein
Stanley Hall, U.C. Berkeley
TMV

14. Bawden and Pirie - 1936
• Crystallized Tomato
bushy stunt virus
(TBSV); find that it
and TMV contain
phosphorous –
conclude that it is
not a protein, but is
a nucleoprotein
TMV
TBSV

15. Markham and Smith - 1949
• Two classes of particles
in purified Turnip yellow
mosaic virus
preparations:
– light ones containing only
protein, which were not
infectious
– heavy ones containing
protein+nucleic acid,
which were infectious
empty full

16. Myron Brakke - 1951
• Development of density
gradient centrifugation
– Isopycnic: particles reach
position of equal density in
gradient
– Rate-zonal: Particles
sediment differentially
through medium as a
function of size, shape, and
density
– Equilibrium zonal:
Combination of the above

17. Fraenkel-Conrat
1955-56
• Complete, infectious TMV
particles can be
reconstituted in vitro from
the RNA and protein
components
• RNA alone is infectious
• RNA can be
“transcapsidated” in
protein from closely
related virus; resulting
virus has properties of
RNA strain
reconstitute
in vitro
Virus Aa:
RNA A
capsid a
RNA
protein
symptoms (A)
extract virus
virus Aa
inoculate plants

18. Fraenkel-Conrat – 1955-56 Transcapsidation
inoculate plants
Virus Aa:
RNA A
capsid a
RNA
protein
symptoms (A)
no
symptoms
extract virus
no virus
virus Aa
inoculate plants
Virus Bb:
RNA B
capsid b
RNA
protein
symptoms (B)
no
symptoms
extract virus
no virus
virus Bb
inoculate plants
Virus Ab:
RNA A
capsid a
RNA
protein
symptoms (A)
no
symptoms
extract virus
no virus
virus Aa

19. Crick and Watson – 1956
• TMV virions are
composed of one
nucleic acid and
many identical protein
subunits: RNA does
not have the coding
capacity to make
many different
subunits

20. Casper and Klug – 1962
• Structure of Tomato bushy stunt
virus solved by X-ray
crystallography, the first
icosahedral virus so determined

21. Heinz Sanger – 1978
• Complete sequence
of Potato spindle
tuber viroid
– First pathogen
sequence to be
determined
– Yielded relatively little
information that was
immediately useful
1 cggaactaaa ctcgtggttc ctgtggttca cacctgacct
cctgagcaaa aaagaaaaaa gataggcggc tcggaggagc
gcttcaggga tccccgggga aacctggagc gaactggcaa
aaaaggacgg tggggagtgc ccagcggccg acaggagtaa
ttcccgccga aacagggttt tcacccttcc tttcttcggg
tgtccttcct cgcgcccgca ggaccacccc tcgccccctt
tgcgctgtcg cttcggctac tacccggtgg aaacaactga
agctcccgag aaccgctttt tctctatctt cttgcttccg
gggcgagggt gtttagccct tggaaccgca gttggttcct 359

22. Paul Ahlquist – 1984
• Infectious viral RNA
transcribed in vitro from
cDNA clones
– Done with Brome mosaic
virus – with 3 RNAs
– Brought reverse genetics
to RNA viruses
RNA
cDNA
RNA Inoculate
plants

23. Roger Beachy – 1986
• Transgenic plants
expressing TMV coat
protein are resistant to
virus infection
• First example of
“pathogen-mediated
resistance”

24. Bill Dougherty – 1991
• RNA was critical component
in resistance in pathogen-
mediated resistance
• All of the hallmarks that later
came to be associated with
PTGS and RNAi were first
observed with Tobacco etch
virus (TEV) (1993 Lindbo et
al., Plant Cell 5:1749-1759)

25. SYMPTOMS

26. Plant Virus Symptoms
• Viruses rarely kill plants
• Most severe disease usually in least well-adapted
host/pathogen systems
• Levels of tissue specificity differ among plant viruses
– Some infect all or most tissues
– Most cause symptoms only in aerial portions
– Some accumulate only in roots
– Symptoms in fruit or flowers may be most harmful
• Systemic symptoms only in developing tissue
• Local necrotic lesions undetectable in natural
infections

27. Plant Virus Symptoms
• Stunting - common
• Mosaics & mottles - common
• Ringspots - common
• Abnormal growth/tumors/enations - rare
• Blights - rare
• Wilts – rare
• Systemic necrotic lesions – relatively rare

28. Blight

30. Stem pitting – usually results in loss of woody perennials

31. Ringspots, oakleaf

32. Deformities
Phyllody – tissue destined to
develop flower parts instead
develops leaves

33. Wilt
Necrotic roots

34. Mosaics – very common
Tomato mosaic
Alfalfa mosaic

35. Mosaics on monocots are streaks or stripes
Maize streak Maize mosaic

36. Necrotic lesions
Systemic
Local
“Local lesion” or “hypersensitive” response is an apoptotic response.
Cells within a short distance of the initially inoculated cell begin to
undergo programmed cell death in advance of virus invasion,
preventing further virus spread.

37. Hypersensitive response – an apoptotic
reaction to infection
May be viral coat
protein or another
viral gene product

38. TAXONOMY AND
NOMENCLATURE

39. Virus taxonomy and nomenclature
• Modified binomial is used
• Taxonomy depends on particle properties, nucleic
acid properties and especially sequence
• Family is the highest taxonomic level that is
commonly used; ends in viridae, e.g., Bromoviridae
• Genus ends in suffix virus, e.g., Bromomovirus
• Species is usually the commonly used virus name;
it is italicized in formal usage, e.g., Brome mosaic
virus
• Small genome sizes, gene shuffling make broad
taxonomic schemes difficult (above Family level)

40. COMPOSITION AND
STRUCTURE

41. Virus properties: Plant viruses are often
simpler than animal viruses
• Genome sizes 0.3 - 300 kb; plant viruses 0.3-30 kb
• May have single-stranded or double-stranded RNA
or DNA genome; most plant viruses ssRNA
• If RNA, may be + or – sense; most plant viruses +
sense ssRNA
• May have one or many proteins in particles; most
plant viruses have 1-2
• May or may not have lipid envelope; most plant
viruses do not

42. Types of plant virus genomes
• double-stranded (ds) DNA (rare)
• single-stranded (ss) DNA (rare)
• ssRNA, negative sense (rare)
• ssRNA, positive sense (common)
• dsRNA (rare)

44. Virus types, by nucleic acid
DNA RNA
ss ds ss ds
env naked env naked env naked env naked
0 5 9 12 9 14 2 5
0 100 200 300 200 600 10 300
Families
Species
Host type
Vertebrate
Invertebrate
Plant
Fungus
Bacteria
- + ++ ++ ++ ++ - ++
- + ++ - ++ ++ - ++
- ++ - + + +++ - +
- - - + + + + +++
- + + +++ - + + -

45. Plant viruses are
diverse, but not as
diverse as animal
viruses – probably
because of size
constraints imposed by
requirement to move
cell-to-cell through
plasmodesmata of host
plants
Plant viruses often
contain divided
genomes spread
among several particles

46. Basic Plant Virus Structures
Helix (rod)
e.g., TMV
Icosahedron
(sphere)
e.g., BMV

47. Helical symmetry
• Tobacco mosaic virus is typical,
well-studied example
• Each particle contains only a single
molecule of RNA (6395 nucleotide
residues) and 2130 copies of the
coat protein subunit (158 amino
acid residues; 17.3 kilodaltons)
– 3 nt/subunit
– 16.33 subunits/turn
– 49 subunits/3 turns
• TMV protein subunits + nucleic
acid will self-assemble in vitro in an
energy-independent fashion
• Self-assembly also occurs in the
absence of RNA
TMV rod is 18 nanometers
(nm) X 300 nm

48. Cubic (icosahedral) symmetry
• Tomato bushy stunt
virus is typical, well-
studied example
• Each particle contains
only a single molecule
of RNA (4800 nt) and
180 copies of the coat
protein subunit (387 aa;
41 kd)
• Viruses similar to TBSV
will self-assemble in
vitro from protein
subunits + nucleic acid
in an energy-
independent fashion
TBSV icosahedron is 35.4
nm in diameter
Protein Subunits Capsomeres
T= 3 Lattice
C
N

49. GENOME ORGANIZATIONS

50. Plant virus genome organizations
• Very compact
• Most are +sense RNA viruses, so translation
regulation very important
• Use various strategies for genome expression
• Only a few genes absolutely required:
– Replicase
– Coat protein
– Cell-to-cell movement protein
• Other genes present in some viruses

51. Plant viruses have members in all 3 supergroups of + strand RNA viruses
From Principles of Virology,
Academic Press 1999

52. Genome expression of + strand RNA viruses
• Most use more than one strategy
– Polyprotein processing
– Subgenomic RNA
– Segmented genome
– Translational readthrough
– Frameshift
– Internal initiation of translation (without scanning)
– Scanning to alternative start site (truncated product)
– Alternative reading frame (gene-within-a-gene)

53. Polyprotein processing
• Post-translational cleavage of viral proteins
may occur in cis or in trans
• Some viruses use polyprotein processing
exclusively to regulate gene expression
• Many viruses use polyprotein processing as
one of several regulation mechanisms
• Examples:
– Potyviruses*
– Comoviruses*
– Closteroviruses
– Carlaviruses

54. Subgenomic RNA
• Similar to traditional mRNA, but synthesized
from an RNA template
• Many viruses use polyprotein processing as
one of several regulation mechanisms
• Examples:
– Tobamoviruses (TMV)*
– Bromoviruses (BMV)*
– Tombusviruses (TBSV)
– Potexviruses (PVX)

55. Segmented genome
• Positive sense RNA genomes are usually
encapsidated in separate particles
• Segmented genomes lend themselves to
recombination
• Examples:
– Bromoviruses (Brome mosaic virus, BMV)*
– Dianthoviruses (Red clover necrotic mosaic virus,
RCNMV)*
– Hordeiviruses (Barley stripe mosaic virus, BSMV)

56. Translational readthrough
• Usually UAG codon is read through using
suppressor tyrosine tRNA
• Common mechanism in plant viruses
• Examples:
– Tobamoviruses (Tobacco mosaic virus, TMV)*
– Dianthoviruses (Red clover necrotic mosaic virus,
RCNMV)*
– Hordeiviruses (Barley stripe mosaic virus, BSMV)

57. Translational frameshift
• Typically +1 or -1
• Common mechanism in plant viruses
• Examples:
– Luteoviruses (Barley yellow dwarf virus, BYBV)*
– Dianthoviruses (Red clover necrotic mosaic virus,
RCNMV)*
– Closteroviruses (Beet yellow vein virus, BYVV)

58. Internal initiation
• Cap-free translation
• Less complex in plant viruses than in
animal viruses
• Examples:
– Potyviruses (Tobacco etch virus, TEV)*
– Sobemoviruses (Southern bean mosaic
virus, (SBMV)*

59. Tobacco mosaic virus is a typical positive-sense RNA
plant virus with a 6.4 kilobase genome

60. INFECTION CYCLE

61. Plant Virus Life Cycle
• Virus entry into host
– no attachment step with plant viruses
– by vector, mechanical, etc. – must be forced
– requires healable wound – delivery into cell
• Uncoating of viral nucleic acid
– may be co-translational for + sense RNA viruses
– poorly understood for many
• Replication
– replication is a complex, multistep process
– viruses encode their own replication enzymes

62. Plant Virus Life Cycle 2
• Cell-to-cell movement
– cell-to-cell movement through plasmodesmata
– move as whole particles or as protein/nucleic
acid complex (no coat protein required)
• Long distance movement in plant
– through phloem
– as particles or protein/nucleic acid complex (coat
protein required)
• Transmission from plant to plant
– requires whole particles

63. Typical RNA-containing plant virus replication cycle
From Shaw, 1996 Ch. 12 in Fundamental Virology (Academic Press)
2. RNA is released;
translates using
host machinery
3. Replication in
cytoplasm
4b. New virus particles
are assembled
1. Virus particle enters
first cell through
healable wound
4a. Infectious TMV
RNA is shuttled to
adjacent cell through
plasmodesmata, by
virus-coded
movement protein

64. Cell-to-Cell Movement of Plant Viruses
• Plant viruses move cell-to-cell slowly through
plasmodesmata
• Most plant viruses move cell-to-cell as
complexes of non-structural protein and
genomic RNA
• The viral protein that facilitates movement is
called the “movement protein” (MP)
• Coat protein is often dispensable for cell-to-cell
movement

65. Cell-to-Cell Movement of Plant Viruses
• Several unrelated lineages of MP proteins have
been described
• MPs act as host range determinants
• MP alone causes expansion of normally
constricted plasmodesmata pores; MPs then
traffic through rapidly
• MPs are homologs of proteins that naturally
traffic mRNAs between cells
• MPs may act as suppressors of gene silencing

66. Plant cells are bound by rigid cell walls and are interconnected
by plasmodesmata, which are too small to allow passage of
whole virus particles.
Plasmodesma

69. Understanding
virus infection
and movement
through plants
requires
understanding
architecture of
dicotyledonous
plants and the
connections
between
different cell
types.
This has been
studied
extensively with
GFP-labeled
virus

70. Some plant viruses radically modify plasmodesmata,
allowing for cell-to-cell movement as whole particles

71. Systemic spread
of plant viruses is
primarily through
vascular tissue,
especially phloem

72. Plant Virus Transmission
• Generally, viruses must enter plant through
healable wounds - they do not enter through
natural openings (no receptors)
• Insect vectors are most important means of
natural spread
• Type of transmission or vector relationship
determines epidemiology
• Seed transmission is relatively common, but
specific for virus and plant

73. Plant Virus Transmission
• Mechanical transmission
– Deliberate – rub-inoculation
– Field – farm tools, etc.
– Greenhouse – cutting tools, plant handling
– Some viruses transmitted only by
mechanical means, others cannot be
transmitted mechanically

74. • Transmission by vectors: general
– Arthropods most important
– Most by insects with sucking mouthparts
• Aphids most important, and most studied
• Leafhoppers next most important
– Some by insects with biting mouthparts
– Nematodes are important vectors
– “Fungi” (protists) may transmit soilborne viruses
– Life cycle of vector and virus/vector relationships
determine virus epidemiology
– A given virus species generally has only a single
type of vector
Plant Virus Transmission by Vectors

75. • Insect transmission (vectors)
– Aphids most important
– Leafhoppers
– Whiteflies
– Thrips
– Mealybugs
– Beetles
– Mites (Arachnidae)
– Ants, grasshoppers, etc. – mechanical
– Bees, other pollinators – pollen transmission

76. Types of vector relationships
Terms apply mainly, but not exclusively, to
aphid transmission
• Non-persistent transmission
– virus acquired quickly, retained short
period (hours), transmitted quickly
– “stylet-borne” transmission
– virus acquired and transmitted during
exploratory probes to epidermis

77. Types of vector relationships
• Persistent transmission
– virus acquired slowly, retained long period
(weeks), transmitted slowly
– circulative or propagative transmission
– virus acquired and transmitted during
feeding probes to phloem

78. Types of vector relationships
• Semi-persistent transmission
– virus acquired fairly quickly, retained
moderate period (days), transmitted fairly
quickly
– virus acquired and transmitted during
exploratory probes

79. Brome mosaic virus
• Relatively little studied prior to 1980
• Relatively narrow host range
• Causes no important disease
• Mechanically transmitted, probably not
vectored
• Similar to Alfalfa mosaic virus and
Cucumber mosaic virus, two important plant
pathogens
• Now most thoroughly understood plant virus
at RNA level

80. BMV structure
• Rigid isometric particles 27 nm
• RNA1 (3.2 kb) and RNA2 (2.9 kb)
packaged alone; RNA3 (2.1 kb) and
RNA4 (1.2 kb) packaged together
• Particle is held together primarily by
protein/RNA interactions
• With RNA, 180-subunit, T=3
particles predominate; without RNA,
120-subunit, T=1, particles

81. Brome mosaic virus genome organization
• 3 genomic RNAs, one
subgenomic RNA
• Only RNAs 1 and 2
required for replication in
protoplasts
• 3’-terminal 200 bases of
segments nearly identical
• All three genomic and
subgenomic RNA are
capped
• Non-templated C and A at
3’-ends
Capping Helicase
Polymerase
Movement Capsid

82. Why is BMV such a powerful system
• 3 RNAs
– only 1 and 2 required for replication
– RNAs can be studied independently
• All three promoter types (+, -, sg) found on RNA
3, which is not required for infection
• Replication is fast in plant protoplasts
• Infects monocot and dicot host plants
• Replicates in yeast, best eukaryotic genetic
system
• Efficient transcriptase/replicase complex has
been isolated for in vitro studies
• Structurally stable particles allow for
encapsidation studies

83. Brome mosaic virus contributions
• 1980
– studies of 3’-terminal pseudoknot (Ahlquist/Kaesburg)
• 1984
– complete BMV sequence (Ahlquist/Kaesburg)
– 3’-terminal replicase recognition site (- strand
promoter) identified (Ahlquist/Hall)
– Nonstructural proteins of BMV, TMV, and alfaviruses
are similar (Haseloff; also Ahlquist)
– First infectious transcripts from a cloned RNA virus
genome (Ahlquist)
• 1985
– Continued dissection of 3’-end functions

84. Brome mosaic virus contributions
• 1986
– CAT gene substituted for CP (French/Ahlquist)
• 1987
– Requirement of intercistronic region of RNA 3 for
replication (French/Ahlquist)
• 1988
– Identification of subgenomic RNA promoter
(French/Ahlquist)
• 1990
– Rescue of CCMV deletion mutants by recombination
(Allison/Ahlquist)

85. Brome mosaic virus contributions
• 1992
– Determination of 5’-terminal sequences involved in transcription
and replication (+ strand promoter (Pogue/Hall)
– TMV movement protein can substitute for BMV MP
(DeJong/Ahlquist)
• 1993
– BMV replication in yeast (Janda/Ahlquist)
– BMV transcription and replication requires compatibility between
polymerase and helicase proteins (Dinant/Ahlquist)
• 1994
– Recombination between viral RNA and transgenic plant transcripts
(Green/Allison)
• 1995
– Formation of RdRp in yeast requires coexpression of RNA 1 and 2
proteins (Quadt/Ahlquist)

86. Brome mosaic virus contributions
• 1997
– Inducible expression of active RNA 3 replicons in yeast from DNA
plasmids (Ishjkawa/Ahlquist)
– Yeast mutations in multiple complementation groups inhibit BMV
RNA replication and transcription (Ishjkawa/Ahlquist)
• 1998
– Specific residues critical for recognition of the 33 nt subgenomic
RNA promoter by the BMV RdRp identified, demonstrating
functional homology of RNA and DNA promoters (Siegel/Kao)
• 1999
– BMV 1a protein functions in in vitro, in yeast, and in plants in
methylation of GTP and cap analogs (Ahola/Ahlquist; Kong/Kao)
– BMV RdRp can use RNA, DNA, or hybrid to initiate RNA
synthesis, suggesting that transition from RNA to DNA world may
have been relatively easy (Siegel/Kao)

87. Brome mosaic virus contributions
• 2000
– Host protein associated with efficient RNA template
selection identified (Diez/Ahlquist)
– BMV protein 2a (RdRp) is directed to ER by
capping/helicase-like 1a protein (Chen/Ahlquist)
• 2001
– Factors regulating template switching during RNA
synthesis by viral RdRps identified using in vitro
assays (Kiml/Kao)
• 2002
– 3’-terminal tRNA-like structure of BMV RNAs mediate
particle assembly (Choi/Dreher/Rao)
– Crystallographic structure of BMV solved
(Lucas/McPherson)

88. Brome mosaic virus contributions
• 2003
– The BMV 3’-terminal core promoter element, stem-loop
C (SLC), functions at different positions on the
template, can initiate RNA synthesis internally, and can
potentiate RNA synthesis from a cellular tRNA
template (Ranjith-Kumarl/Kao)
– Systematic, genome-wide identification of host genes
affecting replication of BMV (Kushner/Ahlquist)
• 2004
– Two distinct types of homologous RNA recombination
in BMV replication (Bujarski)
• 2005
– Gold nanoparticles encapsidated in BMV coat protein
form normal, solid particles (Rao)

89. Some major BMV contributors
• Ahlquist – most prolific, many aspects
• Hall – Cis-acting RNA elements
• Dreher – 3’-terminal structure
• Kao – recent dissection of cis-acting RNA
elements
• Bujarski – Intra-strand and inter-strand RNA
recombination
• Rao – RNA packaging

90. BMV promoters for + strand RNA synthesis
• Promoter sequence for
genome-length plus strand
synthesis is on 3’-end of
minus strand; corresponding
sequences on plus strand are
important for efficient
transcription
• 5’-terminal regions are less
highly conserved than 3’-
terminal regions
• Internal poly(A) tract of
variable length precedes
subgenomic promoter and is
required for efficient sgRNA
transcription
• Core promoter elements

91. Three classes of 3’ termini among + sense RNA viruses
TLS = tRNA-like structure
Het = Heteropolymeric, non-tRNA-
like sequence
An = poly(A) tail
A. Full phylogeny, Supergroups 1-3
B. Supergroup 3
From Dreher 1999, Annu. Rev. Phytopathol. 37: 151-174
Type of 3’-terminal structure
cannot be predicted based on
polymerase phylogeny alone,
supporting the role of interviral
recombination in selection of 3’-
end.

92. Brome mosaic virus 3’-terminal tRNA-like
structure is a multifunctional domain
• Promoter for – strand RNA
synthesis
• RNA protection against
nuclease
• Can be charged with
tyrosine in vitro and in vivo
• (ATP, CTP) tRNA
nucleotidyl transferase
adds terminal C and A
residues
• Involved in RNA packaging
into virions – intact tRNA-
like fold is required
• Relatively little involvement
of 3-end of BMV RNA in
translation regulation

93. Infection of yeast by Brome mosaic virus constructs

94. Brome mosaic virus replication
• 1a protein (capping &
helicase) localizes to ER
• 1a protein recruits 2a
protein (RdRp) during
active translation of 2a
• Viral RNA templates are
recruited to nascent
replication complex by
1a protein
• RNA replication occurs
in membrane-bound,
capsid-like spherules

95. Identification of host genes involved in BMV
replication (Kushner/Ahlquist 2003, PNAS 100:15764)
• Used yeast – genetics
easy, many mutants
available
• Provides information
about replication, not
systemic infection
• Transform with
inducible two plasmid
system requiring
replication for reporter
gene expression
• Screen for replication-
associated genes by
monitoring luciferase
expression
• 4500 yeast mutants examined
(~80% or yeast genes)
• ~100 yeast genes whose deletion
altered BMV-directed expression
of luciferase by > 3 fold

96. Identification of host genes involved in BMV
replication (Kushner/Ahlquist 2003, PNAS 100:15764)
• Mutants that were positive in the luciferase screen were
examined further by northern and western blot

97. Tomato bushy stunt virus genome

98. Comparative properties of BMV and TBSV
• TBSV
– A T=3 icosahedral virus with a 40
kDa capsid subunit and monopartite
RNA genome of 5 kb
– RdRp supergroup 2
– No known helicase domain
– 5’ end of RNA uncapped; 3’ end has
no poly(A) or tRNA-like structure
– Expression of 5 gene products via
cap-independent translation (1)
readthrough (1), subgenomic RNAs
(2), and internal initiation (1)
– Defective-interfering (DI) RNAs
replicate to high levels in permissive
plant host; equally high in yeast
• BMV
– A T=3 icosahedral virus with a 20
kDa capsid protein subunit and
tripartite RNA genome of 8 kb
– RdRp supergroup 3
– No known helicase domain
– 5’ end of RNA capped; 3’ end has
tRNA-like structure aminoacylated
with tyrosine
– Expression of 4 gene products via
normal cap-dependent translation
(3) and subgenomic RNA (1)
– Defective-interfering (DI) RNAs
present, not prevalent

99. TBSV replication
constructs for
yeast assay –
based on trans
replication of a
defective
interfering RNA

100. Yeast genome-wide screen reveals dissimilar sets of
host genes affecting replication of RNA viruses
Tadas Panavas, Elena Serviene, Jeremy Brasher, and Peter D. Nagy*
2005 PNAS 102:7326-7331
• Replication of both BMV and TBSV is suppressed in about 100
out of 4,800 yeast knock-out mutants (YKOs)
• Of those 100 mutants, only 4 were common between the two
viruses: three genes involved in protein metabolism (ubiquitin
pathway), and the fourth a transcription regulator
• Another 10 mutants affecting replication of one or the other
viruses represented genes with known functions in common.
These were involved in: i) protein biosynthesis; ii) protein
metabolism; iii) transcription/DNA remodeling
• Genes involved in protein targeting, membrane association,
vesicle transport, and lipid metabolism affect TBSV, but none in
BMV

101. Viroids

102. Viroids
• Very small, covalently closed, circular RNA molecules
capable of autonomous replication and induction of
disease
• Sizes range from 250-450 nucleotides
• No coding capacity - do not program their own
polymerase
• Use host-encoded polymerase for replication
• Mechanically transmitted; often seed transmitted
• More than 40 viroid species and many variants have
been characterized
• “Classical” viroids have been found only in plants

103. Viroids are divided into two groups, based on site and
details of replication

104. Viroid Diseases
• Potato spindle tuber
viroid (PSTVd)
– May be limiting to potato
growers
– First viroid characterized
– Many variants described
– Control with detection in
mother stock, clean seed
PSTVd in tomato
PSTVd in potato

105. Viroid Diseases
• Citrus exocortis viroid (CEVd)
– Causes stunting of plants,
shelling of bark
– May result in little yield loss
– May be useful to promote
dwarfing for agronomic
advantage
– Transmitted though stock, graft
– Control by removal of infected
plants, detection, clean stock
Citrus exocortis viroid

106. Apple crinkle fruit viroid Avocado sun blotch viroid
Citrus exocortis viroid
Healthy Infected

107. Potato spindle tuber viroid (PSTVd) is the most
thoroughly characterized viroid disease
(From R. Owens, USDA, Beltsville)

108. Viroid structures
-All are covalently closed circular RNAs fold to tightly base-paired structures
-Two main groups of viroids: self-cleaving and non-self-cleaving
-Non-self cleaving viroids replicate in nucleus and fold into “dog bone” or rod-like
structure
-Five domains identifiable in non-self-cleaving
-Left hand (LH) and right hand (RH) domains are non-base-paired loops
-Single mutations to pathogenic domain often alter virulence
-Mutations to conserved central domain are often lethal
-Mutations to variable domain are often permitted

109. Minor variations in viroid sequence, and presumably attendant
RNA structure changes, are associated with virulence differences
(From R. Owens, USDA, Beltsville)

110. Viroid replication
• In nucleus or chloroplasts, depending on class of viroid
• Chloroplast-associated viroids process into monomers by
ribozyme-mediated cleavage; nucleus-associated viroids
process into monomers by using host-derived enzyme
• In both classes, host DNA-dependent RNA polymerase is
the performs RNA polymerization on + and – strand RNA
templates
Ribozyme-mediated Cleavage by host-factor
RZ RZ
RZ RZ HF HF

111. Viroid movement
• Traffic within cell
through nuclear
pores using VirP1, a
nuclear localization
protein that binds
viroid RNA
• Traffic cell-to-cell
through
plasmodesmata
• Traffic long distance
through phloem
• All of these
processes are
associated with host
proteins

112. + and – viroid strands are differentially localized
within the nucleus
• Viroid strands of + polarity localized to nucleolus, as well
as nucleoplasm
• Viroid strands of - polarity localized to only to nucleoplasm
Qi and Bing, 2003, Plant Cell 15:2566

113. Hepatitis delta
• Hepatitis delta virus has many viroid-like properties, but
the RNA is larger (1.7 kb), is encapsidated, and encodes a
virion-associated protein (hepatitis delta antigen)
• Intensifies HBV infection
• HDV requires HBV as helper virus for encapsidation, so it
has satellite-like properties (like a “virusoid”)
• Replicates in nucleus via cellular DNA-dependent RNA
polymerase II