Journal of General Virology (1991), 72, 2895-2903. Printed in Great Britain
2895
Diverse groups of plant RNA and DNA viruses share related movement
proteins that may possess chaperone-like activity
Eugene V. Koonin, 1 Arcady R. Mushegian,2*~ Eugene V. Ryabov 2 and Valerian V. Dolja2:~
~Institute o f Microbiology, U S S R A c a d e m y o f Sciences, Moscow 117811 and ZA. N. Belozersky Laboratory,
Moscow State University, Moscow 119899, Russia
Amino acid sequences of plant virus proteins mediating
cell-to-cell movement were compared to each other and
to protein sequences in databases. Two families of
movement proteins have been identified, the members
of which show statistically significant sequence similarity. The first, larger family (I) encompasses the
movement proteins of tobamo-, tobra-, caulimo- and
comoviruses, apple chlorotic leaf spot virus (ACLSV)
and geminiviruses with bipartite genomes. Thus this
family includes viruses which move by two methods,
those requiring the coat protein for the cell-to-cell
spread (comoviruses) and those not having this requirement (tobamoviruses). The previously unsuspected
relationship between the movement proteins of RNA
and DNA viruses having no RNA stage in their life
cycle (geminiviruses) suggested that their movement
mechanisms might be similar. The second, smaller
family (II) consists of the movement proteins of
tricornaviruses (bromoviruses, cucumoviruses, alfalfa
mosaic virus and tobacco streak virus) and dianthoviruses. Alignment of the sequences of family I
movement proteins highlighted two motifs, centred at
conserved Gly and Asp residues, respectively, which
are assumed to be crucial for the movement protein
function(s). Screening the amino acid sequence database revealed another conserved motif that is shared by
a large subset of family I movement proteins (those of
caulimo- and comoviruses, and ACLSV) and the family
of cellular 90K heat shock proteins (HSP90). Based on
the analogy to HSP90, it is speculated that many plant
virus movement proteins may mediate virus transport
in a chaperone-like manner.
Introduction
infected cells, and more specifically within the plasmodesmata (Tomenius et al., 1987; Moser et al., 1988).
Moreover, an increased size exclusion limit of plasmodesmata has been revealed in transgenic plants expressing the 30K protein (Wolf et al., 1989). These observations appear to constitute conclusive evidence for the
involvement of the 30K protein in virus movement.
In other plant virus systems the evidence for transport
function is less solid. Nevertheless, by the inoculation of
plant cells with subsets of genome segments of multicomponent viruses, artificial mutagenesis of individual
genes and complementation of movement by distantly
related viruses, the movement function has been
assigned to specific gene products of tricornaviruses
(Allison et al., 1989), calulimoviruses (Stratford & Covey,
1989), comoviruses (Wellink & van Kammen, 1989),
hordeiviruses (Petty & Jackson, 1990) and bipartite
geminiviruses (Davies & Stanley, 1989). The complementation experiments seem particularly revealing,
demonstrating that there is probably much in common in
the movement mechanisms of distantly related viruses
(Taliansky et al., 1982; Malyshenko et al., 1989).
Recently, it has been demonstrated that the 30K
protein of TMV and the putative caulimovirus move-
A growing body of evidence indicates that cell-to-cell
spread of virus within the infected plant is mediated by
specific virus-encoded proteins (reviewed by Hull, 1989;
Atabekov & Taliansky, 1990; Citovsky & Zambryski,
1991). The best studied example is the 30K protein of
tobacco mosaic virus (TMV), the participation of which
in virus movement has been suggested by several types of
experiments.
(i) TMV mutants Ni2519 and Lsl, bearing mutations
in the 30K gene (Zimmern & Hunter, 1983; Meshi et al.,
1987), display temperature-sensitive (ts) cell-to-cell
movement, but can be rescued by a wild-type helper
which apparently provides a product acting in trans
(Taliansky et al., 1982; Malyshenko et al., 1989).
(ii) Transgenic plants expressing the 30K protein have
been shown to complement movement-defective TMV
mutants at non-permissive temperatures (Deom et al.,
1987). (iii) The 30K protein is localized to the cell walls of
"~Present address: Department of Plant Pathology,Universityof
Kentucky, Lexington,Kentucky40546, U.S.A.
Presentaddress:Departmentof Biology,TexasA & M University,
College Station, Texas 77843, U.S.A.
0001-0341 © 1991 SGM
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2896
E. V. Koonin and others
m e n t p r o t e i n b i n d s s R N A a n d s s D N A ( C i t o v s k y et al.,
1990, 1991). C i t o v s k y & Z a m b r y s k i (1991) p r o p o s e d t h a t
s i n g l e - s t r a n d e d nucleic a c i d b i n d i n g m i g h t be a c o m m o n
p r o p e r t y o f p l a n t virus m o v e m e n t p r o t e i n s w h i c h is
i n d i s p e n s a b l e for t h e i r function.
C o n s i d e r a b l e effort has b e e n p u t into c o m p a r i n g
a m i n o a c i d sequences o f ( p u t a t i v e ) m o v e m e n t p r o t e i n s o f
p l a n t viruses. M o s t o f this w o r k s t a r t e d w i t h the
s e q u e n c e o f the best s t u d i e d m o v e m e n t p r o t e i n , the 3 0 K
p r o t e i n o f T M V . V a r i o u s degrees of s i m i l a r i t y b e t w e e n
the 30K p r o t e i n a n d the p u t a t i v e m o v e m e n t p r o t e i n s o f
t o b r a v i r u s e s ( B o c c a r a et al., 1986), c o m o v i r u s e s a n d
n e p o v i r u s e s ( M e y e r et al., 1986), a n d u n e x p e c t e d l y
c a u l i m o v i r u s e s (Hull et al., 1986; H a s e g a w a et al., 1989),
h a v e b e e n r e p o r t e d . A t t e m p t s h a v e also b e e n m a d e , w i t h
s o m e w h a t conflicting results, to e s t a b l i s h r e l a t i o n s h i p s
b e t w e e n the p u t a t i v e m o v e m e n t p r o t e i n s of b r o m o - ,
cucumo-, ilar- a n d d i a n t h o v i r u s e s (Savitry & M u r t h y ,
1983; X i o n g & L o m m e l , 1989; A l l i s o n et al., 1989).
Several o b s e r v a t i o n s h a v e b e e n r e p o r t e d on a p p a r e n t
sequence s i m i l a r i t i e s b e t w e e n virus m o v e m e n t p r o t e i n s
a n d v a r i o u s cellular p r o t e i n s ( Z i m m e r n , 1983; Hull et al.,
1986; M a r t i n e z - I z q u i e r d o et al., 1987). It is n o t easy to
assess t h e actual value o f m o s t o f these o b s e r v a t i o n s
b e c a u s e (i) c o m p a r i s o n s usually were m a d e using only
one r e p r e s e n t a t i v e sequence o f the m o v e m e n t p r o t e i n s o f
a virus g r o u p (e.g. t o b a m o v i r u s e s ) a n d / o r a cellular
p r o t e i n family, a n d (ii) in m a n y cases only very s h o r t
s e g m e n t s were aligned.
Recently, M e l c h e r (1990) p u b l i s h e d a m o r e t h o r o u g h
c o m p u t e r - a s s i s t e d analysis of the s e q u e n c e s o f t o b a m o - ,
tobra-, c a u l i m o - a n d t r i c o r n a v i r u s m o v e m e n t proteins,
c o n f i r m i n g a significant s i m i l a r i t y b e t w e e n the p r o t e i n s
o f the first three groups, a n d a m u c h m o r e r e m o t e
r e l a t i o n s h i p b e t w e e n t h e m a n d the t r i c o r n a v i r u s e s .
H o w e v e r , p u t a t i v e c o n s e r v e d m o t i f s t h a t m i g h t be
helpful in further analysis o f virus m o v e m e n t p r o t e i n s
a n d t h e i r t e n t a t i v e cellular h o m o l o g u e s h a v e n o t b e e n
clearly defined. W e sought to e x t e n d this analysis by
s y s t e m a t i c a l l y c o m p a r i n g the sequences o f the v a s t
m a j o r i t y o f p l a n t virus p r o t e i n s i m p l i c a t e d in cell-to-cell
m o v e m e n t , a n d by s c r e e n i n g a m i n o a c i d sequence
d a t a b a s e s for t h e i r possible cellular homologues. O u r
analysis h i g h l i g h t e d an u n e x p e c t e d r e l a t i o n s h i p b e t w e e n
a large collection o f p l a n t virus m o v e m e n t p r o t e i n s a n d
the f a m i l y o f u b i q u i t o u s cellular 9 0 K h e a t shock p r o t e i n s
(HSP90), w h i c h possess m o l e c u l a r c h a p e r o n e activity,
i.e. t h e y are i n v o l v e d in i n t r a c e l l u l a r p r o t e i n trafficking
( W e l c h , 1990). T h e p o s s i b i l i t y o f a s i m i l a r a c t i v i t y for the
virus m o v e m e n t p r o t e i n s is discussed.
Methods
Amino acid sequences. The amino acid sequences of the (putative)
movement proteins of the following viruses were analysed. Tobamo-
viruses: TMV, TMV strain Om (TMVOm), TMV tomato strain
(TMVTo), TMV cowpea strain (TMVCo) and cucumber green mottle
mosaic virus (CGMMV). Tobraviruses: pea early browning virus
(PEBV) and tobacco rattle virus strain SY (TRV). Geminiviruses:
tomato golden mosaic virus (TGMV), bean golden mosaic virus
(BGMV) and cassava latent virus (CLV). Caulimoviruses: cauliflower
mosaic virus strains D/H, Strasbourg and CM1841 (CaMVD, -S and
-C), figwort mosaic virus (FMV), carnation etched ring virus (CERV)
and soybean chlorotic mottle virus (SCMV). Comoviruses: red clover
mottle virus (RCMV) and cowpea mosaic virus (CPMV). Cucomovirus: cucumber mosaic virus (CMV). Apple chlorotic leaf spot virus
(ACLSV; referred to as a clostero virus, but at present included in
capilloviruses) and beet yellows closterovirus (BYV). Bromoviruses:
cowpea chlorotic mottle virus (CCMV), broad bean mosaic virus
(BBMV) and brome mosaic virus (BMV). Ilarviruses: tobacco streak
virus (TSV) and alfalfa mosaic virus (AIMV). Dianthoviruses: red
clover necrotic mosaic virus (RCNMV). Nepoviruses: tobacco black
ring virus (TBRV) and Hungarian grapevine chrome mosaic virus
(GCMV). The sequences were from the SWISSPROT database
(Release 14), except for those of ACLSV (German et al., 1990) and
BYV (Agranovsky et al., 1991a).
Computer-assisted sequence analysis. Initial pairwise comparison of
the sequences was by the DOTHELIX program, generating a diagonal
plot and allowing precise delineation of sequence segments displaying
similarity with the highest deviation from the random expectation
(Leontovich et al., 1990). Sequence database screening was by
the SMART program, implementing a simplified version of the
DOTHELIX algorithm for comparison of a given 'probe' sequence
with all sequences in the database. Programs DOTHELIX and
SMART are modules of the GENEBEE program package for
biopolymer sequence analysis (Brodsky et al., 1991). To determine the
boundaries of the segments to be aligned, which is critical for obtaining
the optimal alignment, the results of DOTHELIX comparisons were
used. Multiple sequence alignments were generated in a stepwise
manner using the OPTAL program (Gorbalenya et al., 1989). The
statistical evaluation of alignment by OPTAL includes calculation of
two values: distance, D = -ln[(S ° - Sr)/(Sm - Sr)] x 100 (Feng et al.,
1985) and the adjusted alignment score, AS = (S° - Sr/a), where S° is
the observed alignment score, Sr is the mean score obtained upon 25
random simulations of the alignment procedure, Sm is the average of
the scores obtained upon comparison of each of the two compared
sequences (alignments) with itself, and cr is the standard deviation of
the score. Generally, D values of less than 200 and AS values of more
than 7 standard deviations (S.D.)can be considered a firm indication of
a genuine relationship between two sequences or alignments; values of
200 to 270 and 4 to 7 S.D. can be considered significant, provided there
is some additional (i.e. functional) evidence of the suspected
relationship (Doolittle, 1986; and unpublished observations). Throughout this study the log odds matrix of Dayhoff (Dayhoffet al., 1983) was
used for calculation of sequence comparison scores. Protein secondary
structure predictions were by the PROTEIN2 program of the
GENEBEE package implementing the modified algorithm of Gamier
(Gibrat et al. 1987).
Results and Discussion
Grouping o f plant virus m o v e m e n t proteins by sequence
similarity
O u r first a p p r o a c h was to g e n e r a t e local s i m i l a r i t y plots
for p a i r s o f (putative) m o v e m e n t p r o t e i n s e n c o d e d b y
viruses o f different g r o u p s to d e l i n e a t e s i m i l a r regions
a n d to e s t a b l i s h t h e a p p r o x i m a t e h i e r a r c h y o f relation-
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Plant virus movement proteins
TMV
TMVOm
Tbg/To
"f~Co
CGMMV
PEBV
TRV
30K
3OK
30K
30K
30K
29K
29K
TGMV
BGMV
CLV
21K
33K
33K
21
21
21
23
24
32
26
K I LPSM f t p V K s V M c S K V D K I -MVHENESL s - E V N L L - K - g V - K L ID ....... sK I LPSMf t pVKs VMcSKVDKI -MVHENESLs-EVNLL-K- gV-KL I D. . . . . . .
sKLLPSM f t pVKsVMVSKVDKI -MVHENESLs - E V N L L - K - g V - K L I E . . . . . . .
gK - F K w K - a p S R v C s I v q s D T I s M t A N G r S L f - T f D V L - K - D V 1 K h a E. . . . . . .
EY
K-LPNyfmILKiLsITdfSvV-KaQsyESLV-pVkLL-R-gV-DLTK
.......
HLMVdGArKrpKYfHRrRETvLsNVAGSLTEh-kLgIfTi-EdvRNVK
.......
sY
LfVdGRrKrpKYfHRPRETvLnhVGGkkSEh-kLDVfDQ-rdyKMIK. . . . . . .
sY
2 S q L A c P p N A F N Y I e S n R - D E y - Q L s h D L T E I - i L Q f p - S - T A S Q L S.a. . . . . .
2 S q L A N P p N A F N Y I e S q R - D E y - Q L s h D L T E I - I L Q f p - S - T A S Q L S.a. . . . . .
CaMVD
CaM'¢S
CaMVC
FMV
ORF1
ORF1
ORF1
ORF1
CERV ORFI
SCMV ORF1
RCMV 49K
CPMV 5SK
70 KaFSRK-NEILYCVSTKELSV-DIHDATgKV-yLPLITKeEINKRLsslkpe-vRR
7 0 KaFSRK-NEILYCVSTKELSV-DIHDATgKV-yLPLITKeEINKRLsslkpe-vRK
70 KaFSRK-NEILYCVSTKELSV-DIHDATgKV-yLPLITReEINKRLsslkpe-vRK
68 TaFT~RK-NEIFYCVSTKEMSV-DIkDvSgQV-yLPLITKqEIQQKLMkidpS-vRs
61KaFSRK-NhIYYhVnYKEfSV-DIcDThgKN-yLPLVTKSEIKKNLDkikdeKvRs
45 nVFSRd-NILKFgLMTgEVQI-pIEQTDgsV-fLAtINKeQITKRIskieeK-Og~R
131 TsLPGG R E V E Y - - K h l D A g h - L L A D T N v V I - D V P L V P Q I a A r t p T D y n f g T s r D K
IS9 S k A A A G - M G F I n - ~ K h m L T g N - I L A Q p T T V L - D I P V t k D k T L a m a S D - f i R K E N L
A C L S V 50K
34 KkASDL-MIhWNeEVFKVNpE~D/AGDgfRLaSIPVIpSsEVQaVI_RK . . . .
:
:
:::
:
"G"
*
TMV
TMVOm
TMVTo
TMVCo
CGP~IV
RL
RF
0 MdtSvPvIsSDYIHSaR-TEy-KLtNDESp[-TLQfp-S-TLERtrV ....... HI
Re
"D"
• ! i !!
30K
30K
30K
30K
30K
29K
29K
g y v C L - a G ]VVt g e w - N L P D N C F G G V s V C L V D K R M - - E R a D E A tL G S Y - y t a A A k k R F q
g y v C L - a G IVVt g e w - N L P D N C r G G V s V C L V D K R M - - E R a D E A t LGSY- y t a A A k R R F q
g y v C L - V G i VVsgew-NLPDNCr GGVs VCMVDKRM - - ERaDEA t LGSY -y £a A A k k R F q
TyvDV - L G v V L s g q w - L L P K G T p G S A E I IL L D S R L - - - K g K a S V L A V F - n c r A A T q E F q
Lyv t L - L G v W s g V w - N V P E s C r G G A t VALVDTRM-- hSVAEG t I cKF- sAPAt v rEFS
Kf i r I-VGiq Ik V t S - h L P R D T S G f LQ IDL IDSRL tO g P d ~ s k V L O R F - V A k A C D n t sV
af i k I-VGvqLVVt S - h L P a D T p G f IQ IDLLDSRLt ekRKKgkt IQ R F - K A F A C D n c s V
TCMV
I~GMV
CLV
21K
33K
33K
SrSCMkIdhcVl e fRqQVPiNaTGSVvVEIhDKRM---TDNESLqASWtFpvrCNiDLh
SrSCMkI dhc VI eyRqQVPi NaTGSVvVEI hDKRM-- -TDNESLqASWt Fp i rCNiDLh
MGKCMkVdhWI eyRnQVPfNaQGSViVTI RDTRL---SDEQqdqAqF t FpiGCNvDLh
CaMVD
CablVS
CaMVC
FMV
CERV
SCMV
ORFI
ORFI
ORF1
ORF1
ORF1
TmSMVIlLGAVk l LLKA(]FRNGID t p Ik I AL IDDR l - - N S R R D c L L G a a - K G N L A y G K F M
T m S M V R L G A V k ILLKAQFRNG iD t p I k IALI DDR I - - N S R R D c L L G a a - K G N L A y G K F M
Im S M V H L G A V k ILLKAQFRNG iDtp Ik IAL IDDR I - - N S R R D c L L G a a - K G N L A y G K F M
K i S M I H L G A V k I L L T A Q F R Q G iD t S V k M A L I D D R I - - v N R K D S L L G a a - R G N L A y G K F M
TISD IH F G A I k V L I K A r E R E G i N s D IkMAL IDDR I - -TDRQDS ILGaa - H G N L v y G K F M
LIRyVH I s tLQVL IKStFIKG-Dt p L E L T L R D N R L - - 1NLEESK I A V g - H G N L K y G K M K
PEBV
. TRV
RCMV
0RF1
49K
SATaLbIVGAI E W I Q S y a s s E C D 1 mAGMMLVDTfh - -SRpEN AI r sVY- I v P IRgGmFM
CPMV 58K
KTSaI HIGAIE I I I QgfaspEgD i mGGfLLVDS1h - -TDLANAI r s I F-VAPMRzGRpV
ACLSV SOK
S T n I VHWGALs I s IDALFRK - - N a G s G V C y V D N R w - - E T f E Q A M L Q K F - H f N L d S G s a T
: ::: : :
:
: :
: :
: :
!
TMV
TMVOm
TMVTo
TMVCo
CGMMV
PEBV
TRV
30K
30K
30K
30K
30K
291(
29K
!
•
!
FkVvPNYAIT%QD-AMkNvWQVLMNIrN~/k-MsAG--Fc
FkVvPNYAITtOD-AMkNvWQVLVNIrNVk-MsAG--Fc
FkVvPNYGITtKD-AekNIWQlrLVNIkNVk-MsAG--Yc
FLISPgYSLTcaD-ALkI~FEIsCNVIDLp-VI(DG--FT
VrFiPNYSWaaD-ALrDPWSLFVRLSNVg-IKDG--FH
VQYkFDYmVStRE-NIaDIWKIGtVaVNVp-VvDd--cy
aQYkVEYSIStQE-NV!DvWKVGCIsEgVp-VcDG--ty
!
~L--sLeF
~L--sLeF
~L--sLeF
~L--sVel
>L--tLeV
~F--sVeV
~F--sleV
109
109
lOS
122
I01
82
82
TC44V 21K
BGMV 33K
CLV
33K
Y-FSSsF-fSLKD-pI--PWKLYYKVcDsN-VhQrtHFakFkGKLkL
43
Y-FSSsF-fSI-KD-pl--PWKLYYRVSDtN-VhQrtHFakFkGKLkL 150
Y - F S a s Y - f S ID D - N V - - P W Q L L Y K V E D s N -VKNG I tFaq IkAKLkL 157
CaMVD
CaMVS
CaMVC
FMV
CERV
SCMV
ORFI
ORF]
ORFI
ORFI
ORFI
ORFI
FtVyPkFGISLNTqrLNQTLSLIhDFENkNLMNkGDkVmtltyIVGY
FtVyPkFGISLNTqrLNQTLSLIhDFENkNLMNkGDkVmtltyVVGY
FtVyPkFGISLNTqrLNQTLSLIhDFENkNLMNkGDkVmtItylVGY
Ft V y P k F A L S L Q S k N L D K T L S f Ih Q F E r k D L M K t G D k V F tVtyL IGY
FtVyPkYTTSILDqrLDRTLaflhhFErNDLMRkGDkVFsItyLVAY
RCMV
CPblV
49K
58K
r aL c F p n T L V p M D s D INNr F K V V F s L p N N D F I ~ G sk 1GHv s I nMaGc 121
r V V T F p n T L a p V S c D L N N r F K L I C s L p N C D I V Q G s Q v A e v s V n V a G c 128
ACLSV 5OK
FDVnLQIGLSLKDIDLDRSIILnYKFLrrNFMKEGNHAFslsyRInY
LVtSPNFpVSLDDpGLsNS]SVAVMFENLNFkfEsyplSvrVGNMcF
102
102
102
i01
102
103
215
Fig. 1. Alignment of conserved domains of family I virus movement
proteins. Protein sequences of distinct virus groups, tobamo-/tobra-,
gemini-, caulimo- and comoviruses, and ACLSV, are separated by
blank lines. The numbers of amino acid residues between the protein
termini and the aligned segments are indicated. Double asterisks
indicate identical or similar residues in all sequences; single asterisks
indicate residues identical or similar in the sequences of tobamo-,
tobra- and geminivirus proteins; colons indicate residues identical or
similar in caulimovirus, comovirus and ACLSV sequences; exclamation marks indicate residues identical or similar in tobamo-, tobra-
2897
ship. Then multiple alignments were generated in a
stepwise manner using this hierarchy. In this way it was
confirmed that the 30/29K movement proteins of
tobamo- and tobraviruses form a tight group, aligning
over their entire length (about 300 amino acid residues)
with a convincing AS of 7-4 S.D.
Quite unexpectedly, it was shown that this group is
related to the BL1 proteins, encoded by the B D N A
components of bipartite geminiviruses and implicated in
virus movement (Etessami et al., 1988; Brough et al.,
1988). A statistically significant alignment (AS about
6-1 S.D.) was obtained for the N-terminal regions of
approximately 140 amino acid residues, which constitute
about 8 0 ~ of the smallest geminivirus protein.
Comparisons of the putative caulimovirus movement
proteins (ORF 1 products) revealed an approximately 180
residue region of significant similarity with the 58/48K
proteins of comoviruses (AS of approximately 7.1 S.D.)
and the 50K protein of ACLSV (AS about 6.9 S.D. upon
comparison of the ACLSV sequence with the alignment
of caulimo- and comoviral proteins). The two alignments
thus obtained were combined to yield a composite
alignment of more than 5-7 S.D. above the random
expectation (Fig. 1). Even more convincing scores (about
6.5 S.D.) could be obtained upon aligning the sequences
of tobamovirus/tobravirus and caulimovirus proteins
separately. We define the set of proteins in the alignment
in Fig. 1 as family I of plant virus movement proteins.
Within this family, the scores for the alignments
reflecting newly observed non-trivial relationships, such
as those between the geminivirus and tobamovirus/
tobravirus proteins, or between caulimovirus and comovirus proteins, were comparable to that for the obvious
alignment between the tobamo- or tobravirus proteins
themselves.
As in other vast protein families, not all of the groups
constituting family I showed convincing similarity to
each other when compared directly. In each of such cases
the relationship could be established by comparison with
a third group. An important example is the comparison
of the movement proteins of tobamo- and tobraviruses,
and comoviruses. Upon direct alignment, these proteins
showed only a marginally significant similarity (4.1 S.D.
when compared within the boundaries shown in Fig. 1).
However, both groups were very similar to the movement proteins of caulimoviruses. Together with the
and caulimovirus proteins (in each case one exception in the
tobamovirus or caulimovirus sequence was allowed). Residues identical or similar in sequences of different groups are shown in upper case.
Amino acid residues were grouped as follows: A, G; S, T; I, L, M, V; F,
Y, W; K, R; D, E, N, Q. The two conserved motifs are designated as
indicated in the text as is the scheme of the alignment generation. The
ASs were over 5.5 s.o. and the distances (see Methods) less than 225 for
each alignment step.
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2898
E. V. Koonin and others
CCMV
BBMV
BMV
CMV
32K 48 GMytNLssNNrlnyiDLV-pkNTGsraHqLFkSEFeE-'Rh'pss-G-vL-IprvLVFL--VrTt
32K 47 GrytELkaNrrlnhlDLV-pkvhGmkmLSMFrSEydk--GnVpss-G-vLnlprlLiYL--VrTs
3ZK 49 GcynyLksNEpRnylDhV-pkShvsawLSw~TSKydk--GeLpsR-G-fMnVpriVcFh--VrT%
32K 40 GrhhwMraDNalsvRpLV-pQvTsnnLLSfFkSgyda--C~LrsK-G-yMsVpqvLcW--trTg
AIMV
TSV
32K 37 sMsI-LgpNQIKICtQLVIsNva~d~VVLSLVSkEkksIIr~IpKIC4~rMy%~hhSalYLIy Mpnl
32K 2g sgsVDLNmg-IBRCaafp-AENTGafLcELtTkEtksFIGKfsdKVrgrVf'DhaVIhMmyIPvl
RCNMV 37K 16 GVaVsLNrytdwKCRs---GvSeAPLIpasMmSKItdY-AKttaK-GNsVaLNyThV~slaPTI
-D,,?
TBRV
GCMV
93K
B4K
617
593
aa
LYafcViMwghssDAEtaslcgagvYlgdnqaavlELPLvcSyl-gnslEd-fda
aa
VYafcViMwghssNAEQaslsgayvYlgdgeasvlQLPLlcgyi-gnalEd~4ea
HSP90
GRP94
HSP82
H~Pg2
HSP90
HSP83
HSP90
human
mouse
D.m.
maize
cabbage
T. crucei
S.C
363
408
347
348
333
337
342
aa
LYvRRVFIND--~sCDELipEYL-NF-IRgVvdSEDLPLNISRE-mLQQsKILkv
aa
LYvRRVFItd---DFHDMmpkYL-NF-VKgVvdSDDLPLNVSRE-TLQQhKLLkv
aa
LYvRRVFIMd---NCEDLIpEYL-NF-NKgVvdSEDLPLNISRE-mLQOJ~VLkv
aa
LYvRRVFIMd---NCEELIpEWL-gF-VKgVvdSDDLPLNISRE-TLQQNKILkv
LYvRRVFIMd---NCEDIlpEYL-gF-VKglvdSEDLPLNISRE-TLQQNKILkv
LYvRRVFIMd---NCEDLcpEWL-gF-LRgVvdSEDLPLNISRE-NLQQNKILkv
LYvRRVFItd---EAEDLIpEWL-SF-VKgVvdSEDLPLNLSRE-mLQQNKIMkv
LYvqRVFIMd--~DAEQfmpNYL-rF-VRgLidSsDLPLNVSRE-ILQDstVtrN
HTPG E . c o l i
CCMV
BBMV
BMV
CMV
tVTeSGSVTIRLYDLisAssveILEpVdGtQEATipissLpAiVcFspSYdcpmQMig---NRhrcf-GLVT
hsSTvGSIT,RLVDtycAsdsclLEAIdG-QEfTvDIssLpcmlgFspTYdcklEMvD---GRrgcf-GIVT
dsAe~SITVsLcDsGkAAragVLEAIdN-QEATIQIs#d~pAIIaltpSYdcpmEVI~dsGRNFcf-GIaT
stdaeGSLkIyLaDLGdkE .... LspldG-QcVTlhnheLpAllsFqpTYdcpmELvg---NRhrcf-AWV
AIMV
TSV
IkSS~SITLKLfNeAtGE---LVDVdTD~hDAI-Qaclf-A-grYprSi-LakDaak---GHDIkl--VVh
InTThAiaeLKLRNLAtGD---eLyGgTk-vNLn-kafIL-t-MtWprSI-faeaVhN---hKEIYIgGtVS
298
aa
aa
aa
aa
CaMV-D ORF1
71
CaMV-S ORFI
71
aa
CaMV-C ORF1
FMV ORFI
C~V
DRF1
71
68
aa
62
aa
.... y C v s - t k E - L - S v d I h d a t g k v y L P L - l T K E - E I - - N l ~ L s s
aFsl~neIL .... yCvs-tkE-L-SvdIhdatgkvyLPL-ITKE-EI--NKRLss
aFsP~neIL .... yCvs-tkE-L-SvdIhdatgkvyLPL-ITRE-EI--NKttLss
aFkRKneIf. . . . yCvs-tkE-M-SvdIKdVsgqvyLPL-IlKQ-EI--QqKLmk
aFsRKnh[y. . . . yhvN-ykE-f-SvdIcdthgkNyLPL-VTKs-EI--kKnLdk
238
aa
LvagiLYdM--cfEYNtLkStYLkNlesfdeflSlyLPL-LSEvfSMNw~papD
BYV
64K
aa
aa
aFsRKnelL
"LPL"
RCNMV gVAipGhVTVeLINpnveG---pfQVMSG~QtLS-wspGa-G-kpclmiFsVhhQLns---DHEpFr--VRI
CCMV
BBMV
BMV
CMV
qLdGvlsSgSTVvMSHAYWSaNfrSKpNnYkQyapmyKYV-epfDrLKr-LSrkq-LKN--YV
eLnGvlgeghTVAIVHAYWkaMfrTKpgnYtRvkpaAKFI-ApfDrLKQ-LSSGq-I/<DafFI
qLSGvVgTtGSVAVtHAYWQaNfkaKpNnYklhGpatiMV-MpfDFI/RQ-LDkks-LKN--YI
erhGylgygGTtAsVcsnWQaQfSSKnNnYthAAaaktLV-LpyNrLAE-hSkps-avA-rLL
69
62
68
112
AIMV
TSV
aVas-tnmn~aVGVLypiWEdELSrK-QileRGADflKFpiAktEpVRDLLNaGK-LtD--FV
caSs-VPahAkIGMwypIWSekVSiK-QIYqNTiD/hK-tEA~etFtpl]41SSdKeM~s--LL
82
78
RCNMV tnTG-IPTkkSyArcHAYWgfDVg~-hrYyKS-EpARLIELevgYqRTLLSSiKaVeA--YV
,22
:
THRV
GCMV
Fig. 2. Alignment of conserved domains of family II virus movement
proteins. The distinct groups (separated by blanks) are represented by
the proteins of the bromo-/cucumo-, liar- and dianthoviruses. Asterisks
indicate identical or similar residues in all sequences. The ASs were
over 6.0 S.D. and the distances less than 220 for each step. The tentative
counterpart of the 'D' motif of family I plant virus movement proteins
is highlighted. Other designations are as in Fig. 1.
HSP90
GRP94
HSP82
HSPg2
HSP90
HSP83
HSP90
93K
B~K
"c"
:
::
yKRsLVlsTV-fFgKsGlfaC~NvfgitavEFTdy-MptsyGgitHERDSw . . . . . qaML l l S
7K~sLV1sTc-fFgtsGlspGQNmfgitavEFTey-LptsyGgitHERDS ..... nOeL 114
human
mouse
D, m
maize
IRKNIVKKcLELFSELA-edKENyK-KFFEAFSKN-LK-LG--IHEDSTN--RRRLsELL
IRKkLVRKTLDMIKKIA-eeKyN-d-TFwkEFgTN-VK-LG--VIEDhSN--RtRLakLL
IRKNLVKKTMELIeeLT-edKENyK-KFyDQFSKN-LK-LG--VHEDSNN--RaKLaDfL
IRKNLVKKcIEMFfelA-enKDDyA-KFyDaFSKN-IK-LG--IHEDSQN--RaKLaDLL
c a b b . IRKNLVKKcMELFfelA-enKEDyA-KFyEaFSKN-LK-LG--IHEDSQN--RtKIaELL
T. c
IRKNIVI</qALELFeeLA-GnKEDyK-KFyEQFSKN-VK-LG--IHEDSSN--RKKLmELL
S.c.
IRKNIVKKIIEaFnelA-edsEQfe-KFysaFSKN-IK-LG--VHEOTQN--R~aLakLL
269
291
268
266
264
265
Z64
HTPG E. c o l i
LRNaL~tKRvL0J~LeKLAkddAEkyq-TF~QQFglv-tH~-E~--paEDfaN--QeaIakLL226
CaMV-D ORFI
CaMV-S ORFI
CaMV-C ORFI
FMV
ORFI
LKpE-V}~RTMsMV-hLG-AvKilIKaqFFNgidTp-IK-IA--LIBDrIN--sRR-dcLL
LKpE-VBKTMsMV-hLG-AvKIIIKaqFrNgldTp-IK-IA--LIDDriN--sRR-dcLL
LKpE-VBKIMsMV-hLG-AvKIIIKaqFrNgidTp-IK-IA--LIDDriN--sRR-dcLL
IDps-VRsklsMl-hLG-AvKilltaqFrOgldTs-VK-MA--LIDDriv--NRK-dsLL
Bh~/
V R l l f e l d A a E L L I K V p - t l n m h d s - I F I - - Y - K N K L R y L e s y f e D D S N E I l K v K V d s L L208
64K
162
162
162
161
"D"
conservation of distinct motifs (discussed below), this
suggests that each of these groups belongs to family I and
are therefore related to each other.
Family II is composed of the putative movement
proteins of tricornaviruses (32K to 33K products of
RNA 3) and dianthoviruses (35K product of R N A 2),
yielding a statistically significant alignment (6.6 S.D.) on
a 200 residue span (Fig. 2). Interestingly, the proteins of
the two subdivisions of tricornaviruses (bromo-/cucumoviruses and A1MV/ilarviruses) were no more similar to
each other than to the dianthovirus protein.
Our attempts to produce an overall alignment of the
movement proteins belonging to families I and II were
not convincing, yielding alignment scores insufficient to
prove that the two families are actually related to each
other ( < 3 S.D.). One more group of plant virus proteins
more or less definitely associated with cell-to-cell
movement included those of nepoviruses (Meyer et al.,
1986). We were unable to produce statistically significant
alignments between the sequences of nepovirus proteins
and any of those included in families I or II.
Search for cellular proteins related to the plant virus
movement proteins
Representative sequences of each group of plant virus
movement proteins were compared with all sequences in
the SWlSSPROT amino acid sequence database
Fig. 3. Alignment of the sequences of (putative) virus movement
proteins with those of a conserved domain of HSP90. Double asterisks
indicate residues identical or conserved in all aligned sequences; single
asterisks indicate identical or conserved residues in HSP90 and the
respective group of virus proteins (one exception in HSP90s or in
caulimovirus proteins was allowed). Identical and similar residues in
cellular and virus protein sequences are in upper case. The adjusted
score for the alignment of the HSP90 sequences with those of
caulimovirus ORF 1 product was 5.4 S.D., with a distance of 184.4.
Including the BYV sequence segment in the alignment yielded a score
of 4 S.D. (distance 264-9). The nepovirus sequences were aligned
separately with those of HSP90, and the resulting alignments were
reconciled by hand. Other designations are as in Fig. I. Abbreviations:
D m . , Drosophila melanogaster; S.c., Saccharomyces cerevisiae; T.
crucei, Trypanosoma crucei; E. coli, Escherichia coil
(Release 14) using the SMART program (see Methods).
No particularly close relationships could be revealed.
The observed modest similarities were subjected to a
detailed evaluation using the programs DOTHELIX and
OPTAL. The only result deserving attention was
obtained for an approximately 100 amino acid residue
conserved segment of HSP90, which yielded a significant
score (5.4 S.D.) upon comparison with the alignment of
the caulimovirus movement proteins. Counterparts to
this segment, though with lower similarity, have been
identified in the putative movement proteins of nepoviruses, and in a partially sequenced 64K protein of
unknown function encoded by BYV (Agranovsky et al.,
1991 a; Fig. 3). The region conserved in HSP90 and virus
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Plant virus movement proteins
MOTIF
"G .
.
VHENESLS--EVNL-L-K
VHENESLS--EVNL-L-K
VHENESLS--EVNL-L-K
TANGRSLF--TFDV-L-K
AQSYESLV--PVKL-L-R
VAGSLTEH--KLGI-FTI
VGGKKSEH--KLDV-FDQ
TGMV
BGHV
CLV
Z1K
331<
33K
23
23
21
LSHDLTEI--ILQF-P-S 12 CM-KID-HCVIEFR
LSHDLTEI--ILQF-P-S 12 ~ - K I D - H C V I E Y R
LTNDESPI--TLQF-P-S 12 (~4-KVD-HVVIEYR
10 W--VE--IHDKRM 103
I0 W--VE--IHDKI~4 211
I0 VI--VT--IRDTRL 220
CaI4VD
CaMVS
CaNVC
YMV
CERV
SC)~V
ORFI
QRF1
ORF1
ORFI
ORF1
ORFI
91
91
91
88
82
66
IHDATGKV--YLPL-ITK
IRBATGKU--YLPL-ITK
IHDATGKV--YLPL-ITR
IKDVSGQV--YLPL-ITK
ICDTHGKN--YLPL-VTK
IEQTDGSV--FLAT-INK
I0
I0
10
i0
10
9
RCMV
CPMV
49K
SSK
ISO
]98
LADTNWI--DVPL-VPQ 20 AL-HVG-AIEVVIQ I0 AG--MM -LVDTFH 189
LAQPTTVL -DIPV-TKD 20 A I - H I G - A I E I I I Q lO GG- FL--LVDSLH 196
ACLSV
50K
55
IAGDGFRL-ASIPV IPS 14 IV-HWG-ALSISID 8 SG--VC -YVDNRW 283
TBRV
GCHV
93K
84K
643
619
LGDNQAAV-LELPLVCSY
LGDQEASV-LQLPLLCGY
BYV
64K
262
ESFDCFLS-LYLPL
385
430
369
370
355
359
364
32Q
IRGVVDSE--DLPLNISR
VKGVVDSD--DLPLNVSR
~GWDSE--DLPLNISR
VKGVVDSD -DLPLNISR
VKGIVDSE--DLPLNISR
LRGVVDSE--DLPLNISR
VKGVVDSE--DLPLNLSR
VRGLIBSS--BLPLNVSR
CONSENSUS
I
VS--VC--LVDKRM
VS--VC--LVDKRM
VS--VC--MVDKRM
AE--II--LLDSRL
AT--VA--LVDTRM
LQ--ID--LIDSRL
IQ--ID--LLDSRL
IK--IA--LIDDRI
~K--IA--LIDDRI
IK--IA--L1DDRI
VK--MA -LIDDRI
IK--MA--LIDDRI
LE--LT--LRDNRL
171
171
167
183
163
150
146
176
176
176
169
170
171
20 FFGKSGLFAGQNVF
21 FFGTSGLSPGQNMF
I0 MPTSYGGITHERDS
I0 LPTSYGGITHERDS
120
119
LSE 25 LLLKVP-TINMHD~
7 LR-YLESYFEDDSN
219
9
9
9
9
9
9
9
9
277
299
276
274
272
373
272
23~
LPL * R
K
MOTIF
MV-HLG-AVKILLK
MU-HLG-EVKILLK
MV-HLG-AVKILLK
MI-HLG-AVKILLT
DI-HFG-AIKVLIK
YV-HIS-TLQVLIK
9
9
9
9
9
9
9
D"
43
43
43
44
45
55
48
19
19
19
19
20
19
CL--AG-LWTGEW
CL--AG-LWTGE~
CL--VG-LWSGEt4
DV--LG-VVL.~QW
TL--LG-VVVSGVW
RI--VG-IQIKVTS
KI--VG-VQLWTS
.
30K
30K
30K
30K
30K
29K
29K
HSP90 human
GRP94 mouse
HSP82 D.m.
HSPSZ maize
HsPgO eabb.
HSP83 T. c,
HsPgo S.c.
~TPG E . c o ] i
I0
I0
10
12
10
12
12
.
TMV
TMVOm
IMVTo
TMVCo
CGbfftV
PEBV
TRV
21LFSELA-EDKENYK
21MIKKIA-EEKYN-D
21LIEELT-EDKENYK
21 MFFEIA-ENKDDYA
21 LFFEIA-ENKEDYN
21LFEELA-GNKEDYK
21 AFNEIA-EDSEQFE
2Z MLEKLKKDDAEKYQ
**
IG II
A
LK--LG--IHEDST
VK--LG--VIEDHS
LK--LG--VHEDSN
IK--LG--IHEDSQ
LK--LG -IHEDSQ
VK -LG--IHEDST
IK- LG--VHEDTQ
LK--EG--PAEDFA
•
OG
A
leD R
E
"LPL"
Fig. 4. Conservedmotifsin (putative)virusmovementproteinsand in
HSP90. CONSENSUSindicatesthe residuesprsentin at least 16ofthe
31 sequencesaligned;asterisksindicatehydrophobicresidues(I, L, V,
M, F, Y, W, C and A).
proteins constitutes a part of the conserved domain of
family I movement proteins (see Fig. 1 and discussion
below).
Conserved motifs in virus movement proteins
Inspection of the alignment of family I movement
proteins revealed at least two short, closely spaced
segments which should be considered conserved motifs
(Fig. 1 and 4). In tobamo- and tobravirus proteins these
motifs were located inside the most conserved
N-terminal region I described by Saito et al. (1988). The
N-proximal 'G' motif included a Gly residue that is
conserved in the vast majority of the sequences of the
family, although it is replaced by Asp in geminivirus
proteins. The flanking regions in all proteins are
significantly enriched in hydrophobic residues. The
distal 'D' motif included the Asp residue, which is the
only invariant residue in the entire alignment; this
residue is flanked by a hydrophobic segment to the
N-terminal side and is separated by one residue from a
highly conserved Arg to the C-terminal side. Despite our
inability to demonstrate any overall similarity between
the two families of movement proteins, one of the
conserved sequence segments of family II resembled the
'D' motif (Fig. 2), also noted by Melcher (1990).
2899
Several mutants of TMV which exhibit the ts
movement phenotype (Zimmern & Hunter, 1983; Meshi
et al., 1987), or overcome the virus spread block in a
tomato Tm2 line (Meshi et al., 1989), have been
characterized. The mutations responsible for these
phenotypes have been mapped to the conserved domain
but fall outside the motifs delineated above. This is
compatible with the apparent modulatory effect of these
mutations on 30K protein activity.
Inspection of the alignment of the amino acid
sequences of HSP90 with caulimovirus and nepovirus
movement proteins and the 64K protein of BYV (Fig. 3)
highlighted another motif with the sequence LPL
(hereafter LPL motif). Secondary structure predictions
for HSP90 and the movement proteins yielded similar
patterns, with a clear preponderance of s-helices and a
loop around the LPL motif (not shown). When HSP90
sequences were compared with the aligned sequences of
the movement proteins of tobamo-, tobra-, caulimo- and
comoviruses, and ACLSV, in boundaries slightly different from those in Fig. 3, an alignment of reasonable
statistical significance (about 5 S.D.) was produced. It
became clear that modified forms of the LPL motif are
present in the putative movement proteins of comoviruses and ACLSV, and degenerate counterparts of this
motif could be tentatively identified in the tobamovirus
and tobravirus sequences (Fig. 4). Thus the degree of
conservation of different motifs varies in virus movement proteins and HSP90; the LPL motif is most
prominent in HSP90 and caulimovirus proteins, whereas
'G' and 'D' motifs are best conserved aong tobamo-,
tobra- and caulimovirus proteins (Fig. 4). Notably,
caulimovirus movement proteins combine all three
motifs in their non-degenerate forms.
The conserved domains delimited above occupy
somewhat different locations in the virus movement
proteins (Fig. 5). In relatively small 22K to 38K proteins
(gemini-, tobamo-, tobra-, caulimo-, tricorna- and
dianthoviruses) these domains reside in N-terminal
regions, with variable C-terminal extensions. Larger
48K to 64K proteins (comoviruses, nepoviruses, ACLSV
and BYV) have both N- and C-terminal extensions.
Functional implications of the alignment of movement
proteins
Recently, two types of plant virus movement, represented by tobamoviruses and comoviruses, were postulated. They differ primarily in that virus capsid proteins
are required for the second, but not for the first type
(Wellink & van Kammen, 1989; van Lent et al., 1990).
By demonstrating that the movement proteins of
tobamoviruses and comoviruses both belong to family I
we have shown that, despite this important distinction,
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2900
E. V. Koonin and others
Tobamo- 'Tobraviruses
'LPL" "G" "D'
..............................
'G ~ 'D"
II II
TGMV
Geminiviruses
I1 iI
I
CLV
BGMV
[J
Caulimoviruses
|
Comoviruses
[
|
II
ACLSV
|
Nepoviruses
|
[I
BYV (Closteroviruses)
|
HSP90 (eukaryotic)
|
HTPG
7_
_._J
E. coli
|
'LPL'
'G'
'D"
Fig. 5. Locationsoftheconserveddomains (black)in the(putative)plant virus movement proteins and HSP90. Movement proteins are
designated by rectangles drawn approximately to scale; the regions aligned in this paper are enclosed in the broken box. The three
conserved motifs described in the text are shown in those proteins in which they could be clearly identified.
the mechanism of movement protein action may be
similar.
Binding of ssRNAs and ssDNAs by the movement
proteins of TMV and CaMV has recently been demonstrated (Citovsky et al., 1990, 1991). Interestingly, the
CaMV protein showed a strong preference for R N A
(Citovsky et al., 1991). Thus in both positive-strand
R N A viruses and caulimoviruses, which replicate by
reverse transcription of the complete RNA transcript
of the viral genome, virus spread may occur in the
R N A [ribonucleoprotein (RNP)] form (Citovsky &
Zambryski, 1991). This is compatible with the significant sequence similarity between the movement proteins
of caulimoviruses and several groups of positive-strand
R N A viruses revealed in this and previous studies. The
relationship identified between the movement proteins
of R N A viruses, caulimoviruses and geminiviruses
suggests that geminivirus proteins may bind ssDNA, and
that the mechanism of movement occurring in the form
of ssRNA and ssDNA may have much in common.
We have searched the sequences of the movement
proteins for known nucleic acid-binding motifs and
compared them to the sequences of single-stranded
nucleic acid-binding proteins. This analysis failed
to reveal any convincing similarity. This may be not
very surprising because, for example, the bacterial and
phage ssDNA-binding proteins themselves show poor
sequence conservation (Prasad & Chiu, 1987).
Initial deletion mutagenesis analysis of the TMV 30K
protein appeared to localize the RNA-binding site to an
approximately 30 amino acid residue segment of this
protein around the 'G' motif (Citovsky et al., 1990).
However, recent detailed studies revealed a more
complex situation, indicating that this site is probably
formed by two non-contiguous regions of the polypeptide. Deletion of short segments encompassing either
the 'G' or 'D' motif did not abolish R N A binding
(V. Citovsky, personal communication). Thus, it is
unlikely that any of these motifs is the primary
determinant of R N A (DNA) binding by the movement
proteins, although their importance for the specificity of
this binding cannot be excluded. Alternatively, one or
both of these motifs might be involved in some quite
different activity of the movement proteins.
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Plant virus movement proteins
The structural similarity between some of the plant
virus movement proteins and HSP90 might imply a
functional analogy. Although the functions of HSP90 are
relatively poorly understood, they are believed to
facilitate intracellular transport of steroid hormone
receptors (Kost et al., 1989) and retroviral tyrosine
protein kinases (Ziemecky et al., 1986). Association of
HSP90 with actin (Koyatsu et al., 1986) and tubulin
(Sanchez et al., 1988) cytoskeletal frameworks may also
indicate that they are involved in intracellular protein
sorting. Thus, it seems likely that HSP90, similarly to the.
more thoroughly studied HSP70, possesses molecular
chaperone activity. Along with intracellular protein
sorting and transport, molecular chaperones mediate cotranslational protein folding (Rothman, 1989; Beckman
et al., 1990) and assembly of multimeric proteins
(Pelham, 1990). They have also been found in association
with various RNPs (Welch, 1990). Plant virus movement
is a complex process and may require any, or all, of these
activities. Obviously, movement includes not only the
passage of virus in the form of virions or other R(D)NPs
through the plasmodesmata, but also their intracellular
routing from the site of assembly to the plasmodesmata. Both stages are plausible targets for chaperone
activity. One attractive possibility is that virus-encoded
chaperones might be involved in promoting assembly of
specific virus R(D)NPs, or of some virus-induced
intraceUular structures (e.g. unusual cytoskeletal and/or
membrane complexes). Various structures of this type
have been described in many virus-plant systems, e.g. in
caulimovirus-infected turnip cells (Linstead et al., 1988).
Recently, tubular structures have been observed in
comovirus-infected cells and their specific association
with the 48/58K putative movement protein has been
demonstrated (van Lent et al., 1990). The chaperones
might also mediate disassembly of the complex structure
of the native plasmodesmata. Obliteration of plasmodesmata fine structure upon virus infection has been
highlighted in several reports (Kitajima & Lauritis,
1969; Weintraub et al., 1976). Functional modification
of plasmodesmata, i.e. an increase in their size exclusion
limit, has been documented in TMV-infected cells (Wolf
et al., 1989).
The relationship of the present results to those o f previous
comparisons
Attempts to establish relationships between the
sequences of movement proteins of various viruses have
been quite numerous. The present results differ from
those reported in many studies in which only short
sequence segments were aligned. On the other hand, the
alignment of tobamo-, tobra- and caulimovirus proteins
is compatible with the alignment published by Melcher
2901
(1990). The latter alignment, however, failed to highlight
the conservation of the 'G' motif in caulimovirus
sequences. We failed to confirm the statistical significance of the alignment of tricornavirus movement
proteins with those of tobamo-, tobra- and/or caulimoviruses, despite extensive efforts to align these sequences
either in the boundaries indicated by Melcher or with
various shifts.
Although we have confirmed the relationship between
the sequences of the putative movement proteins
of ACLSV and caulimoviruses reported previously
(German et al., 1990), our alignment is different. The
divergences probably should be attributed to the fact that
neither Melcher (1990) nor German et al. (1990)
attempted to delineate similar sequence segments by
local similarity searches prior to generation of the
alignment. Rather, these investigators attempted to align
complete protein sequences, a procedure that leads quite
frequently to erratic alignments.
We were unable to confirm claims of similarities
between proteins of some virus groups, in particular the
N-terminal domains of the potyvirus polyproteins
(Domier et al., 1987), and the proteins discussed here.
Zimmern (1983) noticed a degree of similarity between
the 'G' motif and the surrounding sequences of tobamovirus 30K proteins and the sequence of the 'LAGLIDADG' box conserved in yeast mitochondrial intronencoded proteins, the maturases. We failed to confirm
the significance of this observation in statistical terms
using aligned maturase sequences (E. Koonin, unpublished observations) and those of various groups of virus
movement proteins, in accord with the recent analysis by
Melcher (t990). However, because the maturases
mediate splicing by facilitating proper R N A folding
(Burke, 1988), the similarity described by Zimmern may
reflect a common structural feature.
Concluding remarks
The above sequence comparisons suggest that caulimovirus movement proteins are relatively closely related
both to HSP90 and to proteins of several virus groups.
The observed pattern of sequence relationships might be
indicative of 'star' evolution, with the caulimovirus
movement proteins most closely resembling the hypothetical common ancestor constituting the centre of
evolutionary radiation. It is tempting to speculate that
this common ancestor could be a captured cellular
HSP90-related gene.
Of special interest is the unique gene organization in
BYV. One of the putative proteins encoded by this virus
genome displays highly significant sequence similarity
with the HSP70 family (Agranovsky et al., 1991 b). Thus,
as suggested by the present observations, the BYV
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2902
E. V. Koonin and others
genome may encode two HSP homologues in tandem,
and it seems plausible to speculate that they may act in a
concerted manner in virus intra- and/or intercellular
movement. The HSP90-related movement proteins of
other viruses may function in conjunction with cellular
HSP70s. C o n c e r t e d a c t i o n by cellular H S P 7 0 a n d H S P 9 0
has been p o s t u l a t e d r e c e n t l y for r e g u l a t i o n o f steroid
h o r m o n e r e c e p t o r a c t i v i t y ( K o s t et al., 1989).
C o m p a r i s o n o f the two groups of m o v e m e n t p r o t e i n s
identified here to the classification suggested by
A t a b e k o v & T a l i a n s k y (1990) shows t h a t our f a m i l y I
differs s u b s t a n t i a l l y o w i n g to the a d d i t i o n o f g e m i n i viruses a n d A C L S V , a n d the l a c k o f poty- a n d n e p o viruses; f a m i l i e s II are identical. F u r t h e r , we d i d not
include in our c o m p a r i s o n s a set o f t h r e e p r o t e i n s f r o m
potex-, carla-, h o r d e i - a n d furoviruses p r o p o s e d to be
i n v o l v e d in virus cell-to-cell m o v e m e n t ( M o r o z o v et al.,
1989; P e t t y & J a c k s o n , 1990; Beck et al., 1991) b e c a u s e
n o n e o f these p r o t e i n s c o n t a i n s d e t e c t a b l e s e q u e n c e
h o m o l o g y to f a m i l y I or II m o v e m e n t proteins. T h i s fact
m a y be i n d i c a t i v e o f the differences in the m e c h a n i s m o f
m o v e m e n t o f the triple gene b l o c k - c o n t a i n i n g a n d o t h e r
p l a n t viruses.
T h e r e is i n c r e a s i n g e v i d e n c e t h a t m o v e m e n t o f m a n y
p l a n t viruses m a y be affected b y c e r t a i n d o m a i n s o f
p r o t e i n s h a v i n g o t h e r functions in virus m u l t i p l i c a t i o n ,
e.g. the c a p s i d p r o t e i n s o f c o m o v i r u s e s ( W e l l i n k & v a n
K a m m e n , 1989), b r o m o v i r u s e s ( S a c h e r & A h l q u i s t ,
1989; A l l i s o n et al., 1990) a n d m o n o p a r t i t e g e m i n i v i r u s e s
( L a z a r o w i t z et al., 1989), a n d the b r o m o v i r u s R N A
p o l y m e r a s e ( T r a y n o r et aL, 1991). I n s p e c t i o n o f the
a m i n o a c i d sequences o f these d o m a i n s d i d n o t r e v e a l
o b v i o u s c o u n t e r p a r t s to the m o t i f s c o n s e r v e d in the
m o v e m e n t proteins. H o w e v e r , d e t a i l e d a n a l y s e s o f these
sequences were n o t u n d e r t a k e n .
A s w i t h a n y results o f c o m p u t e r - a s s i s t e d c o m p a r i s o n s ,
m u c h c a u t i o n m u s t be e x e r t e d in the f u n c t i o n a l
i n t e r p r e t a t i o n o f the r e l a t i o n s h i p s b e t w e e n p l a n t virus
m o v e m e n t proteins. T h i s should be stressed in view o f
the fact t h a t s o m e o f the a l i g n m e n t s r e p o r t e d here were
o f m o d e r a t e statistical significance. D i r e c t e x t r a p o l a t i o n
o f the o b s e r v a t i o n s m a d e for one p r o t e i n (the 30K
p r o t e i n o f T M V ) to o t h e r p r o t e i n s w i t h d i s t a n t l y r e l a t e d
sequences c a n h a r d l y be e n c o u r a g e d . N e v e r t h e l e s s , it is
o u r hope t h a t the findings p r e s e n t e d here will be helpful
in d e s i g n i n g e x p e r i m e n t s to c h a r a c t e r i z e m o v e m e n t
proteins biochemically.
The authors are grateful to Professor J. G. Atabekov for constant
interest and encouragement, to Drs V. Citovsky and A. E. Gorbalenya
for useful discussions, and to Drs J. C. Carrington, M. E. Taliansky and
A. V. Karasev for critical reading of the manuscript. Thanks are also
due to Drs V. Citovsky, U. Melcher and T. Candresse for
communicating their results prior to publication.
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