Characterisation of wild-type human
adenovirus serotypes Ad4, Ad11, Ad12 and Ad17 and modified viruses Ad5HVR48 and Ad5f35 in comparison to Ad5 for potential use as oncolytic agents
Nick Walters
Degree project in biology, Master of science (2 years), 2011 Examensarbete i biologi 45 hp till masterexamen, 2011
Biology Education Centre and Department of Clinical Immunology, Uppsala University
Supervisor: Professor Magnus Essand
2
!
"#$#!%&$'(#%')*+ ),+ -'./0
%12&+ "34#*+ #/&*)5'$3(+ (&$)%12&(+#
/67
+#
/887
+#
/89
+ #*/+#
/8:
+ #*/+ 4)/','&/+ 5'$3(&(+#
/;"5$6<
+ #*/+#
/;
,=;
+ '*+ !)42#$'()*+ %)+#
/;
+ ,)$+2)%&*%'#.+3(&+#(+)*!).1%'!+#>&*%(
+ +
*
'!?+-
#.%&$(+
! !
%@ABA+ CBA+ DEAB+ FGFHI+ JGKL0HIMA+ NABDHIMAN+ DF+ @OPCQ+ CLAQDEGBON+ R#LS7+ TKCNNGFGAL+ GQHD+ NGU+
NMATGAN+VCNAL+DQ+MBDHAGQ+NHBOTHOBA7+GPPOQDWAQGTGHI+CQL+WAQDPGT+NAXOAQTAY+#L;+CQL+RHD+C+
KANNAB+AUHAQHS+#L9+CBA+H@A+VANH0NHOLGAL+NABDHIMAN+CQL+AUHAQNGEA+BANACBT@+GQHD+H@AGB+ONA+CN+
DQTDKIHGT+CQHG0TCQTAB+CWAQHN+@CN+KACL+HD+ECBGDON+TKGQGTCK+HBGCKNY+%@DOW@+NDPA+DF+H@ANA+CWAQHN+
@CEA+ MBDEAL+ NOTTANNFOK+ J@AQ+ CLPGQGNHABAL+ GQHBC0HOPDOBCKKI7+ GQHBCEAQDON+ GQZATHGDQ+
BAPCGQN+C+T@CKKAQWA7+LOA+HD+BCMGL+TKACBCQTA+DF+H@A+EGBON+VI+ECBGDON+@DNH+GPPOQA+MCH@JCINY+
'QTBACNAL+LDNAN+KACL+HD+@AMCHDHDUGTGHIY+"GW@+WKDVCK+MBAECKAQTA+DF+PCQI+NABDHIMAN+PACQN+
H@CH+ PCQI+ MADMKA+ @CEA+ @GW@+ CQHGVDLI+ HGHBAN+ CWCGQNH+ #LNY+ 5GBONAN+ JGH@+ C+ KDJAB+
GPPOQDWAQGTGHI+CBA+BAXOGBAL+HD+DEABTDPA+H@GN+@OBLKA+GF+#LN+CBA+HD+VA+CLPGQGNHABAL+EGC+H@A+
VKDDLNHBACPY+(HOLI+DF+C+VBDCL+BCQWA+DF+NABDHIMAN+FBDP+ECBGDON+NMATGAN+CQL+PDLGFGTCHGDQ+DF+
EGBONAN+VI+NOVNHGHOHGDQ+DF+NHBOTHOBCK+TDPMDQAQHN+JGH@+MBDHAGQN+FBDP+DH@AB+NABDHIMAN+CBA+
HJD+CBACN+DF+BANACBT@+H@CH+TDOKL+KACL+HD+MBDLOTHGDQ+DF+#LN+H@CH+@CEA+@GW@+DQTDKITGHI+CQL+
KDJ+GPPOQDWAQGTGHIY+
+
'Q+H@GN+NHOLI7+H@A+DQTDKITGHI+DF+@OPCQ+JGKL0HIMA+NABDHIMAN+#L67+#L887+#L89+CQL+#L8:+JCN+
TDPMCBAL+HD+H@CH+DF+#L;+GQ+FDOB+@OPCQ+TCQTAB+TAKK+KGQANY+%@A+QAOHBCKGNCHGDQ+CVGKGHI+DF+FGEA+
@OPCQ+ MKCNPC+ NCPMKAN+ CQL+ DF+ GQHBCEAQDON+ GPPOQDWKDVOKGQN+ R'E'WS+ MDDKAL+ FBDP+ 8[7[[[+
MADMKA+ CWCGQNH+ ACT@+ NABDHIMA+ JCN+ CNNANNALY+ -@GKNH+ #L;+ N@DJAL+ H@A+ @GW@ANH+ DQTDKITGHI+
DEABCKK+ RTKDNAKI+ FDKKDJAL+ VI+ #L88S7+ #L88+ JCN+ H@A+ KACNH+ GPPOQDWAQGT7+ AECLGQW+
QAOHBCKGNCHGDQ+VI+'E'W+CH+@GW@+TDQTAQHBCHGDQY+#L8:+CKND+N@DJAL+KDJ+GPPOQDWAQGTGHI7+JGH@+
@GW@+DQTDKITGHI+GQ+H@BAA+DF+H@A+FDOB+TAKK+KGQAN+HANHALY+
+
#LLGHGDQCKKI7+ H@A+ GPPOQDWAQGTGHI+ DF+ PDLGFGAL+ EGBONAN+ #L;"5$6<R>,2S+ CQL+ #L;F=;R>,2S+
JCN+TDPMCBAL+HD+H@CH+DF+#L;R>,2S+ONGQW+H@A+VKDDL+KDDM+PDLAKY+#L;"5$6<R>,2S+TDQHCGQN+
H@A+@IMAB0ECBGCVKA+BAWGDQ+DF+H@A+NHBOTHOBCK+@AUDQ+MBDHAGQ+DF+#L6<7+J@ABACN+#L;F=;R>,2S+
TDQHCGQN+H@A+FGVBA+DF+#L=;Y+\DH@+#L6<+CQL+#L=;+@CEA+KDJ+MBAECKAQTA+CQL+H@ANA+PDLGFGAL+
EGBONAN+ JABA+ AUMATHAL+ HD+ VA+ KANN+ GPPOQDWAQGTY+ #L;F=;R>,2S+ JCN+ NAXOANHABAL+ VI+
ABIH@BDTIHAN+ HD+ C+ KDJAB+ LAWBAA+ H@CQ+ #L;R>,2S+ CQL+ #L;"5$6<R>,2SY+ "DJAEAB7+ #L;R>,2S+
HBGWWABAL+ H@A+ KDJANH+ TIHD]GQA+ BANMDQNA+ CQL+ N@DJAL+ H@A+ KDJANH+ GPPOQDWAQGTGHI+ DEABCKKY+
%@ANA+BANOKHN+CBA+NOBMBGNGQW+CQL+NOWWANH+H@CH+DH@AB+FCTHDBN+TDOKL+@CEA+C+BDKA+GQ+LAHABPGQGQW+
CLAQDEGBON+GPPOQDWAQGTGHIY+
+
/OA+ HD+ H@AGB+ @GW@+ DQTDKITGHI+ CQL+ KDJ+ GPPOQDWAQGTGHI7+ #L8:+ CQL+ MCBHGTOKCBKI+ #L88+ @CEA+
APABWAL+ CN+ NABDHIMAN+ H@CH+ TDOKL+ MDHAQHGCKKI+ VA+ ONAL+ CN+ DQTDKIHGT+ CWAQHNY+ #L88+ GN+ DF+
MCBHGTOKCB+GQHABANH+LOA+HD+GHN+OQGXOA+BATAMHDB+NMATGFGTGHI7+J@GT@+TDOKL+KAQL+GH+H@A+CVGKGHI+HD+
GQFATH+C+JGLA+BCQWA+DF+TAKK+HIMAN7+GQTKOLGQW+PCQI+TCQTABN7+CQL+MBAEGDON+NHOLGAN+@CEA+N@DJQ+
H@CH+ T@GPABGT+ #L=^88+ CQL+ #L;^88+ EGBONAN+ @CEA+ @GW@AB+ AFFGTCTGAN+ CQL+ PDBA+ FCEDOBCVKA+
NCFAHI+MBDFGKAN+H@CQ+H@AGB+MCBAQH+EGBONANY+
!
3
!
)*%&*%(+
!
! 'QHBDLOTHGDQ+
"#! $%&'()*+,-!./0--*1*203*('4!53+,23,+&!0'%!6*1&!.72/&!
8#! $%&'()*+,-!9::,'(;&'*2*37!
<=#! $%&'()*+0/!>&23(+-!
$%&'()*+,-&-!0-!?'2(/73*2!$;&'3-!
<<#! $*:-!
! ! $ANOKHN+
<@#! A*/%B37C&!D,:0'!0%&'()*+,-!-&+(37C&-!D0)&!)0+7*';!E*//*';!&11*2*&'2*&-!
<"#! A*/%B37C&! 0%&'()*+,-! -&+(37C&-! 0+&! '&,3+0/*-&%! F7! C/0-:0! 0'%! 9)9;! 03! )0+7*';!
&11*2*&'2*&-!
<G#! H&,3+0/*-03*('! (1! $%I! 0'%! :(%*1*&%! )0+*0'3-! F7! F/((%! )0+*&-! -*;'*1*20'3/7! F&3J&&'!
%('(+-!
<K#! $%I1LIMNOPQ! *-! /&--! -,-2&C3*F/&! 3(! -&R,&-3+03*('! F7! 2&//-! 3D0'! $%IMNOPQ! (+!
$%IS>T"8MNOPQ!
<8#! .73(E*'&! +&-C('-&! )0+*&-! F&3J&&'! %('(+-! 0'%! /&)&/-! 0+&! /(J&+! 0;0*'-3! ,':(%*1*&%!
$%IMNOPQ!
!
<U#! /GNTONNGDQ+
! ! 4CHABGCKN+CQL+4AH@DLN+
@@#! .&//!.,/3,+&!
5&+(37C&!.('1*+:03*('!F7!5&R,&'2*';!0'%!T&0/!V*:&!P.T!
O/,(+&-2&'2&!O(+:*';!W'*3!$--07!
@L#! .&//!>*0F*/*37!$--07!
S,:0'!P/0-:0!H&,3+0/*-03*('!$--07!
S,:0'!9'3+0)&'(,-!9::,'(;/(F,/*'!M9)9;Q!H&,3+0/*-03*('!$--07!
V?P?!V$!./('*';!0'%!X'20C-,/03&%!>*+0/!N&'(:&!$--07!
X';*'&&+&%!>*+,-!P+(%,23*('!
@"#! Y/((%!6((C!Z(%&/!
AD(/&!Y/((%!H&,3+0/*-03*('!$--07!
.&//![!P/0-:0!$--(2*03*('!$--07!
!
.73(E*'&!$'0/7-*-!
!
@I#! #T]QDJKALWAPAQHN!
$AFABAQTAN+
4 '
*%$)/3!%')*+
!
!
$%&'()*+,-!./0--*1*203*('4!53+,23,+&!0'%!6*1&!.72/&!
!
$%&'()*+,-&-! M$%Q! 0+&! :&%*,:! -*\&%! M0CC+(]*:03&/7!
U=':! *'! %*0:&3&+Q4! *2(-0D&%+0/4! '('B&')&/(C&%!
)*+,-&-! 2('30*'*';! 0! /*'&0+4! %(,F/&B-3+0'%&%! ^H$!
;&'(:&!(1!@GB"IEF!&'2(%*';!@@B"=!;&'&-#!VD&+&!0+&!
03! /&0-3! I<! 022&C3&%! D,:0'! $%! -&+(37C&-! 2/0--*1*&%!
*'3(! -*]! -C&2*&-! M$BOQ!
_<4! @L`4! F0-&%! ('! *::,'(/(;*20/!
%*-3*'23*)&'&--!0'%!;&'(:*2!-&R,&'2&!M-&&!V0F/&!<Q#!
5C&2*&-! Y! D0-! C+&)*(,-/7! F&&'! 1,+3D&+! %*)*%&%! *'3(!
-,F-C&2*&-! Y<! 0'%! Y@4! F0-&%! ('! ;&'&3*2! %*11&+&'2&-!
0'%!(+;0'!3+(C*-:4!3D(,;D!0!+&2&'34!:(+&!%&-2+*C3*)&!
'(:&'2/03,+&!1(+!-C&2*&-!Y!;+(,C-!J*//!F&!*'3+(%,2&%!
('!C0;&!K#!
!
Z(-3! -&+(37C&-! 20,-&! :*/%! +&-C*+03(+7! *'1&23*('-!
_<4!@=`
4! 022(,'3*';! 1(+! IB<=a! (1! -,2D! *'1&23*('-! *'!
2D*/%+&'!
_G`4! 3D(,;D! $%"! 0'%! $%K! 20'! 20,-&! 02,3&!
+&-C*+03(+7! %*-&0-&!
_@=`#! 5&)&+0/! -&+(37C&-4! -,2D! 0-!
$%@!0'%!$%I!0+&!&'%&:*2!J*3D*'!2&+30*'!+&;*('-!0'%!
0+&! +&;,/0+/7! 02R,*+&%! %,+*';! 2D*/%D((%!
_U`#! 5(:&!
:&:F&+-! (1! -C&2*&-! ^4! *'2/,%*';! $%@@! 0'%! $%LK!
20,-&! &C*%&:*2! E&+03(2('b,'23*)*3*-!
_<`4! 0'!
*'1/0::03*('! (1! F(3D! 3D&! 2(+'&0! 0'%! 3D&!
2('b,'23*)04!0!:,2(,-!:&:F+0'&!3D03!/*'&-!3D&!*''&+!
&7&/*%-!0'%!JD*3&-!(1!3D&!&7&-#!Z&:F&+-!(1!-C&2*&-!
$4! O! 0'%! N! 0+&! 2(::('/7! 0--(2*03&%! J*3D!
;0-3+(&'3&+*3*-!
_<G4! @=`4! JD&+&0-! 3D(-&! 1+(:! Y@! *'1&23!
3D&!E*%'&7-!0'%!,+*'0+7!3+023!
_<U`#!
!
VD&! $%! ;&'(:&! *-! &'20C-,/03&%! F7! 0! 20C-*%!
2('-*-3*';! (1! @"=! D&]('! C+(3&*'-! M<@! ('! &02D! 102&Q!
0'%!<@!C&'3('!F0-&-!M('&!03!&02D!)&+3&]Q!
_<<4!<"4!<84!<U4!@=`
#!S&]('-!MC+(3&*'!99Q!0+&!3+*:&+*24!J*3D!&02D!3+*:&+!
2('-*-3*';!(1!3J(!cdBb&//7!+(//e!%(:0*'-#!X02D!%(:0*'!
2('30*'-! /((C-! 3D03! C+(b&23! (,3J0+%-4! 0'%! *'!
2(:F*'03*('! 3D&! G! %(:0*'-! /&'%! 3D&! D&]('! C+(3&*'!
*3-! D&]0;('0/! -D0C&4! 013&+! JD*2D! 3D&7! 0+&! '0:&%#! VD&! /((C-4! 3&+:&%! 3D& D7C&+B)0+*0F/&!
+&;*('!MS>TQ!0+&!3D&!:(-3!)0+*0F/&!C0+3!(1!3D&!D&]('!0'%!022(,'3!1(+!:,2D!(1!3D&!%*)&+-*37!
F&3J&&'!-&+(37C&-#!X02D!C&'3('!F0-&!MC+(3&*'!999Q!*-!0--(2*03&%!J*3D!0!3+*:&+*2!C&'3('!1*F+&!
MC+(3&*'! 9>Q4! J*3D! 0! E'(F! 03! 3D&! &'%! JD*2D! *-! ,-&%! 1(+! 2&//! &'3+7! 0'%! %&3&+:*'&-! +&2&C3(+!
-C&2*1*2*37!
_<<4!<"4!<8`#!!"#$%&'(!-D(J-!3D&!-3+,23,+&!(1!0'!$%I!C0+3*2/&#!
(MATGAN+ (ABDHIMAN+
#+ <@4!<84!L<!
\8+ L4!K4!<G4!@<4!I=!
\9+ <<4!<"4!L"4!LI!
!+ <4!@4!I4!G!
/+ 84!U4!<=4!<L4!<I4!<K4!<U4!@=4!@@4!
@L4!@"4!@I4!@K4!@84!@U4!L=4!L@4!
LL4!LG4!LK4!L84!LU4!"@4!"L4!""4!
"I4!"G4!"K4!"84!"U4!I<!
&+ "!
,+ "=4!"<!
!"#$%&'()+#LAQDEGBON+TCMNGL+NHBOTHOBA!M$%IQ#!
X02D!20C-*%!-*%&!2('-*-3-!(1!'*'&!D&]('-!MN?HQ#!
$3!&02D!)&+3&]4!('&!C&'3('!F0-&!*-!-,++(,'%&%!
F7!1*)&!D&]('-!MN?5Q#!X02D!C&'3('!F0-&!*-!
2(''&23&%!3(!0!C+(3+,%*';!E'(FF&%!1*F+&#!
9:0;&!1+(:f!S0++*-('4!5#!.#!M@=<=Q#!
!
www.sciencemag.org SCIENCE VOL 329 27 AUGUST 2010
1027
PERSPECTIVES
each subunit has two rather similar parts, giv- ing the trimeric oligomer a pseudo-hexago- nal character. Each of these two modules is a “β–jelly roll” domain, with elaborate out- ward-projecting loops between its strands.
The loops are the most variable regions of the protein; their variation is the basis of sero- type diversity. The subunit of the penton base resembles one half of the bipartite hexon sub- unit, with less elaborate projecting loops ( 6).
Thus, the entire outer shell comprises 25 × 60 such modules, of three different kinds.
Although inserted into the fi vefold symmet- ric penton base, the penton fi ber is a trimer ( 7). Its precise adaptation to the base is not yet evident, even in the present structures.
At its tip is a knob that determines receptor specifi city ( 8).
The hexon trimers form planar, sheet-like structures within each face, and gentle disrup- tion of the particle yields so-called “groups of nine” (GONs), as marked in the fi gure ( 9).
Under other circumstances, the pentons and the fi ve surrounding “peripentonal hexons”
dissociate from an otherwise intact virion, suggesting that these elements form a second subassembly, which Liu et al. ( 4) have called a “group of six” (although it is really a “group of fi ve plus one”). The most important new information from the structures presented here concerns how three different minor pro- teins (designated IIIa, VIII, and IX) serve as
“cement” to hold together each subassembly and to link them to each other.
The fundamental structural module of the adenovirus hexon and penton is present in many other icosahedral viruses, of both eukaryotes and prokaryotes ( 10). In simpler structures, such as those of the picornaviruses (e.g., polioviruses and rhinoviruses) or poly- oma- and papillomaviruses, the geometry of the overall assembly is determined by inter- actions of N- and C-terminal “arms,” exten- sions at the termini of the β–jelly roll domain that link with each other and with neighbor- ing subunits. In the adenovirus particle, these arms are no longer covalent extensions of the β–jelly roll module, but rather independent polypeptides. Their function is nonetheless very similar. The interactions of protein IX, which winds into the crevices between hexons on the outer surface of the virion, gather the nine hexons of the GON into a pinwheel-like assembly and dictate that further hexons do not add onto its rim. One might thus imagine a different and larger “protein IX” that gen- erates a larger hexon pinwheel, thereby cre- ating a larger particle from otherwise similar structural elements. The C-terminal segment of protein IX also participates in a bundle of α helices connecting neighboring GONs. In
a similar manner, protein IIIA links the pen- ton base and peripentonal hexons into a clus- ter (the “GOS”). Protein VIII, like protein IX, helps cement the GON while also participat- ing in contacts that tie the GON to the GOS (particularly through interaction with protein IIIA) and adjacent GONs to each other. Both protein IIIA and protein VIII contact inward- facing surfaces of the hexon and penton.
The antigenic, hypervariable loops of the hexon are largely disordered in crystals of isolated hexons. In the intact virions, some of these segments have contacts, either between hexons or with protein IX, that appear to sta- bilize them. Because a majority of adults, in both developed and developing countries, have circulating antibodies against Ad5,
“loop swaps” between Ad5 and other, rarer serotypes have been attempted in efforts to generate Ad5-based vaccine vectors that escape neutralization ( 11). This “immuno- gen design” has depended on a definition of loop boundaries from positions at which
loops become invisible in the hexon crys- tallographic density map. The new infor- mation defi nes these loops more precisely, and it should therefore facilitate new work on “rationally” designed adenovirus vectors with altered antigenic structure.
Substantial technical achievement under- lies both of these papers. The x-ray crystal- lographic approach required creating a virus with Ad35 penton fi bers—much shorter than those of wild-type Ad5. Moreover, the fi bers appear to have receded into the penton base, leaving only the knobs projecting ( 3). Reddy et al. suggest that this recession represents an entry intermediate, but it could also be a con- sequence of mixing proteins in the recombi- nant virion. The cryo-EM structure is a tour de force of contemporary electron micros- copy. It is not the fi rst in which a polypeptide chain could be traced de novo, but it is a par- ticularly complex and elaborate one. Indeed, the cryo-EM density map of Liu et al. appears to be substantially clearer and more interpre- table than the x-ray density map of Reddy et al. Several other icosahedral virus struc- tures have shown that cryo-EM single-parti- cle analysis now rivals x-ray crystallography when applied to large, homogeneous, highly symmetric objects ( 12– 14). It can be argued that only conquerable computational barri- ers now prevent extension to less symmetric structures, although rigidity and conforma- tional homogeneity (qualities experimentally selected when growing crystals) will continue to be important ( 14). The new results thus use frontier technologies of structural biology to advance basic and applied virology.
References
1. A. J. Berk, in Fields Virology, D. M. Knipe, P. M. Howley, Eds. (Lippincott Williams & Wilkins, Philadelphia, 2007), pp. 2356–2395.
2. S. D. Saban, M. Silvestry, G. R. Nemerow, P. L. Stewart, J.
Virol. 80, 12049 (2006).
3. V. S. Reddy, S. K. Natchiar, P. L. Stewart, G. R. Nemerow, Science 329, 1071 (2010).
4. H. Liu et al., Science 329, 1038 (2010).
5. J. J. Rux, P. R. Kuser, R. M. Burnett, J. Virol. 77, 9553 (2003).
6. C. Zubieta, G. Schoehn, J. Chroboczek, S. Cusack, Mol.
Cell 17, 121 (2005).
7. M. J. van Raaij, A. Mitraki, G. Lavigne, S. Cusack, Nature 401, 935 (1999).
8. D. Xia, L. J. Henry, R. D. Gerard, J. Deisenhofer, Structure 2, 1259 (1994).
9. R. A. Crowther, R. M. Franklin, J. Mol. Biol. 68, 181 (1972).
10. S. C. Harrison, in Fields Virology, D. M. Knipe, P. M. How- ley, Eds. (Lippincott Williams & Wilkins, Philadelphia, 2007), pp. 59–98.
11. D. M. Roberts et al., Nature 441, 239 (2006).
12. X. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 105, 1867 (2008).
13. X. Yu, L. Jin, H. Zhou, Nature 453, 415 (2008).
14. M. Wolf, R. L. Garcea, N. Grigorieff, S. C. Harrison, Proc.
Natl. Acad. Sci. U.S.A. 107, 6298 (2010).
BACKGROUND IMAGE COURTESY OF HONG ZHOU/UNIVERSITY OF CALIFORNIA, LOS ANGELES
II (hexon, GON) II (hexon, GOS) III (penton base) IV (fiber) IIIa VIII IX, IX, IX, IX
A new view. An adenovirus particle, with structural components highlighted. The overall diameter of the particle (not including the spikes) is about 900 Å.
Superposed on a medium-resolution image of the virion surface are triangles showing four groups of nine (GONs)—one in the center, in white, and three surrounding it, in black. The triangles are in orienta- tions that join the tips of the three hexon subunits.
One set of peripentonal hexons is designated by gray triangles. The hexons in the GONs are in light blue;
the peripentonal hexons, in dark blue; the penton bases, in brown; the penton fi bers, in green. The network of protein IX subunits, snaking between the hexons on the outside of the particle, is in yellow, bright green, blue, and red; the positions of some copies of two internal minor proteins, which would not be visible from the outside, are shown by colored lines (proteins IIIa and VIII, in orange and magenta, respectively). The view is directly along a threefold symmetry axis of the particle and hence onto the central GON. The yellow ovals show positions of two- fold axes of symmetry.
10.1126/science.1194922
Published by AAAS
on November 14, 2010 www.sciencemag.org Downloaded from
V0F/&!<#!$%&'()*+,-!-C&2*&-!2/0--*1*203*('#!
5
!"#$%&'*)+!BDNN0NATHGDQ+DF+CQ+CLAQDEGBON+MCBHGTKA4!
-D(J*';!20C-*%!0'%!;&'(:&!0++0';&:&'3#!
9:0;&!1+(:f!T,--&//4!A#!.#!M@==UQ#!
.0C-*%-!0+&!D&/%!3(;&3D&+!F7!)0+*(,-!2&:&'3!C(/7C&C3*%&-!M9990!>94!>999!0'%!9gQ!
_<<4!<"4!<8`4!*'!3J(!
:0*'!37C&-!(1!1(+:03*('4!0-!*//,-3+03&%!*'!!"#$%&'(#!VD&!20C-*%!-*%&-!0+&!(+;0'*-&%!0-!;+(,C-!
(1! '*'&! MN?HQ! h! '*'&! D&]('! C+(3&*'-! M3,+R,(*-&Q4! D&/%! 3(;&3D&+! F7! 3D&! +(C&B/*E&! 2&:&'3!
C(/7C&C3*%&!9g!M+&%Q#!VD&!)&+3*2&-!0+&!(+;0'*-&%!0-!;+(,C-!(1!-*]!MN?5Q!h!1*)&!D&]('!C+(3&*'-!
MF/,&Q! -,++(,'%*';! 0! C&'3('! F0-&! C+(3&*'! MF+(J'Q4! D&/%! 3(;&3D&+! F7! 3D&! D*;D/7! D&/*20/!
2&:&'3! C(/7C&C3*%&! 9990! MC,+C/&Q#! VD&! D&]('-! (1! N?5! 0+&! E'(J'! 0-! C&+*C&'3('0/! D&]('-#!
.&:&'3!C(/7C&C3*%&-!>9!0'%!>999!0+&!C((+/7!2D0+023&+*-&%#!
!
9'-*%&! 3D&! 20C-*%4! 0! 3&+:*'0/! C+(3&*'! *-! 03302D&%! 3(! &02D! Ii! &'%! (1! 3D&! /*'&0+! ;&'(:&! 03!
*')&+3&%! 3&+:*'0/! +&C&03-! M9VTQ4! JD*2D! 0+&! '&2&--0+7! 1(+! &11*2*&'3! )*+0/! +&C/*203*('#! VD&!
;&'(:&!*-!0--(2*03&%!J*3D!D*;D/7!F0-*2!C+(3&*'!>99!0'%!3D&!-:0//!:,!C&C3*%&4!0'%!*-!/*'E&%!
-3+,23,+0//7! 3(! 3D&! 20C-*%! )*0! C+(3&*'! >! 0'%! 2&:&'3! C(/7C&C3*%&! >9#! 9'! 0%%*3*('4! 3D&! )*+,-!
2('30*'-! 0! )*+,-B&'2(%&%! C+(3&0-&4! +&R,*+&%! 1(+! C+(2&--*';! (1! -3+,23,+0/! C+(3&*'-! *'! 3D&!
C+(%,23*('!(1!:03,+&!*'1&23*(,-!C0+3*2/&-!
_<U4!@=`#!$!2+(--B-&23*('!(1!0'!0%&'()*+0/!C0+3*2/&!*-!
*//,-3+03&%!*'!!"#$%&'*#!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
$%&'()*+,-&-!0+&!0F/&!3(!*'1&23!F(3D!%*)*%*';!0'%!
'('B%*)*%*';! 2&//-! (1! 0! J*%&! )0+*&37! (1! 2&//! 37C&-!
0'%! 3*--,&-!
_U4! <G4! <U`#! Z(-3! -&+(37C&-! (3D&+! 3D0'!
3D(-&!1+(:!-C&2*&-!Y!;0*'!2&//!&'3+7!F7!D*;D!011*'*37!
F*'%*';! F&3J&&'! 3D&! 1*F+&! E'(F! 0'%! 3D&!
2(]-02E*&)*+,-! 0'%! 0%&'()*+,-! +&2&C3(+! M.$TQ4! 0'!
*::,'(;/(F,/*'B/*E&! 2&//! -,+102&! +&2&C3(+! 3D03!
:&%*03&-! 2&//B2&//! *'3&+023*('-! 0'%! *-! *')(/)&%! *'!
3D&! &C*3D&/*0/! 2&//! c3*;D3! b,'23*('e!
_@=4! @<4! @G`#! ! $!
-,F-&R,&'3! *'3&+023*('! F&3J&&'! 3D&! $+;BN/7B$-C!
MTN^Q!:(3*1!(1!3D&!C&'3('!F0-&!M-&&!!"#$%&'+Q!0'%!
2&//,/0+! *'3&;+*'-! j)dL! 0'%! j)dI! *'%,2&-!
CD0;(273(-*-! (1! 3D&! )*+*('! *'3(! 2/03D+*'B2(03&%!
&'%(-(:&-!(+!/7-(-(:&-!
_<G4!<U4!@=4!@G`#!
!
!"#$%&'+)+#LAQDEGBON+MAQHDQ+VCNA+MBDHAGQ!(1!$%I4!-D(J*';!TN^!/((C!3D03!*'%,2&-!CD0;(273(-*-!(1!)*+*('#!
5*%&!)*&J4!*:0;&%!F7!2+7(B&/&23+('!:*2+(-2(C7#!
9:0;&!1+(:f!T,--&//4!A#!.#!M@==UQ4!0%0C3&%!1+(:!kD0';!l!Y&+;&/-('!M@==IQ#!
pentonal hexons appear to interact with grooves in the penton base.
The fibre polypeptide comprises 582 aa in Ad2 and binds non-covalently by its N terminus onto the top surface of the penton base (Devaux et al., 1987; Zubieta et al., 2005).
It is found that a sequence near the N terminus (FNPVYPY), which is highly conserved between serotypes (Tarassishin et al., 2000), lies in a relatively hydrophobic groove on the top surface of the base formed between two adjacent monomers (Zubieta et al., 2005). There are also a number of hydrogen bonds and a salt bridge contributing to the stability of the interaction with the penton base. The peptide sequences taking part in this interaction are also well conserved between serotypes. The symmetry mismatch here appears to be a function of co-operative conforma- tional changes following fibre binding, with the result that there is room for only the three conserved fibre peptides.
These are arranged horizontally and radially on the base, allowing the three flexible tails to form the characteristic protruding trimeric fibre. The fibre polypeptide consists of a variable number of pseudorepeats of 15–20 aa connected by a b -turn (Green et al., 1983). These repeats form a shaft of three intertwined strands (van Raaij et al., 1999) that is rigid and stable, but of varying length depending on the number of pseudorepeats (related to the serotype). How- ever, there can be disruptions to the shaft sequences, allowing the fibre to form hinge regions (Chroboczek et al., 1995). A further ~180 aa form the C-terminal globular head or knob. Trimerization is governed by sequences in both the knob and shaft regions (Li et al., 2006). The knob contains eight-stranded b -barrels in each subunit and has a central depression with three symmetry-related valleys.
There are a number of loops emanating from the knob (designated DG, HI and AB) (Xia et al., 1994) and these will be discussed below in the light of their role in receptor
Fig. 2. Facets of the adenovirus icosahedron.
(a) External. The GON hexons are multicol- oured and the H1 peripentonal hexons are either lettered in black when they are on the same plane as the GONs or lettered in orange where they are associated with GONs on a different facet. Similarly, the H2 hexons let- tered in orange are associated with GONs on a different facet. The symbol for protein IX is not to scale. (b) Internal. Hexons are desig- nated in (a). Note symbols for other structural proteins are not to scale. (c) Internal structure at the apex; symbols as above.
Fig. 3. Structure of the capsomeres. (a) Ad5 hexon trimer space-filling model showing seven hypervariable loops in colours with the remainder of the hexon in grey. Top shows side view and bottom the view from the top of the hexon. (b) Penton base from cryo-electron microscopic image reconstruction of the Ad5 capsomere: side view showing one of the RGD loops from the pentameric structure. (c) A ribbon representation of the fibre knob showing protruding loops with the agreed notation. All fibre knobs have the same general structure with variations in the number and amino acid composition. This one is from Ad35. (d) Fibre space-filling model indicating sites for receptor attachment modelled from the atomic structure of Ad2. The figures above were taken with permission from Roberts et al.
(2006) for (a), Zhang & Bergelson (2005) for (b) and (d) and from Wang et al. (2006) for (c).
Adenovirus: update on structure and function
6
transportin (Hindley et al., 2007). There is also evidence that, prior to nuclear import, V and VII interact with the Golgi apparatus via the MTOC and then travel to the endoplasmic reticulum prior to nuclear import (C. Hindley and D. A. Matthews, personal communication). After import, cellular proteins SET and pp32 (components of two multiprotein complexes of cellular chromatin) also play a role in altering the template properties (Xue et al., 2005) and a template-activating factor (TAF-1) binds to VII, leading to remodelling of the virus chromatin (Gyurcsik et al., 2006; Haruki et al., 2006). Protein VII seems to dampen early transcription, but eventually transcription of E1A leads to the release of VII, followed by remodelling of the virus chromatin and late transcrip- tion (Chen et al., 2007; Johnson et al., 2004).
pVII, in addition, participates in virus assembly by interact- ing with protein IVa2 and L1 52/55K, which are both bound to the specific packaging sequences on the virus DNA (Zhang
& Arcos, 2005). It is interesting that protein V, at least in Ad5 infected cells, seems to home to both the nucleus and the nucleolus (Matthews & Russell, 1998b) and induces redistribution of nucleolin and B23 from nucleolus to cytoplasm (Matthews, 2001). B23, an acidic protein and a
component of template-activating factor 3, seems to interact with both V and pre VII and could facilitate dissociation of core proteins (Samad et al., 2007), and thereby reveal the virus template for replication and/or transcription (see above). Furthermore, a nucleolar component ‘upstream binding factor’ appears to be involved in virus DNA replication (Lawrence et al., 2006), but it is not clear whether these effects are directly dependent on protein V. It may be that V facilitates these nucleolar functions without being absolutely necessary. Another possibility is that the small core protein Mu can duplicate these nucleolar functions of V (Ugai et al., 2007). This small peptide can exclusively target the nucleolus (Lee et al., 2004), and appears to play a part as a precursor (preMu) in modulating expression of E2 early proteins, resulting in a shift in late protein expression. PreMu is formed as a precursor molecule, polypeptide X, which presumably condenses the virus prechromatin by virtue of its two basic domains, and following cleavage by the virion protease there is a conformational change to facilitate packaging of the core complex.
There seems to be efficient binding between V, VII and Mu (Chatterjee et al., 1985) to form a complex, although the precise topology of the complex with the virus DNA is Fig. 5. Early events in infection. (a) Adenovirus at the plasma membrane (PM), peripentonal hexons are in red. (b) Binding of pentons to integrins and receptors followed by phagocytosis. (c) A phagocytic vesicle (endo- some/lysosome) and disruption of the adeno- virus particle releasing peripentonal hexons, IIIa and VI and revealing the core. (d) Rupture of endosome/lysosome and release into cyto- plasm of hexon shell with a metastable core (virus DNA with TP, VII, V, Mu and possibly protease). (e) Hexon shell at nuclear pore (NP) with core being released into the nucleus. (f) Core in the nucleus targeting cellular chro- matin. Key steps in the induction of the immune response are noted in red. NM, nuclear membrane.
Adenovirus: update on structure and function
http://vir.sgmjournals.org 9
!!!!!!!!!!!!M;Q!
'
!"#$%&',)+5GBON+VGQLGQW7+M@CWDTIHDNGN7+TCMNGL+
BOMHOBA+CQL+HBCQNMDBH+DF+EGBCK+TDBA+HD+QOTKAONY+
M0Q#!$%&'()*+,-!+&02D&-!C/0-:0!:&:F+0'&#!
MFQ#!P&'3('-!F*'%!3(!*'3&;+*'-!0'%!+&2&C3(+-4!
*'%,2*';!CD0;(273(-*-#!
M2Q#!>*+*('!*'-*%&!CD0;(-(:&#!O*F+&-!0+&!2/&0)&%!
0'%!C&+*C&'3('0/!D&]('-!M+&%Q!0+&!%*-+,C3&%4!
/&0)*';!0!-D&//!3D03!+&)&0/-!3D&!)*+0/!2(+&#!
M%Q#!PD0;(-(:&!+,C3,+&-4!+&/&0-*';!)*+*('#!
M&Q#!>*+0/!2(+&!*-!+&/&0-&%!1+(:!-D&//#!
M1Q#!>*+0/!2(+&!C0--&-!3D+(,;D!',2/&0+!C(+&#!
M;Q#!9:0;&!(1!0!)*+0/!-D&//!2('-*-3*';!(1!D&]('-#!
VD&!C&'3('!F0-&-!0'%!1*F+&-!D0)&!F&&'!
/*F&+03&%!1+(:!3D&!-D&//!0/(';!J*3D!3D&!
C&+*C&'3('0/!D&]('-4!/&0)*';!;0C-!3D+(,;D!
JD*2D!3D&!)*+0/!2(+&!20'!C0--#!
9:0;&-!1+(:f!T,--&//4!A#!.#!M@==UQ#!
recognition (Fig. 3c). Glycosylation of the fibre shaft at a serine residue has been reported, although it is not clear if this has a functional role (Cauet et al., 2005).
Other capsid proteins
The location of polypeptide IIIa (570 aa for Ad2) in the capsid has recently been defined by difference mapping to a position below the penton base (Saban et al., 2006). The N- terminal region is highly helical and as well as binding to the penton base is also associated with hexons and protein VI. Some evidence of binding to core proteins V and VII (see below) has also been reported (Boudin et al., 1980).
There appears to be 60 monomers per virion (Rux &
Burnett, 2004), in agreement with the finding of five helical clusters interacting with the other capsomeres at the apex of the capsid (Figs 1 and 2b, c).
Polypeptide VI (500 aa for Ad2) has two long a -helices and one of these binds to hexon (Matthews & Russell, 1994, 1995) and can be discerned within a cavity at the base of the hexon (Saban et al., 2006). However, a number of reports examining the stoichiometry of VI (Chelius et al., 2002;
Lehmberg et al., 1999) imply that there are only about 360 copies per virion. Since VI also associates with IIIa at the apices, it is not clear if there are multimers of VI or if there is only partial occupancy of the hexon cavity. It has also been suggested that VI is located underneath the peripentonal hexons as trimers of dimers (Stewart et al., 1993), and this has been assumed in formulating Figs 1 and 2 (b, c).
The location of polypeptide VIII (140 aa for Ad2) has been difficult to resolve, but there is now agreement that it can be located to the inner side of the capsid in two non- equivalent positions: viz. five copies in a ring around the peripentonal hexons connecting them to the GONs and three copies in further rings around the threefold axes presumably stabilizing the GONs, giving a copy number of 120 (Fig. 2b). In looking at the internal structure at the apex of the virion (Fig. 2c) it will be noted that VIII provides a bond between the peripentonal hexons and the rest of the capsid. It is possible that this is a relatively weak link which can be ruptured when the penton base is detached during virus entry into the cell, allowing the H1 hexons to be released, leaving a hexon shell (see below).
Polypeptide IX (140 aa for Ad2) has recently been shown to have a great propensity to form coiled coils and it has been proposed that its N terminus is situated at the middle of each facet, while the C terminus forms a four helix bundle, with one helix interacting with the hexon HVR4 loop thus assigning it externally to the adjoining facet edges, in agreement with its copy number of 240 (Marsh et al., 2006;
Saban et al., 2006). However, there are indications that the C termini could be more flexible in their locations and there is some disagreement about these assignments (Marsh et al., 2006) (Fig. 2a).
The remaining structural polypeptides are associated with the virus core, i.e. the virus genome and the core
polypeptides V, VII, Mu as well as terminal protein (TP).
Two other polypeptides, IVa2 and the protease, could also be considered as components of the core (see below). The cores can be visualized by negative staining electron microscopy, but only as rather diffuse entities (Fig. 4c).
There is very little structural information regarding poly- peptide V (368 aa for Ad2); it seems to be associated loosely with polypeptide VII and the virus DNA (Harpst et al., 1977; Russell et al., 1971) and tighter with poly- peptide VI (Matthews & Russell, 1998b), thus providing a bridge between the core and the capsid. Since there are about 160 copies of V and 360 of VI (Chelius et al., 2002;
Rux & Burnett, 2004), there may be multimers of VI involved in the binding to polypeptide V (see above).
Polypeptide VII (174 aa for Ad2) is highly basic and binds tightly to DNA (Russell & Precious, 1982) and since there are over 800 copies per virion these appear to be spread along the length of the virus DNA, although there is some indication of the formation of particulate structures and supercoiling on treatment with nucleases (Goding &
Russell, 1983b; Nermut et al., 1975; Wong & Hsu, 1989).
Another very basic component of the nucleoprotein core is Mu (36 aa), with about 100 copies per virion. Mu has
Fig. 4. Hexon shells and cores. On heating adenovirus hexon (a) shells (b) and cores (c) can be viewed by negative staining. The bar represents 500 A˚ (50 nm; Russell et al., 1967b).
W. C. Russell
4 Journal of General Virology 90
VD&!1*F+&-!0+&!2/&0)&%4!+&/&0-*';!3D&!20C-*%!1+(:!3D&!)&-*2/&!0'%!3D&!)*+,-B&'2(%&%!C+(3&0-&!
%*-+,C3-! 20C-*%! -30F*/*37! F7! C+(3&(/7-*-! (1! 3D&! C&+*C&'3('0/! D&]('-!
_@=`#! VD&! +&-,/3*';! -D&//!
&]C(-&-!3D&!)*+0/!2(+&4!JD*2D!2('-*-3-!(1!3D&!;&'(:&4!3&+:*'0/!C+(3&*'-4!C+(3&*'-!>!0'%!>99!
0'%!:,!C&C3*%&-#!V+0'-C(+303*('!(1!3D&!-D&//!0/(';!:*2+(3,F,/&-!3(!3D&!',2/&,-!*-!:&%*03&%!
F7!2&//,/0+!C+(3&*'!CL@!
_U4!<U4!@=`#!VD&!)*+0/!2(+&!C0--&-!(,3!(1!3D&!-D&//!0'%!&'3&+-!3D&!D(-3!2&//!
',2/&,-!3D+(,;D!0!',2/&0+!C(+&4!<B@!D(,+-!013&+!*'1&23*('#!!"#$%&',!*//,-3+03&-!3D*-!C+(2&--#!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
VD&! )0+*0F*/*37! (1! 2&//! -C&2*1*2*37! F&3J&&'! -&+(37C&-! *-! /&'3! F7! )0+*03*('! *'!
3D&! 1*F+&-! 0'%! 3D&! +&2&C3(+-! 3D&7! 0+&! 0F/&! 3(! &]C/(*3#! VD&! 1*F+&-! (1! -(:&!
-&+(37C&-! 0+&! 0F/&! 3(! F*'%! 3(! +&2&C3(+-! 0%%*3*('0/! 3(! .$T4! *'2/,%*';! 3D&!
*::,'(;/(F,/*'! -,C&+10:*/7! M-C&2*&-! .Q! 0'%! -*0/(;/72(C+(3&*'! +&2&C3(+-!
M-(:&! :&:F&+-! (1! -C&2*&-! ^Q!
_U4! <G4! <U4! @=`#! 5C&2*&-! O! -&+(37C&-! M$%"=! 0'%!
$%"<Q4! JD*2D! *'1&23! ;0-3+(*'3&-3*'0/! 2&//-! D0)&! 3J(! 1*F+&-! (1! %*11&+&'3!
/&';3D-#!VD&!/(';!1*F+&-!F*'%!.$T!0'%!3D&!-D(+3!1*F+&-!0+&!,'0F/&!3(!
_<U4!@=`#!
VD&!1*F+&!(1!$%"!M3D&!('/7!:&:F&+!(1!-C&2*&-!XQ!*-!)&+7!-*:*/0+!3(!3D03!(1!
-C&2*&-! .! :&:F&+-4! 3D(,;D! 0'3*F(%*&-! &-30F/*-D&%! F7! :*2&! ('! *'1&23*('!
J*3D!$%"!%(!'(3!2+(--B+&023!J*3D!1*F+&-!1+(:!(3D&+!-&+(37C&-!
_@@`#!!"#$%&'-!
-D(J-!$%@!1*F+&#!
!
!"#$%&'-)+#LAQDEGBON+FGVBA!(1!$%@4!-D(J*';!+&2&C3(+!F*'%*';!-*3&-!03!1*F+&!E'(F#!5*%&!)*&J4!-C02&B1*//*';!:(%&/#!
9:0;&!1+(:f!T,--&//4!A#!.#!M@==UQ4!0%0C3&%!1+(:!kD0';!l!Y&+;&/-('!M@==IQ#!
pentonal hexons appear to interact with grooves in the penton base.
The fibre polypeptide comprises 582 aa in Ad2 and binds non-covalently by its N terminus onto the top surface of the penton base (Devaux et al., 1987; Zubieta et al., 2005).
It is found that a sequence near the N terminus (FNPVYPY), which is highly conserved between serotypes (Tarassishin et al., 2000), lies in a relatively hydrophobic groove on the top surface of the base formed between two adjacent monomers (Zubieta et al., 2005). There are also a number of hydrogen bonds and a salt bridge contributing to the stability of the interaction with the penton base. The peptide sequences taking part in this interaction are also well conserved between serotypes. The symmetry mismatch here appears to be a function of co-operative conforma- tional changes following fibre binding, with the result that there is room for only the three conserved fibre peptides.
These are arranged horizontally and radially on the base, allowing the three flexible tails to form the characteristic protruding trimeric fibre. The fibre polypeptide consists of a variable number of pseudorepeats of 15–20 aa connected by a
b-turn (Green et al., 1983). These repeats form a shaft of three intertwined strands (van Raaij et al., 1999) that is rigid and stable, but of varying length depending on the number of pseudorepeats (related to the serotype). How- ever, there can be disruptions to the shaft sequences, allowing the fibre to form hinge regions (Chroboczek et al., 1995). A further ~180 aa form the C-terminal globular head or knob. Trimerization is governed by sequences in both the knob and shaft regions (Li et al., 2006). The knob contains eight-stranded
b-barrels in each subunit and has a central depression with three symmetry-related valleys.
There are a number of loops emanating from the knob (designated DG, HI and AB) (Xia et al., 1994) and these will be discussed below in the light of their role in receptor
Fig. 2. Facets of the adenovirus icosahedron.(a) External. The GON hexons are multicol- oured and the H1 peripentonal hexons are either lettered in black when they are on the same plane as the GONs or lettered in orange where they are associated with GONs on a different facet. Similarly, the H2 hexons let- tered in orange are associated with GONs on a different facet. The symbol for protein IX is not to scale. (b) Internal. Hexons are desig- nated in (a). Note symbols for other structural proteins are not to scale. (c) Internal structure at the apex; symbols as above.
Fig. 3. Structure of the capsomeres. (a) Ad5 hexon trimer space-filling model showing seven hypervariable loops in colours with the remainder of the hexon in grey. Top shows side view and bottom the view from the top of the hexon. (b) Penton base from cryo-electron microscopic image reconstruction of the Ad5 capsomere: side view showing one of the RGD loops from the pentameric structure. (c) A ribbon representation of the fibre knob showing protruding loops with the agreed notation. All fibre knobs have the same general structure with variations in the number and amino acid composition. This one is from Ad35. (d) Fibre space-filling model indicating sites for receptor attachment modelled from the atomic structure of Ad2. The figures above were taken with permission from Roberts et al.
(2006) for (a), Zhang & Bergelson (2005) for (b) and (d) and from Wang et al. (2006) for (c).
Adenovirus: update on structure and function
http://vir.sgmjournals.org 3
7
9'!@==G4!V,)&4!5#!&3!0/#!C+(C(-&%!0!:(+&!%&-2+*C3*)&!;+(,C*';!(1!-C&2*&-!Y!-&+(37C&-!F0-&%!('!
3D&*+!+&2&C3(+!-C&2*1*2*374!-,::0+*-&%!*'!V0F/&!@!
_@L`#!O+(:!D&+&!('!*'!3D*-!,C%03&%!-C&2*&-!Y!
'(:&'2/03,+&!-D0//!F&!,-&%4!J+*33&'!0-!$%YB<4!$%YB@!0'%!$%YBL#!$%YB<[L!(+!$%Y@[L!-D0//!F&!
,-&%!3(!+&1&+!3(!3D&!2(++&-C('%*';!;+(,C-!2(//&23*)&/7#!
!
!
!
!
!
!
!
$%L! M$%YB@Q! 0'%! $%LI! M$%YB<Q! D0)&! F(3D! F&&'! -D(J'! 3(! &'3&+! D(-3! 2&//-! C+*:0+*/7! F7!
:02+(C*'(273(-*-!
_@4!<=4!@=`#!$/-(!0'!&'%(273(3*2!C+(2&--4!:02+(C*'(273(-*-!*')(/)&-!3D&!(13&'!
'('B-&/&23*)&! ,C30E&! (1! 1/,*%! 0'%! :&:F+0'&4! 3+*;;&+&%! *'! 3D&! 20-&! (1! $%! F7! *'3&+023*('-!
F&3J&&'! 3D&! 1*F+&! 0'%! 2(:C/&:&'3! +&;,/03(+7! C+(3&*'! .^"G#! $+(,'%! <a! (1! )*+0/! C0+3*2/&-!
-3*//! &'3&+! F7! 2/03D+*'B:&%*03&%! &'%(273(-*-!
_<=`#! $-! +&2&'3/7! 0-! ^&2&:F&+! @=<=4! 3D&!
C+&)*(,-/7! ,'*%&'3*1*&%! +&2&C3(+! E'(J'! 0-! c+&2&C3(+! ge! J0-! *%&'3*1*&%! 0-! %&-:(;/&*'B@!
M^5NB@Q!
_@"`#!^5NB@!*-!0!:&:F+0'&!C+(3&*'!0F,'%0'3!('!F(3D!D&0/3D7!&C*3D&/*0/!2&//-!0'%!('!0!
J*%&!+0';&!(1!&C*3D&/*0/!:0/*;'0'2*&-#!$%YB@[L!-&+(37C&-!,-&!^5NB@!1(+!2&//!&'3+7#!$%YB<[L!
-&+(37C&-!&]C/(*3!.^"G!1(+!2&//!&'3+74!0!C+(3&*'!&]C+&--&%!,F*R,*3(,-/7!('!:(-3!2&//!37C&-#!
$%<<4!3D&!('/7!:&:F&+!(1!$%YBL!C+*:0+*/7!,-&-!.^"G!1(+!2&//!&'3+74!F,3!*-!0/-(!20C0F/&!(1!
,-*';!^5NB@!*1!.^"G!*-!F/(2E&%!
_@L4!@"`4!:0E*';!*3!('&!(1!3D&!:(-3!*'3&+&-3*';!-&+(37C&-!%,&!3(!
*3-!%*)&+-*37!0'%!*3-!3D&(+&3*20/!C(3&'3*0/!3(!*'1&23!0!J*%&!+0';&!(1!2&//!37C&-#!
!
VD&! $%! ;&'(:&! 2('-*-3-! (1! &0+/74! *'3&+:&%*03&! 0'%! /03&! 3+0'-2+*C3*('0/! ,'*3-4! (+;0'*-&%!
022(+%*';!3(!(+%&+!(1!+&C/*203*('!
_<U4!@=`!0'%!-,::0+*-&%!*'!V0F/&!L#!V+0'-2+*C3*('!(22,+-!('!
F(3D! -3+0'%-! (1! 3D&! )*+0/! ;&'(:&#! Z(-3! ;&'&-! D0)&! :,/3*C/&! (C&'! +&0%*';! 1+0:&-! 0'%!
:,/3*C/&!-C/*2&!)0+*0'3-#!X<$!*-!3D&!1*+-3!$%!;&'&!3(!F&!&]C+&--&%!0'%!*3!+&;,/03&-!0!J*%&!D(-3!
(1!:&2D0'*-:-!3D03!0//(J!1(+!&]C+&--*('!0'%!+&C/*203*('!(1!(3D&+!$%!;&'&-#!
!
V0F/&!L#!$%&'()*+,-!;&'&!C+(%,23-!0'%!1,'23*('-#!
+ >AQA+ 2BDLOTHN+_+,OQTHGDQN+
&0+/7!
X<$! P+(3&*'-!3D03!3+0'-023*)03&!(3D&+!+&;*('-!(1!)*+0/!;&'(:&#!
9'%,2&!5!CD0-&!(1!2&//!272/&4!JD*2D!20'!/&0%!3(!0C(C3(-*-#!
Z(%,/03&!2&//,/0+!:&30F(/*-:4!:0E*';!D(-3!2&//!:(+&!-,-2&C3*F/&!3(!)*+0/!+&C/*203*('#!
X<Y! P+(3&*'-!3D03!C+(:(3&!-,+)*)0/!(1!*'1&23&%!2&//-!3D+(,;D!*'D*F*3*('!(1!0C(C3(-*-#!
VD&!X<Y!<UE^0!C+(3&*'!*-!0'0/(;(,-!3(!Y2/B@4!*'D*F*3*';!0C(C3(-*-!F7!*'3&+023*';!J*3D!3D&!Y0]!10:*/7#!
VD&!X<Y!IIE^0!C+(3&*'!*'3&+023-!%*+&23/7!J*3D!CIL!3(!*'D*F*3!0C(C3(-*-#!
X@$! 5*';/&B-3+0'%&%!^H$!F*'%*';!C+(3&*'!M^YPQ#!
9')(/)&%!*'!)*+0/!+&C/*203*('#!
X@Y! P+&B3&+:*'0/!C+(3&*'!MCVPQ!0'%!^H$!C(/7:&+0-&!MC(/Q#!
9')(/)&%!*'!)*+0/!+&C/*203*('#!
XL! P+(3&*'-!3D03!-,F)&+3!D(-3!*::,'&!+&-C('-&!0'%!0+&!'(3!+&R,*+&%!F7!)*+,-!1(+!+&C/*203*('!!"#$!%&'#!
VD&!XL!<UE^0!;/72(C+(3&*'!*'D*F*3-!ZS.!2/0--!9!&]C+&--*('!('!2&//!-,+102&#!
VD&!<=#"[<"#IE^0!MT9^!2(:C/&]Q!0'%!<"#KE^0!C+(3&*'-!*'D*F*3!O0-!/*;0'%B!(+!3,:(,+!'&2+(-*-!1023(+!
MVHOQB:&%*03&%!0C(C3(-*-#!
$%&'()*+,-!%&03D!C+(3&*'!M$^PQ!20,-&-!/03&!273(/7-*-!(1!3D&!D(-3!2&//!0'%!)*+0/!+&/&0-&#!
X"! P+(3&*'-! 3D03! 102*/*303&! )*+0/! :TH$! :&30F(/*-:! 0'%! C+(:(3&! )*+0/! ^H$! +&C/*203*('4! 0/(';! J*3D!
*'D*F*3*('!(1!D(-3!C+(3&*'!-7'3D&-*-#!
(MATGAN+\+ (ABDHIMAN+ $ATAMHDB+(MATGFGTGHI+
#L\08+ <G!MY<Q4!@<!MY<Q4!LI!MY@Q4!I=!MY<Q! .^"G!
#L\09+ L!MY<Q4!K!MY<Q4!<"!MY@Q! %&-:(;/&*'B@!
#L\0=+ <<!MY@Q! .^"G!0'%!%&-:(;/&*'B@!
+ L"!MY@Q! ,'-C&2*1*&%!
V0F/&!@#!WC%03&%!0%&'()*+,-!Y!-,F-C&2*&-!;+(,C*';!!_@L`4!J*3D!3D&!C+&)*(,-!-,F-C&2*&-!',:F&+!*'!F+02E&3-#!
8
*'3&+:&%#! 9>0@! X]C+&--&%! 03! D*;D! /&)&/-! 0'%! 3(;&3D&+! -C&2*1*20//7! 023*)03&! 3D&! :0b(+! /03&! C+(:(3&+! MZ6PQ4! JD*2D!
+&;,/03&-!/03&!;&'&-#!
9g!
>$9! >*+,-!0--(2*03&%!TH$!3D03!C+(:(3&-!C+(3&*'!-7'3D&-*-!F7!*'D*F*3*';!CD(-CD(+7/03*('!(1!E*'0-&!PG8#!
>$99! >*+,-!0--(2*03&%!TH$!3D03!*'D*F*3-!*'3&+1&+('!M9OHQ!0'3*)*+0/!+&-C('-&#!
/03&! 6<B6I! .(:C/&]! -C/*2*';! (1! 3+0'-2+*C3-! /&0%-! 3(! C+(%,23*('! (1! -3+,23,+0/! 20C-*%! C+(3&*'-4! -,2D! 0-! D&]('-4!
C&'3('!F0-&-!0'%!C&'3('!1*F+&-!0'%!(1!C+(3&*'-!3D03!0--*-3!20C-*%!0--&:F/74!-,2D!0-!9>0@4!>99!0'%!9g#!
!
$13&+! )*+0/! ;&'(:&! +&C/*203*('! 0'%! C+(3&*'! C+(%,23*('! D0-! (22,++&%4! 0'! $V! +*2D! C02E0;*';!
-&R,&'2&!*'%,2&-!&'20C-*%03*('!
_<=4!<U`#!.0C-*%-!0--&:F/&!0+(,'%!;&'(:&-!0'%!1(+:!C+(;&'7!
)*+*('-#!VD&!D(-3!',2/&0+!:&:F+0'&!*-!C&+:&0F*/*-&%4!+&/&0-*';!/0+;&!',:F&+-!(1!)*+*('-!*'3(!
3D&! 273(C/0-:#! O*'0//74! 3D&! 2&//! :&:F+0'&! %*-*'3&;+03&-4! E*//*';! 3D&! 2&//! 0'%! +&/&0-*';! 3D&!
)*+,-!*'3(!3D&!-,++(,'%*';!3*--,&#!
!
!
$%&'()*+,-!9::,'(;&'*2*37!
!
VD&! /(J! :(+F*%*37! *'%,2&%! F7! :(-3! 0%&'()*+,-&-!
_<U`4! 2(:F*'&%! J*3D! 3D&! :*/%! -7:C3(:-!
3D03!:(-3!-&+(37C&-!20,-&!:0E&-!3D&:!-&&:!/*E&!*%&0/!0;&'3-!1(+!)*+(3D&+0C7#!S(J&)&+4!3D&!
*::,'&! :&2D0'*-:-! 0;0*'-3! $%! :,-3! F&! 30E&'! *'3(! 2/(-&! 2('-*%&+03*('! JD&'! %&)&/(C*';!
3D&:!0-!('2(/73*2!0;&'3-#!S*;D!*::,'*37!0;0*'-3!-(:&!-&+(37C&-!+&-,/3-!*'!C((+!%&/*)&+7!3(!
3D&!3,:(,+!-*3&!JD&'!*'b&23&%!*'3+0)&'(,-/7!
_<G4!<U`#!5*;'*1*20'3/7!*'2+&0-*';!3D&!%(-&!20'!/&0%!
3(!D&C03(3(]*2*37!0'%!D0-!/&0%!3(!3D&!%&03D!(1!3J(!C03*&'3-!,'%&+;(*';!2/*'*20/!3+*0/-!
_<G4!<U`#!
!
9::,'*37!20'!F&!%*)*%&%!*'3(!3D&!*''03&!M*::&%*03&4!'('B-C&2*1*24!-D(+3B3&+:Q!0'%!0%0C3*)&!
M-/(J&+!F,3!-3+(';&+4!D*;D/7B-C&2*1*24!/(';B/0-3*';Q!+&-C('-&-#!Y(3D!C+(2&--&-!0+&!*')(/)&%!*'!
%&1&'2&!0;0*'-3!$%#!9'!3&+:-!(1!*''03&!*::,'*374!-(:&!&C*3D&/*0/!2&//-!+&/&0-&!0'3*B:*2+(F*0/!
%&1&'-*'! C&C3*%&-! 3D03! C+(3&23! 0;0*'-3! $%! *'1&23*('!
_<K4! <U`#! 5(:&! 3*--,&-! *'%,2&! 0'!
*'1/0::03(+7! +&-C('-&! 3D+(,;D! 3D&! +&2+,*3:&'3! (1! '&,3+(CD*/-4! )*0! 3D&*+! +&/&0-&! (1!
2D&:(E*'&-!M-:0//!273(E*'&-!*'2/,%*';!VHO4!96B<d4!96BG4!96B<@4!9PB<=!0'%!Z.PB<Q!
_L4!U4!<I4!<U4!@@`#!
Z02+(CD0;&! 0'%! '03,+0/! E*//&+! 2&//! +&2+,*3:&'3! 0+&! 0/-(! 1(+:-! (1! *''03&! *::,'*37! 3D03!
C+(3&23!0;0*'-3!$%!!"#$!$'#
_<=4!<U`#!V+0'-2+*C3*('!1023(+!HOBmY!*-!0!E&7!+&;,/03(+!(1!3D*-!+&-C('-&!
3D+(,;D! *3-! 0F*/*37! 3(! 023*)03&! 273(E*'&! 0'%! 0%D&-*('! :(/&2,/&! 3+0'-2+*C3*('! -(('! 013&+!
*'1&23*('#!Y*'%*';!F&3J&&'!X<$!0'%!HOBmY!2(,/%!3+*;;&+!3D*-!+&-C('-&4!3D(,;D!X<Y!023-!3(!
-,CC+&--!3D*-!023*)03*('#!9'3&+023*('-!F&3J&&'!3D&!C&'3('!F0-&!C+(3&*'!0'%!2&//,/0+!*'3&;+*'-!
:07!0/-(!023*)03&!HOBmY!(+!/&0%!3(!2D&:(E*'&!(+!*'3&+1&+('!+&/&0-&!
_<U`#!VD&!3D+&&!C03DJ07-!
(1!3D&!2(:C/&:&'3!20-20%&!0+&!0/-(!*')(/)&%!*'!$%!*'D*F*3*('!
_84!<=4!<U`#!9;NB!(+!9;ZBF(,'%!$%!
3+*;;&+-!3D&!2/0--*20/!2(:C/&:&'3!C03DJ07!F7!*'3&+023*('!J*3D!2(:C/&:&'3!.<B2(:C/&]#!VD&!
/&23*'!C03DJ07!*-!D(:(/(;(,-!3(!3D*-4!&]2&C3!1(+!0!-*';/&!2(:C('&'34!JD*/-3!3D&!0/3&+'03*)&!
C03DJ07!20'!F&!3+*;;&+&%!F7!.L!D7%+(/7-*-#!
!
9'3&+1&+('-! 0+&! 2&//,/0+! C+(3&*'-! 3D03! 20'! F&! +&/&0-&%! -(('! 013&+! )*+0/! *'1&23*('#! $%-! 20'!
3+*;;&+! 3D&*+! +&/&0-&! &)&'! F&1(+&! )*+0/! C+(3&*'! -7'3D&-*-#! 9'3&+1&+('-! 023*)03&! n0E[5V$V!
C03DJ07-!F7!F*'%*';!3(!2&//!+&2&C3(+-#!5V$V!2(:C/&]&-!+&/(203&!3(!3D&!',2/&,-!0'%!F*'%!3(!
*'3&+1&+('B-3*:,/03&%! +&-C('-&! &/&:&'3-! M95TXQ! ('! 3D&! D(-3! ;&'(:&4! JD*2D! +&;,/03&!
3+0'-2+*C3*('!(1!)0+*(,-!C+(3&*'-4!*'2/,%*';!0!C+(3&*'!E*'0-&!MPToQ#!S(J&)&+4!$%-!0+&!0F/&!3(!
2(,'3&+!3D*-!*'3&+1&+('!023*)*37!3D+(,;D!PTo!*'023*)03*('!MF7!F*'%*';!(1!>$!TH$-Q!0'%!%(J'B
+&;,/03*('!(1!5V$V!023*)03(+-!F7!X<$!C+(%,23-!
_I4!<U`#!
9
V,:(,+!-,CC+&--(+!;&'&!CIL!+&;,/03&-!3+0'-2+*C3*('!(1!0C(C3(3*2!1023(+-4!-,2D!0-!:&:F&+-!
(1!3D&!Y0]!10:*/74!JD*2D!*'%,2&-!0C(C3(-*-!3D+(,;D!+&2+,*3:&'3!(1!20-C0-&-#!VD&!X<Y!IIE^0!
C+(3&*'! *'3&+023-! %*+&23/7! J*3D! CIL! 3(! *'D*F*3! 0C(C3(-*-! 0'%! 3D&! X<Y! <UE^0! C+(3&*'! *-!
0'0/(;(,-!3(!Y2/B@4!JD*2D!*'D*F*3-!20-C0-&!+&/&0-&#!VD&!/033&+!D(J&)&+!/&0%-!3(!+&/&0-&!(1!HOB mY!3(!3D&!',2/&,-4!*'%*203*';!3D03!3D&+&!*-!0!2(:C/&]!*'3&+C/07!F&3J&&'!3D&!)*+,-!0'%!D(-3!
2&//!()&+!0C(C3(3*2!+&;,/03*('!
_<U`#!
!
Y(3D!.^8
p!273(3(]*2!VB/7:CD(273&-!M.V6-Q!0'%!.^"
p!VBD&/C&+!2&//-!C+()*%&!0%0C3*)&!2&//,/0+!
+&-C('-&-! 0;0*'-3! $%-!
_<U`#! .V6-! +&2(;'*-&! )*+,-! 0'3*;&'-! M;&'&! C+(%,23-Q! *'! 2(:C/&]! J*3D!
ZS.!2/0--!9!:(/&2,/&-!('!3D&!2&//!-,+102&4!3+*;;&+*';!+&/&0-&!(1!C&+1(+*'!0'%!-,F-&R,&'3!2&//!
/7-*-#!VD*-!*-!%&C&'%&'3!('!3D&!1(+:03*('!(1!0!2(:C/&]!F&3J&&'!3D&!0'3*;&'!0'%!3D&!ZS.!
:(/&2,/&!J*3D*'!3D&!XT!:&:F+0'&4!1(//(J&%!F7!3+0'-C(+303*('!(1!3D&!2(:C/&]!3(!3D&!C/0-:0!
:&:F+0'&#!VD&!XL!;C<UE^0!C+(3&*'!2(,'3&+-!3D*-!%&1&'2&!-7-3&:!F7!+&30*'*';!3D&!ZS.!*'!
3D&!XT#!VD&!.^"
p!D&/C&+!2&//-!J(+E!*'!0!-*:*/0+!J074!+&2(;'*-*';!ZS.!2/0--!99!:(/&2,/&-!*'!
2(:C/&]! J*3D! )*+0/! 0'3*;&'-! MC(--*F/7! 9990! (+! 1*F+&Q#! VD&7! -,F-&R,&'3/7! -3*:,/03&! Y! 2&//!
C+(/*1&+03*('!1(+!*::,'(;/(F,/*'!C+(%,23*('#!VD*-!*'3&+023*('!0CC&0+-!3(!F&!/&--!-&+(37C&B -C&2*1*2!0'%!:07!C/07!0!+(/&!*'!*::,'*37!0;0*'-3!:,/3*C/&!-&+(37C&-#!H&,3+(CD*/-4!JD*2D!0+&!
C+&-&'3!*'!3D&!F/((%!03!D*;D!/&)&/-!013&+!*'1/0::03*('!0'%!0+&!C+*:0+7!&11&23(+-!(1!3D&!*''03&!
*::,'&!+&-C('-&4!*'3&+'0/*-&!$%!0'%!0+&!+&2+,*3&%!3(!3D&!/*)&+!
_L4!<U`#!
!
VD&!D,:(,+0/!+&-C('-&!*-!%&C&'%&'3!('!3D&!%*-C/07!(1!*::,'(;/(F,/*'-!M9;Q!('!3D&!-,+102&!
(1! Y! /7:CD(273&-4! 0-! -3*:,/03&%! F7! V! D&/C&+! 2&//-4! 0'%! 3D&! +&/&0-&! (1! 3D&-&! 9;-! *'3(! 3D&!
C/0-:0#! Z(-3! $%B'&,3+0/*-*';! 0'3*F(%*&-! M$FQ! 0+&! -C&2*1*2! 0;0*'-3! 1*F+&! 0'%! C&'3('! F0-&!
C+(3&*'-4! %,&! 3(! 3D&*+! *:C(+30'2&! *'! +&2&C3(+! F*'%*';4! 0-! J&//! 0-! 3D&! &]3&+'0/! C0+3! (1! 3D&!
D&]('!C+(3&*'#!5&+(37C&B-C&2*1*2!$F-!3&'%!3(!+&2(;'*-&!%*11&+&'3!C0+3-!(1!3D&!1*F+&!-3&:!(+!
E'(F#! VD&! )0+*0F*/*37! (1! 3D&! D&]('! S>T! F&3J&&'! -&+(37C&-! *-! /*E&/7! 3(! D0)&! &)(/)&%! 0-! 0!
:&2D0'*-:!3(!0)(*%!+&2(;'*3*('!F7!$F-!
_84!<U4!@=`#!9'!2('3+0-34!3D&!*'3&+'0/!C0+3!(1!3D&!D&]('!
C+(3&*'!*-!D*;D/7!2('-&+)&%!F&3J&&'!-&+(37C&-!0'%!*-!2+*3*20/!3(!)*+*('!-30F*/*37!
_<K4!<U`#!Y*'%*';!
F&3J&&'! $%! 0'%! C/0-:0! C+(3&*'-! -,2D! 0-! )*30:*'! oB%&C&'%&'3! 2(0;,/03*('! \7:(;&'-!
M&'\7:&! C+&2,+-(+-Q! *'! 3D&*+! *'023*)&! 1(+:! D0-! 0/-(! F&&'! -D(J'! 3(! *'2+&0-&! 3D&*+!
3+0'-%,23*('!0F*/*37!(1!D&C03(:0!2&//-!
_L4!<=`#!
!
VD&! *'3&+023*('-! (1! $%! J*3D! D(-3! C/0-:0! C+(3&*'-4! F/((%! 2&//-! 0'%! C/03&/&3-! 0+&! 2+*3*20/! 3(!
*'3+0)&'(,-! %&/*)&+7! (1! ('2(/73*2! $%! 0;&'3-#! 5,2D! *'3&+023*('-! (22,+! *::&%*03&/7! 013&+!
0%:*'*-3+03*('!0'%!0+&!3D&!E&7!/*:*3*';!1023(+!(1!3D&!0F*/*37!(1!$%-!3(!+&02D!3D&*+!30+;&3!-*3&#!
()#$!$'!-3,%*&-!,-*';!D,:0'!2&//-!D0)&!-D(J'!3D03!()&+!U=a!(1!$%!F*'%-!3(!&+73D+(273&-#!$!
D,:0'! F/((%! -0:C/&! C(-3B2/*'*20/! 3+*0/! 0/-(! -D(J&%! 3D03! ()&+! U8a! (1! $%! ;&'(:&-! J&+&!
0--(2*03&%! J*3D! &+73D+(273&-!
_L`#! $%:*'*-3+03*('! (1! $%! 3(! F/((%! 2('30*'*';! D*;D! /&)&/-! (1!
'&,3+0/*-*';!$F!1,+3D&+!*'D*F*3-!$%!*'1&23*('#!6(J!/&)&/-!(1!$F!0//(J!1(+!*'2+&0-&%!*'1&23*('!(1!
:('(273&-4!C(3&'3*0//7!+&-,/3*';!*'!*'2+&0-&%!0'3*;&'!C+&-&'303*('#!VD&!-&R,&-3&+*';!(1!$%I!
F7!F/((%!2&//-!*'!D,:0'-!MF,3!'(3!:*2&Q!D0-!F&&'!-D(J'!3(!F&!%,&!3(!3D&!C+&-&'303*('!(1!
.$T!0'%!2(:C/&:&'3!+&2&C3(+!<!('!3D&!-,+102&!(1!&+73D+(273&-!
_"`#!P/03&/&3-!0+&!2('-30'3/7!
C+&-&'3! *'! D*;D! ',:F&+-! *'! 3D&! F/((%! 0'%! 3+*;;&+! -&R,&-3&+*';! (1! $%! 3(! 3D&! /*)&+! 3D+(,;D!
2(0;,/03*('!J*3D!/&,E(273&-!
_L4!84!@=4!@@`#!9'!3D&!/*)&+4!-C&2*0/*-&%!:02+(CD0;&-!E'(J'!0-!o,C11&+!
2&//-! 30E&! ,C! 3D&! 0;;+&;03&-! 0'%! %&;+0%&! 3D&:!
_<G4! <K4! @<4! @@`#! P/03&/&3! %&C/&3*('! F&1(+&!
0%:*'*-3+03*('! 3(! :*2&! !"# $!$'! +&-,/3-! *'! +&%,2&%! -&R,&-3+03*('! F7! )0+*(,-! (+;0'-4!
D*;D/*;D3*';!3D&!-3+(';!*'D*F*3(+7!&11&23!/&0'3!F7!3D&*+!0F,'%0'2&#!
10
$%&'()*+0/!>&23(+-!
!
$%&'()*+,-&-!0+&!0F/&!3(!*'1&23!0!J*%&!+0';&!(1!2&//!37C&-!0'%!3*--,&-4!J*3D(,3!*'3&;+03*('!(1!
3D&*+!;&'(:&-!*'3(!3D&!D(-3!;&'(:&!
_84!U4!<I`#!VD&*+!/73*2!0F*/*37!+&-,/3-!*'!%&03D!(1!D(-3!2&//-!
_U4!<U`
