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`IPR2018-00123
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`122
`Infectious Disorders - Drug Targets 2006, Vol. 6, N0. 2
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`Fig. (1). Genetic organization and protein products of HCV. Above: the HCV genome, showing the 5’ and 3’ untranslated regions. The
`^_`aÿb caÿd%0%$/-ÿ#"=&0/Z&$/#0ÿ&01ÿ!"#$%/0ÿ!"#1,-$'ÿ#EÿF3G(ÿBH#7%eÿ$+%ÿF3Gÿ=%0#J%5ÿ'+#I/0=ÿ$+%ÿDfÿ&01ÿ@fÿ,0$"&0'.&$%1ÿ"%=/#0'(ÿ*+%
`proposed secondary structure for the CRE domain (cis-response element) is also shown. IRES-dependent translation generates a 3011 amino
`!"#!#'%1ÿ'%-#01&"<ÿ'$",-$,"%ÿE#"ÿ$+%ÿ3N8ÿ1#J&/0ÿ2ghi;"%'!#0'%ÿ%.%J%0$4ÿ/'ÿ&.'#ÿ'+#I0(ÿSN8?;1%!%01%0$ÿ$"&0'.&$/#0ÿ=%0%"&$%'ÿ&ÿ@j99ÿ&J/0#
`acid-long polyprotein product (below) which is subsequently processed into structural and non-structural proteins. The proteolysis products
`&-/1;.#0=ÿ!#.<!"#$%/0ÿ!"#1,-$ÿ2H%.#I4ÿI+/-+ÿ/'ÿ',H'%k,%0$.<ÿ!"#-%''%1ÿ/0$#ÿ'$",-$,"&.ÿ&01ÿ0#0;'$",-$,"&.ÿ!"#$%/0'(ÿ*+%ÿ!"#$%#.<'/'ÿ!"#1,-$'
`and their functions are indicated.
`&01ÿ$+%/"ÿE,0-$/#0'ÿ&"%ÿ/01/-&$%1(
`
`proteases. The structural proteins include the capsid protein
`!"#$%&'%'(ÿ*+%ÿ'$",-$,"&.ÿ!"#$%/0'ÿ/0-.,1%ÿ$+%ÿ-&!'/1ÿ!"#$%/0
`(C), p7 and the envelope proteins E1 and E2. The non-
`2345ÿ!6ÿ&01ÿ$+%ÿ%07%.#!%ÿ!"#$%/0'ÿ89ÿ&01ÿ8:(ÿ*+%ÿ0#0;
`structural products are involved in polyprotein processing
`'$",-$,"&.ÿ!"#1,-$'ÿ&"%ÿ/07#.7%1ÿ/0ÿ!#.<!"#$%/0ÿ!"#-%''/0=
`(NS2, NS3, NS4A) and replication (NS4B, NSSA and
`2>?:5ÿ>?@5ÿ>?AB4ÿ&01ÿ"%!./-&$/#0ÿ2>?AC5ÿ>?DBÿ&01
`NSSB).
`>?DC4(
`
`The success of HCV infection is conditioned by how well
`*+%ÿ',--%''ÿ#EÿF3Gÿ/0E%-$/#0ÿ/'ÿ-#01/$/#0%1ÿH<ÿ+#IÿI%..
`the virus avoids the immune host response. The initial
`$+%ÿ7/",'ÿ&7#/1'ÿ$+%ÿ/JJ,0%ÿ+#'$ÿ"%'!#0'%(ÿ*+%ÿ/0/$/&.
`defense reaction depends on the viral pathogen-associated
`1%E%0'%ÿ"%&-$/#0ÿ1%!%01'ÿ#0ÿ$+%ÿ7/"&.ÿ!&$+#=%0;&''#-/&$%1
`molecular pattern (PAMP). For HCV, the PAMP includes
`J#.%-,.&"ÿ!&$$%"0ÿ2KBLK4(ÿM#"ÿF3G5ÿ$+%ÿKBLKÿ/0-.,1%'
`the genomic RNA, the NSSA protein, the core protein, and
`$+%ÿ=%0#J/-ÿN>B5ÿ$+%ÿ>?DBÿ!"#$%/05ÿ$+%ÿ-#"%ÿ!"#$%/05ÿ&01
`even entire viral particles, all of which can be presented by
`%7%0ÿ%0$/"%ÿ7/"&.ÿ!&"$/-.%'5ÿ&..ÿ#EÿI+/-+ÿ-&0ÿH%ÿ!"%'%0$%1ÿH<
`dendritic cells (for a review, see [22]). PAMP presentation
`1%01"/$/-ÿ-%..'ÿ2E#"ÿ&ÿ"%7/%I5ÿ'%%ÿO::P4(ÿKBLKÿ!"%'%0$&$/#0
`initiates the production of interferon and cytokines [23] in an
`/0/$/&$%'ÿ$+%ÿ!"#1,-$/#0ÿ#Eÿ/0$%"E%"#0ÿ&01ÿ-<$#Q/0%'ÿO:@Pÿ/0ÿ&0
`autoregulatory fashion ([24]; reviewed in [22]). It has been
`&,$#"%=,.&$#"<ÿE&'+/#0ÿ2O:APRÿ"%7/%I%1ÿ/0ÿO::P4(ÿS$ÿ+&'ÿH%%0
`reported that the NS3/4A protein acts as an antagonist of
`"%!#"$%1ÿ$+&$ÿ$+%ÿ>?@TABÿ!"#$%/0ÿ&-$'ÿ&'ÿ&0ÿ&0$&=#0/'$ÿ#E
`certain key factors in the induction of the immune response.
`-%"$&/0ÿQ%<ÿE&-$#"'ÿ/0ÿ$+%ÿ/01,-$/#0ÿ#Eÿ$+%ÿ/JJ,0%ÿ"%'!#0'%(
`This mainly involves a reduction in IFN production, thus
`*+/'ÿJ&/0.<ÿ/07#.7%'ÿ&ÿ"%1,-$/#0ÿ/0ÿSM>ÿ!"#1,-$/#05ÿ$+,'
`inducing alterations in the pathways and complexes whose
`/01,-/0=ÿ&.$%"&$/#0'ÿ/0ÿ$+%ÿ!&$+I&<'ÿ&01ÿ-#J!.%U%'ÿI+#'%
`functioning is dependent on it. For example, antigen
`E,0-$/#0/0=ÿ/'ÿ1%!%01%0$ÿ#0ÿ/$(ÿM#"ÿ%U&J!.%5ÿ&0$/=%0
`presentation is greatly reduced, meaning T cells cannot be
`!"%'%0$&$/#0ÿ/'ÿ="%&$.<ÿ"%1,-%15ÿJ%&0/0=ÿ*ÿ-%..'ÿ-&00#$ÿH%
`stimulated. NS3/4A has also been related to a prolonged
`'$/J,.&$%1(ÿ>?@TABÿ+&'ÿ&.'#ÿH%%0ÿ"%.&$%1ÿ$#ÿ&ÿ!"#.#0=%1
`blocking of pro-apoptotic factor activity, providing a
`H.#-Q/0=ÿ#Eÿ!"#;&!#!$#$/-ÿE&-$#"ÿ&-$/7/$<5ÿ!"#7/1/0=ÿ&
`molecular link between HCV infection and the development
`J#.%-,.&"ÿ./0QÿH%$I%%0ÿF3Gÿ/0E%-$/#0ÿ&01ÿ$+%ÿ1%7%.#!J%0$
`
`of hepatocellular carcinoma [25]. Similarly,
`the NSSA
`#Eÿ+%!&$#-%..,.&"ÿ-&"-/0#J&ÿO:DP(ÿ?/J/.&".<5ÿ$+%ÿ>?DB
`protein may activate IL-8 production, which interferes with
`!"#$%/0ÿJ&<ÿ&-$/7&$%ÿSV;Wÿ!"#1,-$/#05ÿI+/-+ÿ/0$%"E%"%'ÿI/$+
`IFN-mediated response pathways, and in conjunction with
`SM>;J%1/&$%1ÿ"%'!#0'%ÿ!&$+I&<'5ÿ&01ÿ/0ÿ-#0X,0-$/#0ÿI/$+
`E2 it may block protein kinase R (PKR), an enzyme involved
`8:ÿ/$ÿJ&<ÿH.#-Qÿ!"#$%/0ÿQ/0&'%ÿNÿ2KYN45ÿ&0ÿ%0Z<J%ÿ/07#.7%1
`in the amplification of the immune response. The intensity of
`/0ÿ$+%ÿ&J!./E/-&$/#0ÿ#Eÿ$+%ÿ/JJ,0%ÿ"%'!#0'%(ÿ*+%ÿ/0$%0'/$<ÿ#E
`these effects differs depending on the genetic diversity of the
`$+%'%ÿ%EE%-$'ÿ1/EE%"'ÿ1%!%01/0=ÿ#0ÿ$+%ÿ=%0%$/-ÿ1/7%"'/$<ÿ#Eÿ$+%
`infecting viral pool. Finally, HCV genotypes 1a and lb have
`/0E%-$/0=ÿ7/"&.ÿ!##.(ÿM/0&..<5ÿF3Gÿ=%0#$<!%'ÿ9&ÿ&01ÿ9Hÿ+&7%
`a smaller number of UU and AU sites than genotypes 2 or 3,
`&ÿ'J&..%"ÿ0,JH%"ÿ#Eÿ[[ÿ&01ÿB[ÿ'/$%'ÿ$+&0ÿ=%0#$<!%'ÿ:ÿ#"ÿ@5
`impeding RNase L-mediated degradation of the HCV
`/J!%1/0=ÿN>&'%ÿV;J%1/&$%1ÿ1%="&1&$/#0ÿ#Eÿ$+%ÿF3G
`genome (this enzyme specifically cleaves nucleic acids at
`=%0#J%ÿ2$+/'ÿ%0Z<J%ÿ'!%-/E/-&..<ÿ-.%&7%'ÿ0,-.%/-ÿ&-/1'ÿ&$
`these sites;
`[26]). This provides another possibility for
`$+%'%ÿ'/$%'RÿO:\P4(ÿ*+/'ÿ!"#7/1%'ÿ&0#$+%"ÿ!#''/H/./$<ÿE#"
`variation in terms of resistance to IFN since RNase L is
`7&"/&$/#0ÿ/0ÿ$%"J'ÿ#Eÿ"%'/'$&0-%ÿ$#ÿSM>ÿ'/0-%ÿN>&'%ÿVÿ/'
`activated by this molecule.
`&-$/7&$%1ÿH<ÿ$+/'ÿJ#.%-,.%(
`
`The identification of HCV in 1989 by Houghton and co-
`*+%ÿ/1%0$/E/-&$/#0ÿ#EÿF3Gÿ/0ÿ9]W]ÿH<ÿF#,=+$#0ÿ&01ÿ-#;
`workers [15] led to numerous studies aimed at deciphering
`I#"Q%"'ÿO9DPÿ.%1ÿ$#ÿ0,J%"#,'ÿ'$,1/%'ÿ&/J%1ÿ&$ÿ1%-/!+%"/0=
`its molecular biology, but advances in our knowledge
`/$'ÿJ#.%-,.&"ÿH/#.#=<5ÿH,$ÿ&17&0-%'ÿ/0ÿ#,"ÿQ0#I.%1=%
`regarding the viral cycle have been hindered by the lack of
`"%=&"1/0=ÿ$+%ÿ7/"&.ÿ-<-.%ÿ+&7%ÿH%%0ÿ+/01%"%1ÿH<ÿ$+%ÿ.&-Qÿ#E
`an efficient and stable culture system. However, recent
`&0ÿ%EE/-/%0$ÿ&01ÿ'$&H.%ÿ-,.$,"%ÿ'<'$%J(ÿF#I%7%"5ÿ"%-%0$
`advances in replicon and pseudoparticle production methods
`&17&0-%'ÿ/0ÿ"%!./-#0ÿ&01ÿ!'%,1#!&"$/-.%ÿ!"#1,-$/#0ÿJ%$+#1'
`[27, 28] have allowed a more detailed understanding of HCV
`O:65ÿ:WPÿ+&7%ÿ&..#I%1ÿ&ÿJ#"%ÿ1%$&/.%1ÿ,01%"'$&01/0=ÿ#EÿF3G
`spread and replication to be grasped. In addition, the latest
`'!"%&1ÿ&01ÿ"%!./-&$/#0ÿ$#ÿH%ÿ="&'!%1(ÿS0ÿ&11/$/#05ÿ$+%ÿ.&$%'$
`virion production strategies provide the starting point for the
`7/"/#0ÿ!"#1,-$/#0ÿ'$"&$%=/%'ÿ!"#7/1%ÿ$+%ÿ'$&"$/0=ÿ!#/0$ÿE#"ÿ$+%
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`2
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`BerzaI-Herranz et al.
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`E'UTR
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`".4-.I
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`
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`
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`
`lim clupc
`
`l’rnleuscs‘hcl least:
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`Fig. (1). Genetic organization and protein products of HCV. Above: the HCV genome, showing the 5’ and 3’ untranslated regions. The
`^_`aÿb caÿd%0%$/-ÿ#"=&0/Z&$/#0ÿ&01ÿ!"#$%/0ÿ!"#1,-$'ÿ#EÿF3G(ÿBH#7%eÿ$+%ÿF3Gÿ=%0#J%5ÿ'+#I/0=ÿ$+%ÿDfÿ&01ÿ@fÿ,0$"&0'.&$%1ÿ"%=/#0'(ÿ*+%
`proposed secondary structure for the CRE domain (cis-response element) is also shown. IRES-dependent translation generates a 3011 amino
`!"#!#'%1ÿ'%-#01&"<ÿ'$",-$,"%ÿE#"ÿ$+%ÿ3N8ÿ1#J&/0ÿ2ghi;"%'!#0'%ÿ%.%J%0$4ÿ/'ÿ&.'#ÿ'+#I0(ÿSN8?;1%!%01%0$ÿ$"&0'.&$/#0ÿ=%0%"&$%'ÿ&ÿ@j99ÿ&J/0#
`acid-long polyprotein product (below) which is subsequently processed into structural and non-structural proteins. The proteolysis products
`&-/1;.#0=ÿ!#.<!"#$%/0ÿ!"#1,-$ÿ2H%.#I4ÿI+/-+ÿ/'ÿ',H'%k,%0$.<ÿ!"#-%''%1ÿ/0$#ÿ'$",-$,"&.ÿ&01ÿ0#0;'$",-$,"&.ÿ!"#$%/0'(ÿ*+%ÿ!"#$%#.<'/'ÿ!"#1,-$'
`and their functions are indicated.
`&01ÿ$+%/"ÿE,0-$/#0'ÿ&"%ÿ/01/-&$%1(
`
`proteases. The structural proteins include the capsid protein
`!"#$%&'%'(ÿ*+%ÿ'$",-$,"&.ÿ!"#$%/0'ÿ/0-.,1%ÿ$+%ÿ-&!'/1ÿ!"#$%/0
`(C), p7 and the envelope proteins E1 and E2. The non-
`2345ÿ!6ÿ&01ÿ$+%ÿ%07%.#!%ÿ!"#$%/0'ÿ89ÿ&01ÿ8:(ÿ*+%ÿ0#0;
`structural products are involved in polyprotein processing
`'$",-$,"&.ÿ!"#1,-$'ÿ&"%ÿ/07#.7%1ÿ/0ÿ!#.<!"#$%/0ÿ!"#-%''/0=
`(NS2, NS3, NS4A) and replication (NS4B, NSSA and
`2>?:5ÿ>?@5ÿ>?AB4ÿ&01ÿ"%!./-&$/#0ÿ2>?AC5ÿ>?DBÿ&01
`NSSB).
`>?DC4(
`
`The success of HCV infection is conditioned by how well
`*+%ÿ',--%''ÿ#EÿF3Gÿ/0E%-$/#0ÿ/'ÿ-#01/$/#0%1ÿH<ÿ+#IÿI%..
`the virus avoids the immune host response. The initial
`$+%ÿ7/",'ÿ&7#/1'ÿ$+%ÿ/JJ,0%ÿ+#'$ÿ"%'!#0'%(ÿ*+%ÿ/0/$/&.
`defense reaction depends on the viral pathogen-associated
`1%E%0'%ÿ"%&-$/#0ÿ1%!%01'ÿ#0ÿ$+%ÿ7/"&.ÿ!&$+#=%0;&''#-/&$%1
`molecular pattern (PAMP). For HCV, the PAMP includes
`J#.%-,.&"ÿ!&$$%"0ÿ2KBLK4(ÿM#"ÿF3G5ÿ$+%ÿKBLKÿ/0-.,1%'
`the genomic RNA, the NSSA protein, the core protein, and
`$+%ÿ=%0#J/-ÿN>B5ÿ$+%ÿ>?DBÿ!"#$%/05ÿ$+%ÿ-#"%ÿ!"#$%/05ÿ&01
`even entire viral particles, all of which can be presented by
`%7%0ÿ%0$/"%ÿ7/"&.ÿ!&"$/-.%'5ÿ&..ÿ#EÿI+/-+ÿ-&0ÿH%ÿ!"%'%0$%1ÿH<
`dendritic cells (for a review, see [22]). PAMP presentation
`1%01"/$/-ÿ-%..'ÿ2E#"ÿ&ÿ"%7/%I5ÿ'%%ÿO::P4(ÿKBLKÿ!"%'%0$&$/#0
`initiates the production of interferon and cytokines [23] in an
`/0/$/&$%'ÿ$+%ÿ!"#1,-$/#0ÿ#Eÿ/0$%"E%"#0ÿ&01ÿ-<$#Q/0%'ÿO:@Pÿ/0ÿ&0
`autoregulatory fashion ([24]; reviewed in [22]). It has been
`&,$#"%=,.&$#"<ÿE&'+/#0ÿ2O:APRÿ"%7/%I%1ÿ/0ÿO::P4(ÿS$ÿ+&'ÿH%%0
`reported that the NS3/4A protein acts as an antagonist of
`"%!#"$%1ÿ$+&$ÿ$+%ÿ>?@TABÿ!"#$%/0ÿ&-$'ÿ&'ÿ&0ÿ&0$&=#0/'$ÿ#E
`certain key factors in the induction of the immune response.
`-%"$&/0ÿQ%<ÿE&-$#"'ÿ/0ÿ$+%ÿ/01,-$/#0ÿ#Eÿ$+%ÿ/JJ,0%ÿ"%'!#0'%(
`This mainly involves a reduction in IFN production, thus
`*+/'ÿJ&/0.<ÿ/07#.7%'ÿ&ÿ"%1,-$/#0ÿ/0ÿSM>ÿ!"#1,-$/#05ÿ$+,'
`inducing alterations in the pathways and complexes whose
`/01,-/0=ÿ&.$%"&$/#0'ÿ/0ÿ$+%ÿ!&$+I&<'ÿ&01ÿ-#J!.%U%'ÿI+#'%
`functioning is dependent on it. For example, antigen
`E,0-$/#0/0=ÿ/'ÿ1%!%01%0$ÿ#0ÿ/$(ÿM#"ÿ%U&J!.%5ÿ&0$/=%0
`presentation is greatly reduced, meaning T cells cannot be
`!"%'%0$&$/#0ÿ/'ÿ="%&$.<ÿ"%1,-%15ÿJ%&0/0=ÿ*ÿ-%..'ÿ-&00#$ÿH%
`stimulated. NS3/4A has also been related to a prolonged
`'$/J,.&$%1(ÿ>?@TABÿ+&'ÿ&.'#ÿH%%0ÿ"%.&$%1ÿ$#ÿ&ÿ!"#.#0=%1
`blocking of pro-apoptotic factor activity, providing a
`H.#-Q/0=ÿ#Eÿ!"#;&!#!$#$/-ÿE&-$#"ÿ&-$/7/$<5ÿ!"#7/1/0=ÿ&
`molecular link between HCV infection and the development
`J#.%-,.&"ÿ./0QÿH%$I%%0ÿF3Gÿ/0E%-$/#0ÿ&01ÿ$+%ÿ1%7%.#!J%0$
`
`of hepatocellular carcinoma [25]. Similarly,
`the NSSA
`#Eÿ+%!&$#-%..,.&"ÿ-&"-/0#J&ÿO:DP(ÿ?/J/.&".<5ÿ$+%ÿ>?DB
`protein may activate IL-8 production, which interferes with
`!"#$%/0ÿJ&<ÿ&-$/7&$%ÿSV;Wÿ!"#1,-$/#05ÿI+/-+ÿ/0$%"E%"%'ÿI/$+
`IFN-mediated response pathways, and in conjunction with
`SM>;J%1/&$%1ÿ"%'!#0'%ÿ!&$+I&<'5ÿ&01ÿ/0ÿ-#0X,0-$/#0ÿI/$+
`E2 it may block protein kinase R (PKR), an enzyme involved
`8:ÿ/$ÿJ&<ÿH.#-Qÿ!"#$%/0ÿQ/0&'%ÿNÿ2KYN45ÿ&0ÿ%0Z<J%ÿ/07#.7%1
`in the amplification of the immune response. The intensity of
`/0ÿ$+%ÿ&J!./E/-&$/#0ÿ#Eÿ$+%ÿ/JJ,0%ÿ"%'!#0'%(ÿ*+%ÿ/0$%0'/$<ÿ#E
`these effects differs depending on the genetic diversity of the
`$+%'%ÿ%EE%-$'ÿ1/EE%"'ÿ1%!%01/0=ÿ#0ÿ$+%ÿ=%0%$/-ÿ1/7%"'/$<ÿ#Eÿ$+%
`infecting viral pool. Finally, HCV genotypes 1a and lb have
`/0E%-$/0=ÿ7/"&.ÿ!##.(ÿM/0&..<5ÿF3Gÿ=%0#$<!%'ÿ9&ÿ&01ÿ9Hÿ+&7%
`a smaller number of UU and AU sites than genotypes 2 or 3,
`&ÿ'J&..%"ÿ0,JH%"ÿ#Eÿ[[ÿ&01ÿB[ÿ'/$%'ÿ$+&0ÿ=%0#$<!%'ÿ:ÿ#"ÿ@5
`impeding RNase L-mediated degradation of the HCV
`/J!%1/0=ÿN>&'%ÿV;J%1/&$%1ÿ1%="&1&$/#0ÿ#Eÿ$+%ÿF3G
`genome (this enzyme specifically cleaves nucleic acids at
`=%0#J%ÿ2$+/'ÿ%0Z<J%ÿ'!%-/E/-&..<ÿ-.%&7%'ÿ0,-.%/-ÿ&-/1'ÿ&$
`these sites;
`[26]). This provides another possibility for
`$+%'%ÿ'/$%'RÿO:\P4(ÿ*+/'ÿ!"#7/1%'ÿ&0#$+%"ÿ!#''/H/./$<ÿE#"
`variation in terms of resistance to IFN since RNase L is
`7&"/&$/#0ÿ/0ÿ$%"J'ÿ#Eÿ"%'/'$&0-%ÿ$#ÿSM>ÿ'/0-%ÿN>&'%ÿVÿ/'
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`124
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` !ÿ#$%!ÿ&'()*')(+,ÿ./01,ÿ2(/2/310ÿ4/(ÿ'51ÿ678ÿ9:;<=ÿ31>.1?'@ÿ<51ÿ0A441(1?'ÿ0/.+A?3ÿ/4ÿ'51ÿ1?'A(1ÿ31*/?0+(Bÿ3'()*')(1ÿ+(1ÿA?0A*+'10@
`
`5+C1ÿA01?'A4A10ÿ'5(11ÿ0A441(1?'ÿ0/.+A?3Dÿ'51ÿ8=ÿ(1>A/?Eÿ'51
`2/,B;F7ÿ'(+*'ÿ+?0ÿ9:Gÿ(1>A/?ÿHIJEÿIKL@
`
`<51ÿ9:;<=ÿM1>A?3ÿNA'5ÿ+ÿ35/('ÿ+?0ÿ5A>5,BÿC+(A+M,1
`31O)1?*1Eÿ+,'5/)>5ÿA'ÿ+,3/ÿ*/?'+A?3ÿ+ÿ*/?31(C10ÿ./'A4ÿ+?0
`;Pÿ0A?)*,1/'A01ÿHI9L@ÿ<5A3ÿ0/.+A?ÿA3ÿ?+.10ÿ8=ÿQC+(A+M,1
`(1>A/?Rÿ+?0ÿA'ÿ5+3ÿM11?ÿ35/N?ÿA'3ÿA.2,A*+'A/?ÿA?ÿCA(+,ÿ=ST
`3B?'513A3ÿ)3A?>ÿ3)M>1?/.A*ÿ(12,A*/?ÿ3B3'1.3ÿHKJL@
`
`Tÿ2/,B;F7U(A*5ÿ'(+*'ÿ/4ÿC+(A+M,1ÿ,1?>'5ÿ+?0ÿ*/.2/3A'A/?
`4/,,/N3ÿ'51ÿ8=ÿ31O)1?*1@ÿ<5A3ÿ*+?ÿA?'1(+*'ÿNA'5ÿ5/3'
`2(/'1A?3ÿ+?0ÿ(1>),+'1ÿCA(+,ÿ'(+?3,+'A/?ÿHIVEÿIWL@ÿ&/.1ÿ/4ÿ'5131
`5/3'ÿ2(/'1A?3ÿ5+C1ÿM11?ÿA01?'A4A10Eÿ1@>@Eÿ2/,B2B(A.A0A?1ÿ'(+*'U
`MA?0A?>ÿ2(/'1A?ÿQX<YREÿ51'1(/>1?1/)3ÿ?)*,1+(ÿ(AM/?)U
`*,1/2(/'1A?ÿ7ÿQ5?=SXÿ7Rÿ+?0ÿ>,B*1(+,015B01U9U25/325+'1
`015B0(/>1?+31ÿQPTZX6REÿ+./?>ÿ/'51(3ÿHIIUI[L@
`
`\A?+,,BEÿ'51ÿ9:;<=ÿ(1>A/?ÿ*/?'+A?3ÿ+ÿ][ÿ?'U,/?>ÿ0/.+A?
`^?/N?ÿ+3ÿ'51ÿ9:Gÿ(1>A/?ÿ_ÿ/?1ÿ/4ÿ'51ÿ./3'ÿ*/?31(C10ÿ./'A43
`NA'5A?ÿ'5A3ÿ(1>A/?@ÿ`'ÿ4/,03ÿA?'/ÿ'5(11ÿ0A441(1?'ÿ3'1.U,//2
`./'A43ÿ_ÿ&aJEÿ&aKÿ+?0ÿ&a9ÿHW]Lÿ_ÿ+?0ÿA3ÿA?C/,C10ÿA?ÿCA(+,
`=STÿ(12,A*+'A/?ÿHKJEÿI]UbJLÿ+?0ÿCA(A/?ÿA?41*'ACA'Bÿcdÿfcfg
`HIhL@ÿ`'3ÿA?'1(+*'A/?ÿNA'5ÿ5).+?ÿ(AM/3/.+,ÿ*/.2,1i13ÿ5+3
`
`+,3/ÿM11?ÿ(12/('10Eÿ3)>>13'A?>ÿ+ÿ2/'1?'A+,ÿ(/,1ÿA?ÿ'51
`./0),+'A/?ÿ/4ÿ`=j&U0121?01?'ÿ'(+?3,+'A/?ÿHbKL@ÿ`'ÿA3
`A.2/('+?'ÿ'/ÿ?/'1ÿ'5+'ÿ'5A3ÿ0/.+A?ÿA3ÿ1i*,)3AC1ÿ'/ÿ678
`Q(1CA1N10ÿA?ÿHb9LRÿ+?0ÿA3ÿ+?ÿ1i*1,,1?'ÿ*+?0A0+'1ÿ'+(>1'ÿ4/(
`?1Nÿ+?'ACA(+,ÿ0()>3@
`
`klmÿno$ÿpqrkmsn
`
`t?1ÿ/4ÿ'51ÿ./3'ÿ1i'1?3AC1,Bÿ3')0A10ÿ+?0ÿM13'ÿ)?01(3'//0
`'+(>1'3ÿA?ÿ+?'AU678ÿ'51(+2BÿA3ÿ'51ÿS&9ÿ2(/'1A?ÿUÿ+
`.),'A4)?*'A/?+,ÿ./,1*),1ÿ*/.2/310ÿ/4ÿ'N/ÿ0/.+A?3ÿNA'5
`31(A?1ÿ2(/'1+31ÿ+?0ÿ51,A*+31ÿ+*'ACA'BÿHbVEÿbWL@
`
`X(/'1+31ÿ+*'ACA'BÿA3ÿ/?1ÿ/4ÿ'51ÿ2(141((10ÿ'+(>1'3ÿ4/(ÿ'51
`013A>?1(3ÿ/4ÿ?1Nÿ0()>3@ÿX(/'1+31ÿ5+3ÿ+ÿ^1Bÿ(/,1ÿA?ÿ678
`A?41*'A/?ÿ3A?*1ÿA'ÿA3ÿ(132/?3AM,1ÿ4/(ÿ2/,B2(/'1A?ÿ2(/*133A?>
`0)(A?>ÿCA()3ÿ.+')(+'A/?@ÿ<51ÿ5/3'ÿ2(/'1+313ÿ4A(3'ÿ*,1+C1ÿ'51
`2(1*)(3/(ÿ'/ÿ(1,1+31ÿ'51ÿ3'()*')(+,ÿ2(/'1A?3@ÿ&1*/?0,BEÿCA(+,,BU
`1?*/010ÿS&KFS&9ÿ2(/'1+313ÿBA1,0ÿ'51ÿ.+')(1ÿS&9ÿ2(/'1A?
`HbILÿN5A*5ÿA?A'A+'13ÿ'51ÿ*,1+C+>1ÿ/4ÿ'51ÿ(1.+A?A?>ÿ'+(>1'ÿ3A'13
`A?ÿ+?ÿu>KvU0121?01?'ÿ(1+*'A/?ÿHbbLDÿ4A(3'Eÿ*/'(+?3,+'A/?+,,Bÿ+'
`'51ÿS&9FS&VTÿw)?*'A/?Eÿ+?0ÿ'51?ÿA?ÿ'51ÿ/(01(ÿS&WTUWYE
`S&VTUVYÿ+?0ÿS&VYUWTÿHb[L@ÿ<51ÿxcyÿ*,1+C+>1ÿA3ÿ>/C1(?10
`
`5
`
`
`
`126
`Infectious Disorders - Drug Targets 2006, Vol. 6, N0. 2
` ÿÿÿÿ
`
ÿ
` ÿÿ
` ÿÿ
ÿ
ÿ ÿ ÿÿ ÿ
`
`by the intrinsic folding of the polyprotein, which brings the
`!"ÿ$%&ÿ'($)'(*'+ÿ,-./'(0ÿ-,ÿ$%&ÿ1-."1)-$&'(2ÿ3%'+%ÿ!)'(0*ÿ$%&
`protease domain and its substrate close together. The trans-
`1)-$&4*&ÿ/-54'(ÿ4(/ÿ'$*ÿ*6!*$)4$&ÿ+.-*&ÿ$-0&$%&)7ÿ8%&ÿ9:;<=>
`reaction has more stringent
`requirements including the
`)&4+$'-(ÿ%4*ÿ5-)&ÿ*$)'(0&($ÿ)&?6')&5&($*ÿ'(+.6/'(0ÿ$%&
`correct positioning of the protease and its polyprotein
`+-))&+$ÿ1-*'$'-('(0ÿ-,ÿ$%&ÿ1)-$&4*&ÿ4(/ÿ'$*ÿ1-."1)-$&'(
`substrate (reviewed in [79]).
`*6!*$)4$&ÿ@)&A'&3&/ÿ'(ÿBCDEF7
`
`The proteolytic processing mediated by NS3 is dependent
`8%&ÿ1)-$&-."$'+ÿ1)-+&**'(0ÿ5&/'4$&/ÿ!"ÿGHIÿ'*ÿ/&1&(/&($
`on NS4A, a small polypeptide composed of only 54
`-(ÿGHJK2ÿ4ÿ*54..ÿ1-."1&1$'/&ÿ+-51-*&/ÿ-,ÿ-(."ÿLJ
`aminoacids [80, 81]. The interaction between them maps to
`45'(-4+'/*ÿBMN2ÿMOE7ÿ8%&ÿ'($&)4+$'-(ÿ!&$3&&(ÿ$%&5ÿ541*ÿ$-
`the 22 residues most N—terminal of the NS3 protein [82, 83]
`$%&ÿPPÿ)&*'/6&*ÿ5-*$ÿG>$&)5'(4.ÿ-,ÿ$%&ÿGHIÿ1)-$&'(ÿBMP2ÿMIE
`and induces several conformational changes affecting the
`4(/ÿ'(/6+&*ÿ*&A&)4.ÿ+-(,-)54$'-(4.ÿ+%4(0&*ÿ4,,&+$'(0ÿ$%&
`catalytic residues, giving rise to a highly stable complex
`+4$4."$'+ÿ)&*'/6&*2ÿ0'A'(0ÿ)'*&ÿ$-ÿ4ÿ%'0%."ÿ*$4!.&ÿ+-51.&Q
`containing NS4A [84]. NS3 has no membrane anchoring
`+-($4'('(0ÿGHJKÿBMJE7ÿGHIÿ%4*ÿ(-ÿ5&5!)4(&ÿ4(+%-)'(0
`domains itself, but its cofactor NS4A is an arnphipathic
`/-54'(*ÿ'$*&.,2ÿ!6$ÿ'$*ÿ+-,4+$-)ÿGHJKÿ'*ÿ4(ÿ451%'14$%'+
`peptide that can interact with membranes via a highly
`1&1$'/&ÿ$%4$ÿ+4(ÿ'($&)4+$ÿ3'$%ÿ5&5!)4(&*ÿA'4ÿ4ÿ%'0%."
`hydrophobic stretch of aminoacids to form a transmembrane
`%"/)-1%-!'+ÿ*$)&$+%ÿ-,ÿ45'(-4+'/*ÿ$-ÿ,-)5ÿ4ÿ$)4(*5&5!)4(&
`helix. This allows the NS3/4A complex to be immobilized
`%&.'Q7ÿ8%'*ÿ4..-3*ÿ$%&ÿGHIRJKÿ+-51.&Qÿ$-ÿ!&ÿ'55-!'.'S&/
`on the surface of the endoplasmic reticulum (ER; [85]), a
`-(ÿ$%&ÿ*6),4+&ÿ-,ÿ$%&ÿ&(/-1.4*5'+ÿ)&$'+6.65ÿ@TUVÿBMLEF2ÿ4
`requirement for the establishment of the replication complex.
`)&?6')&5&($ÿ,-)ÿ$%&ÿ&*$4!.'*%5&($ÿ-,ÿ$%&ÿ)&1.'+4$'-(ÿ+-51.&Q7
`
`The structure of the protease-NS4A complex has been
`8%&ÿ*$)6+$6)&ÿ-,ÿ$%&ÿ1)-$&4*&>GHJKÿ+-51.&Qÿ%4*ÿ!&&(
`resolved by X-ray crystallography. This information has
`)&*-.A&/ÿ!"ÿW>)4"ÿ+)"*$4..-0)41%"7ÿ8%'*ÿ'(,-)54$'-(ÿ%4*
`given rise to intensive efforts to identify potential targets for
`0'A&(ÿ)'*&ÿ$-ÿ'($&(*'A&ÿ&,,-)$*ÿ$-ÿ'/&($',"ÿ1-$&($'4.ÿ$4)0&$*ÿ,-)
`new antiviral drugs. As a result, a chymotrypsin—like folding
`(&3ÿ4($'A')4.ÿ/)60*7ÿK*ÿ4ÿ)&*6.$2ÿ4ÿ+%"5-$)"1*'(>.'X&ÿ,-./'(0
`with an exposed Zn-binding site located on the surface of the
`3'$%ÿ4(ÿ&Q1-*&/ÿY(>!'(/'(0ÿ*'$&ÿ.-+4$&/ÿ-(ÿ$%&ÿ*6),4+&ÿ-,ÿ$%&
`complex was detected [86]. Zinc ions (a requisite for
`+-51.&Qÿ34*ÿ/&$&+$&/ÿBMZE7ÿY'(+ÿ'-(*ÿ@4ÿ)&?6'*'$&ÿ,-)
`catalytic activity; [87]), are coordinated via three cysteine
`+4$4."$'+ÿ4+$'A'$"VÿBMCEF2ÿ4)&ÿ+--)/'(4$&/ÿA'4ÿ$%)&&ÿ+"*$&'(&
`residues in a long loop region that acts as a linker between
`)&*'/6&*ÿ'(ÿ4ÿ.-(0ÿ.--1ÿ)&0'-(ÿ$%4$ÿ4+$*ÿ4*ÿ4ÿ.'(X&)ÿ!&$3&&(
`two B-barrel domains and which contributes to the confor-
`$3-ÿ[>!4))&.ÿ/-54'(*ÿ4(/ÿ3%'+%ÿ+-($)'!6$&*ÿ$-ÿ$%&ÿ+-(,-)>
`mational integrity of the molecule as a whole (reviewed in
`54$'-(4.ÿ'($&0)'$"ÿ-,ÿ$%&ÿ5-.&+6.&ÿ4*ÿ4ÿ3%-.&ÿ@)&A'&3&/ÿ'(
`[88]). This structural feature clearly differentiates NS3/4A
`BMMEF7ÿ8%'*ÿ*$)6+$6)4.ÿ,&4$6)&ÿ+.&4)."ÿ/',,&)&($'4$&*ÿGHIRJK
`from other members of
`the
`serine-protease
`family.
`,)-5ÿ-$%&)ÿ5&5!&)*ÿ-,ÿ$%&ÿ*&)'(&>1)-$&4*&ÿ,45'."7
`Consequently, it is plausible that compounds affecting the
`\-(*&?6&($."2ÿ'$ÿ'*ÿ1.46*'!.&ÿ$%4$ÿ+-51-6(/*ÿ4,,&+$'(0ÿ$%&
`coordination sphere of the metal ions could act as effective
`+--)/'(4$'-(ÿ*1%&)&ÿ-,ÿ$%&ÿ5&$4.ÿ'-(*ÿ+-6./ÿ4+$ÿ4*ÿ&,,&+$'A&
`and specific inhibitors of protease activity. Research into the
`4(/ÿ*1&+','+ÿ'(%'!'$-)*ÿ-,ÿ1)-$&4*&ÿ4+$'A'$"7ÿU&*&4)+%ÿ'($-ÿ$%&
`three dimensional structure of the protease-NS4A complex
`$%)&&ÿ/'5&(*'-(4.ÿ*$)6+$6)&ÿ-,ÿ$%&ÿ1)-$&4*&>GHJKÿ+-51.&Q
`has also contributed to the identification of the substrate-
`%4*ÿ4.*-ÿ+-($)'!6$&/ÿ$-ÿ$%&ÿ'/&($','+4$'-(ÿ-,ÿ$%&ÿ*6!*$)4$&>
`binding site. This is positioned in a wide, very exposed cleft
`!'(/'(0ÿ*'$&7ÿ8%'*ÿ'*ÿ1-*'$'-(&/ÿ'(ÿ4ÿ3'/&2ÿA&)"ÿ&Q1-*&/ÿ+.&,$
`(reviewed in [89]) and can bind a minimum 10 amino acid-
`@)&A'&3&/ÿ'(ÿBMDEFÿ4(/ÿ+4(ÿ!'(/ÿ4ÿ5'('565ÿONÿ45'(-ÿ4+'/>
`long substrate. Competitive inhibitors based on the structure
`.-(0ÿ*6!*$)4$&7ÿ\-51&$'$'A&ÿ'(%'!'$-)*ÿ!4*&/ÿ-(ÿ$%&ÿ*$)6+$6)&
`of the cleavable substrate have been designed (reviewed in
`-,ÿ$%&ÿ+.&4A4!.&ÿ*6!*$)4$&ÿ%4A&ÿ!&&(ÿ/&*'0(&/ÿ@)&A'&3&/ÿ'(
`[90]). The enzyme is also clearly inhibited by its reaction
`BDNEF7ÿ8%&ÿ&(S"5&ÿ'*ÿ4.*-ÿ+.&4)."ÿ'(%'!'$&/ÿ!"ÿ'$*ÿ)&4+$'-(
`products [91], which opens a broad range of possibilities for
`1)-/6+$*ÿBDOE2ÿ3%'+%ÿ-1&(*ÿ4ÿ!)-4/ÿ)4(0&ÿ-,ÿ1-**'!'.'$'&*ÿ,-)
`the design of mimetic peptide drugs.
`$%&ÿ/&*'0(ÿ-,ÿ5'5&$'+ÿ1&1$'/&ÿ/)60*7
`
`The helicase activity of NS3 maps to its C-tenninal end.
`8%&ÿ%&.'+4*&ÿ4+$'A'$"ÿ-,ÿGHIÿ541*ÿ$-ÿ'$*ÿ\>$&)5'(4.ÿ&(/7
`This domain mediates the RNA duplex unwinding promoted
`8%'*ÿ/-54'(ÿ5&/'4$&*ÿ$%&ÿUGKÿ/61.&Qÿ6(3'(/'(0ÿ1)-5-$&/
`by NTP hydrolysis during Viral replication, suggesting that
`!"ÿG8]ÿ%"/)-."*'*ÿ/6)'(0ÿA')4.ÿ)&1.'+4$'-(2ÿ*600&*$'(0ÿ$%4$
`this helicase also has NTPase activity [92]. RNA duplex
`$%'*ÿ%&.'+4*&ÿ4.*-ÿ%4*ÿG8]4*&ÿ4+$'A'$"ÿBDPE7ÿUGKÿ/61.&Q
`*&14)4$'-(ÿ)&?6')&*ÿ^0P_ÿ4(/ÿ'*ÿ*&A&)&."ÿ'(%'!'$&/ÿ!"
`separation requires Mg2+ and is
`severely inhibited by
`monovalent ions [77].
`5-(-A4.&($ÿ'-(*ÿBCCE7
`
`Crystallography studies and mutational assays of the
`\)"*$4..-0)41%"ÿ*$6/'&*ÿ4(/ÿ56$4$'-(4.ÿ4**4"*ÿ-,ÿ$%&
`helicase domain have shown it to have a unique Y shape in
`%&.'+4*&ÿ/-54'(ÿ%4A&ÿ*%-3(ÿ'$ÿ$-ÿ%4A&ÿ4ÿ6('?6&ÿ`ÿ*%41&ÿ'(
`which three domains are defined by each arm of the Y [93-
`3%'+%ÿ$%)&&ÿ/-54'(*ÿ4)&ÿ/&,'(&/ÿ!"ÿ&4+%ÿ4)5ÿ-,ÿ$%&ÿ`ÿBDI>
`97]. Although separated by clefis, the different domains may
`DCE7ÿK.$%-60%ÿ*&14)4$&/ÿ!"ÿ+.&,$*2ÿ$%&ÿ/',,&)&($ÿ/-54'(*ÿ54"
`interact with each other. This finding has led to the design of
`'($&)4+$ÿ3'$%ÿ&4+%ÿ-$%&)7ÿ8%'*ÿ,'(/'(0ÿ%4*ÿ.&/ÿ$-ÿ$%&ÿ/&*'0(ÿ-,
`new helicase inhibitors
`that affect different
`functions
`(&3ÿ%&.'+4*&ÿ'(%'!'$-)*ÿ$%4$ÿ4,,&+$ÿ/',,&)&($ÿ,6(+$'-(*
`simultaneously.
`*'56.$4(&-6*."7
`
`The different NS3 activities show a high level of
`8%&ÿ/',,&)&($ÿGHIÿ4+$'A'$'&*ÿ*%-3ÿ4ÿ%'0%ÿ.&A&.ÿ-,
`interdependence. It seems clear that NTPase capacity is
`'($&)/&1&(/&(+&7ÿa$ÿ*&&5*ÿ+.&4)ÿ$%4$ÿG8]4*&ÿ+414+'$"ÿ'*
`necessary but not sufficient for helicase activity [97-100].
`(&+&**4)"ÿ!6$ÿ(-$ÿ*6,,'+'&($ÿ,-)ÿ%&.'+4*&ÿ4+$'A'$"ÿBDC>ONNE7
`Moreover, NS3 helicase and protease activities are activated
`^-)&-A&)2ÿGHIÿ%&.'+4*&ÿ4(/ÿ1)-$&4*&ÿ4+$'A'$'&*ÿ4)&ÿ4+$'A4$&/
`
`BerzaI-Herranz et at.
`
` ÿ ÿ
`
`by polynucleotides, preferentially by poly(U) [101]. These
`!"ÿ1-."(6+.&-$'/&*2ÿ1)&,&)&($'4.."ÿ!"ÿ1-."@bFÿBONOE7ÿ8%&*&
`observations suggest
`that genomic RNA modulates the
`-!*&)A4$'-(*ÿ*600&*$ÿ$%4$ÿ0&(-5'+ÿUGKÿ5-/6.4$&*ÿ$%&
`interplay between the different functions of NS3 to help form
`'($&)1.4"ÿ!&$3&&(ÿ$%&ÿ/',,&)&($ÿ,6(+$'-(*ÿ-,ÿGHIÿ$-ÿ%&.1ÿ,-)5
`a stable replication complex [101]. Recently, it has also been
`4ÿ*$4!.&ÿ)&1.'+4$'-(ÿ+-51.&QÿBONOE7ÿU&+&($."2ÿ'$ÿ%4*ÿ4.*-ÿ!&&(
`suggested that a relationship exists between the helicase and
`*600&*$&/ÿ$%4$ÿ4ÿ)&.4$'-(*%'1ÿ&Q'*$*ÿ!&$3&&(ÿ$%&ÿ%&.'+4*&ÿ4(/
`protease domains, and that this interaction positively modu-
`1)-$&4*&ÿ/-54'(*2ÿ4(/ÿ$%4$ÿ$%'*ÿ'($&)4+$'-(ÿ1-*'$'A&."ÿ5-/6>
`lates duplex unwinding and viral polyprotein processing
`.4$&*ÿ/61.&Qÿ6(3'(/'(0ÿ4(/ÿA')4.ÿ1-."1)-$&'(ÿ1)-+&**'(0
`[102, 103].
`BONP2ÿONIE7
`
`The above features have led to the proposal that NS3 may
`8%&ÿ4!-A&ÿ,&4$6)&*ÿ%4A&ÿ.&/ÿ$-ÿ$%&ÿ1)-1-*4.ÿ$%4$ÿGHIÿ54"
`be an excellent target for the inactivation of HCV. The
`!&ÿ4(ÿ&Q+&..&($ÿ$4)0&$ÿ,-)ÿ$%&ÿ'(4+$'A4$'-(ÿ-,ÿc\d7ÿ8%&
`existence of
`several domains with different catalytic
`&Q'*$&(+&ÿ-,ÿ*&A&)4.ÿ/-54'(*ÿ3'$%ÿ/',,&)&($ÿ+4$4."$'+
`activities essential for HCV infection provides a good target
`4+$'A'$'&*ÿ&**&($'4.ÿ,-)ÿc\dÿ'(,&+$'-(ÿ1)-A'/&*ÿ4ÿ0--/ÿ$4)0&$
`series for possible drugs or combinations of drugs with
`*&)'&*ÿ,-)ÿ1-**'!.&ÿ/)60*ÿ-)ÿ+-5!'(4$'-(*ÿ-,ÿ/)60*ÿ3'$%
`different specificities.
`/',,&)&($ÿ*1&+','+'$'&*7
`THE NSSB PROTEIN
`efgÿhijkÿlmnegoh
`
`The NSSB protein is a key enzyme in the viral cycle
`8%&ÿGHLpÿ1)-$&'(ÿ'*ÿ4ÿX&"ÿ&(S"5&ÿ'(ÿ$%&ÿA')4.ÿ+"+.&
`because of its RNA-dependent RNA polymerase activity
`!&+46*&ÿ-,ÿ'$*ÿUGK>/&1&(/&($ÿUGKÿ1-."5&)4*&ÿ4+$'A'$"
`(RdRp). This enzyme is required for the synthesis of the
`@U/U1F7ÿ8%'*ÿ&(S"5&ÿ'*ÿ)&?6')&/ÿ,-)ÿ$%&ÿ*"($%&*'*ÿ-,ÿ$%&
`negative and positive RNA strands during the replication of
`(&04$'A&ÿ4(/ÿ1-*'$'A&ÿUGKÿ*$)4(/*ÿ/6)'(0ÿ$%&ÿ)&1.'+4$'-(ÿ-,
`the genome. The NS5B protein is highly conserved across
`$%&ÿ0&(-5&7ÿ8%&ÿGHLpÿ1)-$&'(ÿ'*ÿ%'0%."ÿ+-(*&)A&/ÿ4+)-**
`different HCV genotypes (and their subtypes and isolates),
`/',,&)&($ÿc\dÿ0&(-$"1&*ÿ@4(/ÿ$%&')ÿ*6!$"1&*ÿ4(/ÿ'*-.4$&*F2
`rendering it a potential target for the design of antiviral
`)&(/&)'(0ÿ'$ÿ4ÿ1-$&($'4.ÿ$4)0&$ÿ,-)ÿ$%&ÿ/&*'0(ÿ-,ÿ4($'A')4.
`compounds with minimal side effects (reviewed in [104]).
`+-51-6(/*ÿ3'$%ÿ5'('54.ÿ*'/&ÿ&,,&+$*ÿ@)&A'&3&/ÿ'(ÿBONJEF7
`
`NSSB is produced as part of the polyprotein HCV
`GHLpÿ'*ÿ1)-/6+&/ÿ4*ÿ14)$ÿ-,ÿ$%&ÿ1-."1)-$&'(ÿc\d
`precursor. Once it is processed by the NS3/NS4A complex, it
`1)&+6)*-)7ÿq(+&ÿ'$ÿ'*ÿ1)-+&**&/ÿ!"ÿ$%&ÿGHIRGHJKÿ+-51.&Q2ÿ'$
`is transported to the ER membranes and anchored there
`'*ÿ$)4(*1-)$&/ÿ$-ÿ$%&ÿTUÿ5&5!)4(&*ÿ4(/ÿ4(+%-)&/ÿ$%&)&
`through the highly hydrophobic C-tenninal end,
`the N-
`$%)-60%ÿ$%&ÿ%'0%."ÿ%"/)-1%-!'+ÿ\>$&)5'(4.ÿ&(/2ÿ$%&ÿG>
`terminal domain facing the cytosol [105]. Here, NS5B is
`$&)5'(4.ÿ/-54'(ÿ,4+'(0ÿ$%&ÿ+"$-*-.ÿBONLE7ÿc&)&2ÿGHLpÿ'*
`involved in the recruitment of non-structural HCV proteins
`'(A-.A&/ÿ'(ÿ$%&ÿ)&+)6'$5&($ÿ-,ÿ(-(>*$)6+$6)4.ÿc\dÿ1)-$&'(*
`for
`the
`formation of
`the
`replication
`complex,
`an
`,-)ÿ$%&ÿ,-)54$'-(ÿ-,ÿ$%&ÿ)&1.'+4$'-(ÿ+-51.&Q2ÿ4(
`indispensable step [106-108]. This association of HCV
`'(/'*1&(*4!.&ÿ*$&1ÿBONZ>ONME7ÿ8%'*ÿ4**-+'4$'-(ÿ-,ÿc\d
`structural and non-structural proteins plus genomic RNA
`*$)6+$6)4.ÿ4(/ÿ(-(>*$)6+$6)4.ÿ1)-$&'(*ÿ1.6*ÿ0&(-5'+ÿUGK
`causes a deformation of the lipid layer in the ER membranes
`+46*&*ÿ4ÿ/&,-)54$'-(ÿ-,ÿ$%&ÿ.'1'/ÿ.4"&)ÿ'(ÿ$%&ÿTUÿ5&5!)4(&*
`that can be appreciated by electron microscopy [106, 108,
`$%4$ÿ+4(ÿ!&ÿ411)&+'4$&/ÿ!"ÿ&.&+$)-(ÿ5'+)-*+-1"ÿBONZ2ÿONM2
`109]. This alteration, known as a membranous web,
`is
`ONDE7ÿ8%'*ÿ4.$&)4$'-(2ÿX(-3(ÿ4*ÿ4ÿ5&5!)4(-6*ÿ3&!2ÿ'*
`similar to the “sponge-like inclusions” detected in the liver
`*'5'.4)ÿ$-ÿ$%&ÿr*1-(0&>.'X&ÿ'(+.6*'-(*sÿ/&$&+$&/ÿ'(ÿ$%&ÿ.'A&)
`of infected chimpanzees
`[110]. These macromolecular
`-,ÿ'(,&+$&/ÿ+%'514(S&&*ÿBOONE7ÿ8%&*&ÿ54+)-5-.&+6.4)
`structures are co
`
`by the intrinsic folding of the polyprotein, which brings the
`!"ÿ$%&ÿ'($)'(*'+ÿ,-./'(0ÿ-,ÿ$%&ÿ1-."1)-$&'(2ÿ3%'+%ÿ!)'(0*ÿ$%&
`protease domain and its substrate close together. The trans-
`1)-$&4*&ÿ/-54'(ÿ4(/ÿ'$*ÿ*6!*$)4$&ÿ+.-*&ÿ$-0&$%&)7ÿ8%&ÿ9:;<=>
`reaction has more stringent
`requirements including the
`)&4+$'-(ÿ%4*ÿ5-)&ÿ*$)'(0&($ÿ)&?6')&5&($*ÿ'(+.6/'(0ÿ$%&
`correct positioning of the protease and its polyprotein
`+-))&+$ÿ1-*'$'-('(0ÿ-,ÿ$%&ÿ1)-$&4*&ÿ4(/ÿ'$*ÿ1-."1)-$&'(
`substrate (reviewed in [79]).
`*6!*$)4$&ÿ@)&A'&3&/ÿ'(ÿBCDEF7
`
`The proteolytic processing mediated by NS3 is dependent
`8%&ÿ1)-$&-."$'+ÿ1)-+&**'(0ÿ5&/'4$&/ÿ!"ÿGHIÿ'*ÿ/&1&(/&($
`on NS4A, a small polypeptide composed of only 54
`-(ÿGHJK2ÿ4ÿ*54..ÿ1-."1&1$'/&ÿ+-51-*&/ÿ-,ÿ-(."ÿLJ
`aminoacids [80, 81]. The interaction between them maps to
`45'(-4+'/*ÿBMN2ÿMOE7ÿ8%&ÿ'($&)4+$'-(ÿ!&$3&&(ÿ$%&5ÿ541*ÿ$-
`the 22 residues most N—terminal of the NS3 protein [82, 83]
`$%&ÿPPÿ)&*'/6&*ÿ5-*$ÿG>$&)5'(4.ÿ-,ÿ$%&ÿGHIÿ1)-$&'(ÿBMP2ÿMIE
`and induces several conformational changes affecting the
`4(/ÿ'(/6+&*ÿ*&A&)4.ÿ+-(,-)54$'-(4.ÿ+%4(0&*ÿ4,,&+$'(0ÿ$%&
`catalytic residues, giving rise to a highly stable complex
`+4$4."$'+ÿ)&*'/6&*2ÿ0'A'(0ÿ)'*&ÿ$-ÿ4ÿ%'0%."ÿ*$4!.&ÿ+-51.&Q
`containing NS4A [84]. NS3 has no membrane anchoring
`+-($4'('(0ÿGHJKÿBMJE7ÿGHIÿ%4*ÿ(-ÿ5&5!)4(&ÿ4(+%-)'(0
`domains itself, but its cofactor NS4A is an arnphipathic
`/-54'(*ÿ'$*&.,2ÿ!6$ÿ'$*ÿ+-,4+$-)ÿGHJKÿ'*ÿ4(ÿ451%'14$%'+
`peptide that can interact with membranes via a highly
`1&1$'/&ÿ$%4$ÿ+4(ÿ'($&)4+$ÿ3'$%ÿ5&5!)4(&*ÿA'4ÿ4ÿ%'0%."
`hydrophobic stretch of aminoacids to form a transmembrane
`%"/)-1%-!'+ÿ*$)&$+%ÿ-,ÿ45'(-4+'/*ÿ$-ÿ,-)5ÿ4ÿ$)4(*5&5!)4(&
`helix. This allows the NS3/4A complex to be immobilized
`%&.'Q7ÿ8%'*ÿ4..-3*ÿ$%&ÿGHIRJKÿ+-51.&Qÿ$-ÿ!&ÿ'55-!'.'S&/
`on the surface of the endoplasmic reticulum (ER; [85]), a
`-(ÿ$%&ÿ*6),4+&ÿ-,ÿ$%&ÿ&(/-1.4*5'+ÿ)&$'+6.65ÿ@TUVÿBMLEF2ÿ4
`requirement for the establishment of the replication complex.
`)&?6')&5&($ÿ,-)ÿ$%&ÿ&*$4!.'*%5&($ÿ-,ÿ$%&ÿ)&1.'+4$'-(ÿ+-51.&Q7
`
`The structure of the protease-NS4A complex has been
`8%&ÿ*$)6+$6)&ÿ-,ÿ$%&ÿ1)-$&4*&>GHJKÿ+-51.&Qÿ%4*ÿ!&&(
`resolved by X-ray crystallography. This information has
`)&*-.A&/ÿ!"ÿW>)4"ÿ+)"*$4..-0)41%"7ÿ8%'*ÿ'(,-)54$'-(ÿ%4*
`given rise to intensive efforts to identify potential targets for
`0'A&(ÿ)'*&ÿ$-ÿ'($&(*'A&ÿ&,,-)$*ÿ$-ÿ'/&($',"ÿ1-$&($'4.ÿ$4)0&$*ÿ,-)
`new antiviral drugs. As a result, a chymotrypsin—like folding
`(&3ÿ4($'A')4.ÿ/)60*7ÿK*ÿ4ÿ)&*6.$2ÿ4ÿ+%"5-$)"1*'(>.'X&ÿ,-./'(0
`with an exposed Zn-binding site located on the surface of the
`3'$%ÿ4(ÿ&Q1-*&/ÿY(>!'(/'(0ÿ*'$&ÿ.-+4$&/ÿ-(ÿ$%&ÿ*6),4+&ÿ-,ÿ$%&
`complex was detected [86]. Zinc ions (a requisite for
`+-51.&Qÿ34*ÿ/&$&+$&/ÿBMZE7ÿY'(+ÿ'-(*ÿ@4ÿ)&?6'*'$&ÿ,-)
`catalytic activity; [87]), are coordinated via three cysteine
`+4$4."$'+ÿ4+$'A'$"VÿBMCEF2ÿ4)&ÿ+--)/'(4$&/ÿA'4ÿ$%)&&ÿ+"*$&'(&
`residues in a long loop region that acts as a linker between
`)&*'/6&*ÿ'(ÿ4ÿ.-(0ÿ.--1ÿ)&0'-(ÿ$%4$ÿ4+$*ÿ4*ÿ4ÿ.'(X&)ÿ!&$3&&(
`two B-barrel domains and which contributes to the confor-
`$3-ÿ[>!4))&.ÿ/-54'(*ÿ4(/ÿ3%'+%ÿ+-($)'!6$&*ÿ$-ÿ$%&ÿ+-(,-)>
`mational integrity of the molecule as a whole (reviewed in
`54$'-(4.ÿ'($&0)'$"ÿ-,ÿ$%&ÿ5-.&+6.&ÿ4*ÿ4ÿ3%-.&ÿ@)&A'&3&/ÿ'(
`[88]). This structural feature clearly differentiates NS3/4A
`BMMEF7ÿ8%'*ÿ*$)6+$6)4.ÿ,&4$6)&ÿ+.&4)."ÿ/',,&)&($'4$&*ÿGHIRJK
`from other members of
`the
`serine-protease
`family.
`,)-5ÿ-$%&)ÿ5&5!&)*ÿ-,ÿ$%&ÿ*&)'(&>1)-$&4*&ÿ,45'."7
`Consequently, it is plausible that compounds affecting the
`\-(*&?6&($."2ÿ'$ÿ'*ÿ1.46*'!.&ÿ$%4$ÿ+-51-6(/*ÿ4,,&+$'(0ÿ$%&
`coordination sphere of the metal ions could act as effective
`+--)/'(4$'-(ÿ*1%&)&ÿ-,ÿ$%&ÿ5&$4.ÿ'-(*ÿ+-6./ÿ4+$ÿ4*ÿ&,,&+$'A&
`and specific inhibitors of protease activity. Research into the
`4(/ÿ*1&+','+ÿ'(%'!'$-)*ÿ-,ÿ1)-$&4*&ÿ4+$'A'$"7ÿU&*&4)+%ÿ'($-ÿ$%&
`three dimensional structure of the protease-NS4A complex
`$%)&&ÿ/'5&(*'-(4.ÿ*$)6+$6)&ÿ-,ÿ$%&ÿ1)-$&4*&>GHJKÿ+-51.&Q
`has also contributed to the identification of the substrate-
`%4*ÿ4.*-ÿ+-($)'!6$&/ÿ$-ÿ$%&ÿ'/&($','+4$'-(ÿ-,ÿ$%&ÿ*6!*$)4$&>
`binding site. This is positioned in a wide, very exposed cleft
`!'(/'(0ÿ*'$&7ÿ8%'*ÿ'*ÿ1-*'$'-(&/ÿ'(ÿ4ÿ3'/&2ÿA&)"ÿ&Q1-*&/ÿ+.&,$
`(reviewed in [89]) and can bind a minimum 10 amino acid-
`@)&A'&3&/ÿ'(ÿBMDEFÿ4(/ÿ+4(ÿ!'(/ÿ4ÿ5'('565ÿONÿ45'(-ÿ4+'/>
`long substrate. Competitive inhibitors based on the structure
`.-(0ÿ*6!*$)4$&7ÿ\-51&$'$'A&ÿ'(%'!'$-)*ÿ!4*&/ÿ-(ÿ$%&ÿ*$)6+$6)&
`of the cleavable substrate have been designed (reviewed in
`-,ÿ$%&ÿ+.&4A4!.&ÿ*6!*$)4$&ÿ%4A&ÿ!&&(ÿ/&*'0(&/ÿ@)&A'&3&/ÿ'(
`[90]). The enzyme is also clearly inhibited by its reaction
`BDNEF7ÿ8%&ÿ&(S"5&ÿ'*ÿ4.*-ÿ+.&4)."ÿ'(%'!'$&/ÿ!"ÿ'$*ÿ)&4+$'-(
`products [91], which opens a broad range of possibilities for
`1)-/6+$*ÿBDOE2ÿ3%'+%ÿ-1&(*ÿ4ÿ!)-4/ÿ)4(0&ÿ-,ÿ1-**'!'.'$'&*ÿ,-)
`the design of mimetic peptide drugs.
`$%&ÿ/&*'0(ÿ-,ÿ5'5&$'+ÿ1&1$'/&ÿ/)60*7
`
`The helicase activity of NS3 maps to its C-tenninal end.
`8%&ÿ%&.'+4*&ÿ4+$'A'$"ÿ-,ÿGHIÿ541*ÿ$-ÿ'$*ÿ\>$&)5'(4.ÿ&(/7
`This domain mediates the RNA duplex unwinding promoted
`8%'*ÿ/-54'(ÿ5&/'4$&*ÿ$%&ÿUGKÿ/61.&Qÿ6(3'(/'(0ÿ1)-5-$&/
`by NTP hydrolysis during Viral replication, suggesting that
`!"ÿG8]ÿ%"/)-."*'*ÿ/6)'(0ÿA')4.ÿ)&1.'+4$'-(2ÿ*600&*$'(0ÿ$%4$
`this helicase also has NTPase activity [92]. RNA duplex
`$%'*ÿ%&.'+4*&ÿ4.*-ÿ%4*ÿG8]4*&ÿ4+$'A'$"ÿBDPE7ÿUGKÿ/61.&Q
`*&14)4$'-(ÿ)&?6')&*ÿ^0P_ÿ4(/ÿ'*ÿ*&A&)&."ÿ'(%'!'$&/ÿ!"
`separation requires Mg2+ and is
`severely inhibited by
`monovalent ions [77].
`5-(-A4.&($ÿ'-(*ÿBCCE7
`
`Crystallography studies and mutational assays of the
`\)"*$4..-0)41%"ÿ*$6/'&*ÿ4(/ÿ56$4$'-(4.ÿ4**4"*ÿ-,ÿ$%&
`helicase domain have shown it to have a unique Y shape in
`%&.'+4*&ÿ/-54'(ÿ%4A&ÿ*%-3(ÿ'$ÿ$-ÿ%4A&ÿ4ÿ6('?6&ÿ`ÿ*%41&ÿ'(
`which three domains are defined by each arm of the Y [93-
`3%'+%ÿ$%)&&ÿ/-54'(*ÿ4)&ÿ/&,'(&/ÿ!"ÿ&4+%ÿ4)5ÿ-,ÿ$%&ÿ`ÿBDI>
`97]. Although separated by clefis, the different domains may
`DCE7ÿK.$%-60%ÿ*&14)4$&/ÿ!"ÿ+.&,$*2ÿ$%&ÿ/',,&)&($ÿ/-54'(*ÿ54"
`interact with each other. This finding has led to the design of
`'($&)4+$ÿ3'$%ÿ&4+%ÿ-$%&)7ÿ8%'*ÿ,'(/'(0ÿ%4*ÿ.&/ÿ$-ÿ$%&ÿ/&*'0(ÿ-,
`new helicase inhibitors
`that affect different
`functions
`(&3ÿ%&.'+4*&ÿ'(%'!'$-)*ÿ$%4$ÿ4,,&+$ÿ/',,&)&($ÿ,6(+$'-(*
`simultaneously.
`*'56.$4(&-6*."7
`
`The different NS3 activities show a high level of
`8%&ÿ/',,&)&($ÿGHIÿ4+$'A'$'&*ÿ*%-3ÿ4ÿ%'0%ÿ.&A&.ÿ-,
`interdependence. It seems clear that NTPase capacity is
`'($&)/&1&(/&(+&7ÿa$ÿ*&&5*ÿ+.&4)ÿ$%4$ÿG8]4*&ÿ+414+'$"ÿ'*
`necessary but not sufficient for helicase activity [97-100].
`(&+&**4)"ÿ!6$ÿ(-$ÿ*6,,'+'&($ÿ,-)ÿ%&.'+4*&ÿ4+$'A'$"ÿBDC>ONNE7
`Moreover, NS3 helicase and protease activities are activated
`^-)&-A&)2ÿGHIÿ%&.'+4*&ÿ4(/ÿ1)-$&4*&ÿ4+$'A'$'&*ÿ4)&ÿ4+$'A4$&/
`
`BerzaI-Herranz et at.
`
` ÿ ÿ
`
`by polynucleotides, preferentially by poly(U) [101]. These
`!"ÿ1-."(6+.&-$'/&*2ÿ1)&,&)&($'4.."ÿ!"ÿ1-."@bFÿBONOE7ÿ8%&*&
`observations suggest
`that genomic RNA modulates the
`-!*&)A4$'-(*ÿ*600&*$ÿ$%4$ÿ0&(-5'+ÿUGKÿ5-/6.4$&*ÿ$%&
`interplay between the different functions of NS3 to help form
`'($&)1.4"ÿ!&$3&&(ÿ$%&ÿ/',,&)&($ÿ,6(+$'-(*ÿ-,ÿGHIÿ$-ÿ%&.1ÿ,-)5
`a stable replication complex [101]. Recently, it has also been
`4ÿ*$4!.&ÿ)&1.'+4$'-(ÿ+-51.&QÿBONOE7ÿU&+&($."2ÿ'$ÿ%4*ÿ4.*-ÿ!&&(
`suggested that a relationship exists between the helicase and
`*600&*$&/ÿ$%4$ÿ4ÿ)&.4$'-(*%'1ÿ&Q'*$*ÿ!&$3&&(ÿ$%&ÿ%&.'+4*&ÿ4(/
`protease domains, and that this interaction positively modu-
`1)-$&4*&ÿ/-54'(*2ÿ4(/ÿ$%4$ÿ$%'*ÿ'($&)4+$'-(ÿ1-*'$'A&."ÿ5-/6>
`lates duplex unwinding and viral polyprotein processing
`.4$&*ÿ/61.&Qÿ6(3'(/'(0ÿ4(/ÿA')4.ÿ1-."1)-$&'(ÿ1)-+&**'(0
`[102, 103].
`BONP2ÿONIE7
`
`The above features have led to the proposal that NS3 may
`8%&ÿ4!-A&ÿ,&4$6)&*ÿ%4A&ÿ.&/ÿ$-ÿ$%&ÿ1)-1-*4.ÿ$%4$ÿGHIÿ54"
`be an excellent target for the inactivation of HCV. The
`!&ÿ4(ÿ&Q+&..&($ÿ$4)0&$ÿ,-)ÿ$%&ÿ'(4+$'A4$'-(ÿ-,ÿc\d7ÿ8%&
`existence of
`several domains with different catalytic
`&Q'*$&(+&ÿ-,ÿ*&A&)4.ÿ/-54'(*ÿ3'$%ÿ/',,&)&($ÿ+4$4."$'+
`activities essential for HCV infection provides a good target
`4+$'A'$'&*ÿ&**&($'4.ÿ,-)ÿc\dÿ'(,&+$'-(ÿ1)-A'/&*ÿ4ÿ0--/ÿ$4)0&$
`series for possible drugs or combinations of drugs with
`*&)'&*ÿ,-)ÿ1-**'!.&ÿ/)60*ÿ-)ÿ+-5!'(4$'-(*ÿ-,ÿ/)60*ÿ3'$%
`different specificities.
`/',,&)&($ÿ*1&+','+'$'&*7
`THE NSSB PROTEIN
`efgÿhijkÿlmnegoh
`
`The NSSB protein is a key enzyme in the viral cycle
`8%&ÿGHLpÿ1)-$&'(ÿ'*ÿ4ÿX&"ÿ&(S"5&ÿ'(ÿ$%&ÿA')4.ÿ+"+.&
`because of its RNA-dependent RNA polymerase activity
`!&+46*&ÿ-,ÿ'$*ÿUGK>/&1&(/&($ÿUGKÿ1-."5&)4*&ÿ4+$'A'$"
`(RdRp). This enzyme is required for the synthesis of the
`@U/U1F7ÿ8%'*ÿ&(S"5&ÿ'*ÿ)&?6')&/ÿ,-)ÿ$%&ÿ*"($%&*'*ÿ-,ÿ$%&
`negative and positive RNA strands during the replication of
`(&04$'A&ÿ4(/ÿ1-*'$'A&ÿUGKÿ*$)4(/*ÿ/6)'(0ÿ$%&ÿ)&1.'+4$'-(ÿ-,
`the genome. The NS5B protein is highly conserved across
`$%&ÿ0&(-5&7ÿ8%&ÿGHLpÿ1)-$&'(ÿ'*ÿ%'0%."ÿ+-(*&)A&/ÿ4+)-**
`different HCV genotypes (and their subtypes and isolates),
`/',,&)&($ÿc\dÿ0&(-$"1&*ÿ@4(/ÿ$%&')ÿ*6!$"1&*ÿ4(/ÿ'*-.4$&*F2
`rendering it a potential target for the design of antiviral
`)&(/&)'(0ÿ'$ÿ4ÿ1-$&($'4.ÿ$4)0&$ÿ,-)ÿ$%&ÿ/&*'0(ÿ-,ÿ4($'A')4.
`compounds with minimal side effects (reviewed in [104]).
`+-51-6(/*ÿ3'$%ÿ5'('54.ÿ*'/&ÿ&,,&+$*ÿ@)&A'&3&/ÿ'(ÿBONJEF7
`
`NSSB is produced as part of the polyprotein HCV
`GHLpÿ'*ÿ1)-/6+&/ÿ4*ÿ14)$ÿ-,ÿ$%&ÿ1-."1)-$&'(ÿc\d
`precursor. Once it is processed by the NS3/NS4A complex, it
`1)&+6)*-)7ÿq(+&ÿ'$ÿ'*ÿ1)-+&**&/ÿ!"ÿ$%&ÿGHIRGHJKÿ+-51.&Q2ÿ'$
`is transported to the ER membranes and anchored there
`'*ÿ$)4(*1-)$&/ÿ$-ÿ$%&ÿTUÿ5&5!)4(&*ÿ4(/ÿ4(+%-)&/ÿ$%&)&
`through the highly hydrophobic C-tenninal end,
`the N-
`$%)-60%ÿ$%&ÿ%'0%."ÿ%"/)-1%-!'+ÿ\>$&)5'(4.ÿ&(/2ÿ$%&ÿG>
`terminal domain facing the cytosol [105]. Here, NS5B is
`$&)5'(4.ÿ/-54'(ÿ,4+'(0ÿ$%&ÿ+"$-*-.ÿBONLE7ÿc&)&2ÿGHLpÿ'*
`involved in the recruitment of non-structural HCV proteins
`'(A-.A&/ÿ'(ÿ$%&ÿ)&+)6'$5&($ÿ-,ÿ(-(>*$)6+$6)4.ÿc\dÿ1)-$&'(*
`for
`the
`formation of
`the
`replication
`complex,
`an
`,-)ÿ$%&ÿ,-)54$'-(ÿ-,ÿ$%&ÿ)&1.'+4$'-(ÿ+-51.&Q2ÿ4(
`indispensable step [106-108]. This association of HCV
`'(/'*1&(*4!.&ÿ*$&1ÿBONZ>ONME7ÿ8%'*ÿ4**-+'4$'-(ÿ-,ÿc\d
`structural and non-structural proteins plus genomic RNA
`*$)6+$6)4.ÿ4(/ÿ(-(>*$)6+$6)4.ÿ1)-$&'(*ÿ1.6*ÿ0&(-5'+ÿUGK
`causes a deformation of the lipid layer in the ER membranes
`+46*&*ÿ4ÿ/&,-)54$'-(ÿ-,ÿ$%&ÿ.'1'/ÿ.4"&)ÿ'(ÿ$%&ÿTUÿ5&5!)4(&*
`that can be appreciated by electron microscopy [106, 108,
`$%4$ÿ+4(ÿ!&ÿ411)&+'4$&/ÿ!"ÿ&.&+$)-(ÿ5'+)-*+-1"ÿBONZ2ÿONM2
`109]. This alteration, known as a membranous web,
`is
`ONDE7ÿ8%'*ÿ4.$&)4$'-(2ÿX(-3(ÿ4*ÿ4ÿ5&5!)4(-6*ÿ3&!2ÿ'*
`similar to the “sponge-like inclusions” detected in the liver
`*'5'.4)ÿ$-ÿ$%&ÿr*1-(0&>.'X&ÿ'(+.6*'-(*sÿ/&$&+$&/ÿ'(ÿ$%&ÿ.'A&)
`of infected chimpanzees
`[110]. These macromolecular
`-,ÿ'(,&+$&/ÿ+%'514(S&&*ÿBOONE7ÿ8%&*&ÿ54+)-5-.&+6.4)
`structures are co
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