New insights regarding HCV-NS5A structure/function and indication of genotypic differences.

0.43 MB·English

by  Yamasaki, Lilian HT

Yamasakietal.VirologyJournal2012,9:14
http://www.virologyj.com/content/9/1/14
RESEARCH Open Access
New insights regarding HCV-NS5A structure/
function and indication of genotypic differences
Lilian HT Yamasaki1,4*, Helen A Arcuri2, Ana Carolina G Jardim1, Cintia Bittar1, Isabel Maria VG de Carvalho-Mello3
and Paula Rahal1
Abstract
Background: HCV is prevalent throughout the world. It is a major cause of chronic liver disease. There is no
effective vaccine and the most common therapy, based on Peginterferon, has a success rate of ~50%. The
mechanisms underlying viral resistance have not been elucidated but it has been suggested that both host and
virus contribute to therapy outcome. Non-structural 5A (NS5A) protein, a critical virus component, is involved in
cellular and viral processes.
Methods: The present study analyzed structural and functional features of 345 sequences of HCV-NS5A genotypes
1 or 3, using in silico tools.
Results: There was residue type composition and secondary structure differences between the genotypes. In
addition, second structural variance were statistical different for each response group in genotype 3. A motif search
indicated conserved glycosylation, phosphorylation and myristoylation sites that could be important in structural
stabilization and function. Furthermore, a highly conserved integrin ligation site was identified, and could be linked
to nuclear forms of NS5A. ProtFun indicated NS5A to have diverse enzymatic and nonenzymatic activities,
participating in a great range of cell functions, with statistical difference between genotypes.
Conclusion: This study presents new insights into the HCV-NS5A. It is the first study that using bioinformatics
tools, suggests differences between genotypes and response to therapy that can be related to NS5A protein
features. Therefore, it emphasizes the importance of using bioinformatics tools in viral studies. Data acquired herein
will aid in clarifying the structure/function of this protein and in the development of antiviral agents.
Keywords: Hepatitis C virus, Non-structural 5A protein, Bioinformatics, Genotype, Quasispecies, IFN response
Introduction addition, an infected patient will harbour different but
Hepatitis C is a major health problem; it is highly preva- related viral genomes known as quasispecies. This high
lent worldwide and has a high probability of persistence variability can be explained by a combination of three
[1,2]. Chronic persistence can result in cirrhosis and factors: (1) viral RNA polymerase acts without proof-
hepatocellular carcinoma [3,4]. Hepatitis C virus (HCV) reading [7]; (2) HCV has co-evolved with human popu-
is member of the Flaviviridae family within the Hepaci- lations for millions of years [8]; (3) the viral life cycle is
virus genus, although many of its features are distinct fast, resulting in the production of approximately 1.3 ×
from other family members including the structural 1012 virions per patient per day [9].
organization of the protein and the 5’-cap independent The HCV RNA genome translates a polyprotein that
translation [5]. is cleaved by viral and host proteases to generate ten
On the basis of viral variability, HCV is classified into structural and non-structural proteins [10-12]. Among
seven genotypes and more than 50 subtypes [6]. In the non-structural proteins, NS5A is a phosphoprotein
critical for the HCV life cycle. It is composed of
approximately 447 amino acids and may participate in
*Correspondence:[email protected]
1DepartmentofBiology,SaoPauloStateUniversity–UNESP,SaoJosedoRio viral RNA replication, modulation of cell signaling path-
Preto,SP,Brazil ways, interferon response, pathogenesis and apoptosis
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regulation. Its enzymatic functions and its complete resulted in different variation behaviour comparing the
structure have yet to be elucidated. However, evidence threeoutcomeresponsessequencesingenotype3(Figure
suggests that it functions through interaction with other 2A-C).Thesametestappliedforanalysisintra-response
HCV and host cell proteins [13-16]. group indicates that even when sequences are from dif-
NS5A is divided into three domains [17,18]. Domain I ferent patients, if they are from the same therapy out-
contains a membrane binding domain [19,20] and a zinc come group, they are notsignificantly different (p >.05)
finger domain that are essential for HCV replication (Table1).
[17]. Domain II and III are naturally unstructured, per-
forming function by interacting with several proteins Transmembrane regions and pattern/motif search
[15,21]. MEMSAT3 analysis demonstrated that all NS5A and
The importance of NS5A protein in disease caused by reference sequences contained a possible transmem-
hepatitis C is unquestioned. However, difficulties with brane region between residues 32 and 51, and Prosite
experimental methods used to determine the structures recognized seven functional patterns in each NS5A
of highly flexible proteins have resulted in a poor under- sequence. No relationship was observed between therapy
standing of the overall structure and functions of NS5A. response or genotype and pattern number or motif loca-
Such difficulties have led to the development of bioin- tion. Table 2 presents a summary of each conserved pat-
formatic tools that are helpful in obtaining reliable data tern/motif encountered in the NS5A analysis. Between
for these types of proteins. Ab initio tools are also two and seven N-glycosylation sites were present within
important for studying proteins with low or no homol- the same sequence. In two positions (69 and 268), this
ogy, and can be used to compare them with experimen- pattern was conserved in 90% of sequences; this motif
tally determined structures. was absent at these positions in 4% of cases. cAMP- and
In the present study, complete sequences from HCV cGMP-dependent protein kinase phosphorylation sites
NS5A genotypes 1 and 3 were analyzed. These were identified; 90% of sequences had a minimum of
sequences were obtained from Brazilian patients who one such motif at position 357, but 4% of sequences
showed different responses to Peginterferon (PegIFN) from one patient lacked this motif. Protein kinase C
therapy. Using these sequences, the aims were to ana- (PKC) phosphorylation sites were present in all
lyze structural and functional features. The knowledge sequences at positions 71 and 238. The number of these
obtained should aid in the design of new drugs and vac- sites in the same sequence ranged from five to eight.
cines, and in developing other resources to improve Casein kinase II phosphorylation sites were recognized
HCV therapy. in non-conserved positions, being present between 4
and 8 times in the same sequence. Tyrosine kinase
Results phosphorylation sites were identified in a conserved
Amino acid composition and secondary structure of NS5A position (residue 122 or 123). N-myristoylation sites
Therapy response did not differ according to the amino were present in various numbers and several positions,
acidscomposition or the secondary structure type com- with NS5A sequences possessing between four and eight
position. However, considering the genotypes, the aver- of these motifs in the same sequence. A cell attachment
agepercentagesofalanine,glutamicacid,glutamineand sequence (RGD sequence) was present in all sequences,
tyrosine present in the NS5A protein were different conserved in number and position; NS5A sequences
betweenthegenotypes1a,1band3.Theaveragepercen- possessed this motif at position 48. Figure 3 summarizes
tages of cysteine, valine and threonine differed between the results of the MEMSAT3 and Prosite analyses in the
genotype1and3.Allsequencesobtainedfromgenotype reference sequence AF009606.
1bpresentedwith2%tyrosine(datanotshown).Second-
ary structure analysis demonstrated that distribution of Prediction of functional features
each secondary structure type followed a normal distri- ProtFun analysis results are detailed in Table 3. It indi-
bution. Statistical analysis(t-test)suggeststhatthethree cated that the complete NS5A has properties similar to
secondary arrangements are high significant factors (p- proteins which play role in regulatory functions, replica-
value < .001) to differ the genotypes. Composition of tion and transcription, central intermediary metabolism
helix,sheetorcoilinpercentagedidnotresultinsignifi- or purines/pyrimidines. All sequences were classified as
cant difference when we compared these three arrange- an enzyme of unknown class, with the transcription reg-
ments the therapy outcome responses (Figure 1A-C). In ulation as the gene ontology category. Calculated chi-
contrast,whenwecomparedthevariationofthesecom- square test showed significant difference (p < 0.05)
positions among the response groups, there is a great between therapy outcome groups and between geno-
(andstatistical)difference,especiallyingenotype3.Test types related to functional category predicted, with sta-
for equal variances (Bartlett’s test and Levene’s test) tistical power level varying from 0.617 to 1.
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A (cid:43)(cid:76)(cid:86)(cid:87)(cid:82)(cid:74)(cid:85)(cid:68)(cid:80)(cid:3)(cid:82)(cid:73)(cid:3)(cid:302)(cid:68)(cid:16) (cid:43)(cid:72)(cid:79)(cid:76)(cid:91)
(cid:49)(cid:82)(cid:85)(cid:80)(cid:68)(cid:79)(cid:3)
(cid:20)(cid:23) (cid:20)(cid:25) (cid:20)(cid:27) (cid:21)(cid:19) (cid:21)(cid:21) (cid:21)(cid:23) (cid:21)(cid:25) (cid:21)(cid:27)
(cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:20) (cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:22) (cid:40)(cid:55)(cid:53)
(cid:19)(cid:17)(cid:26) (cid:49)(cid:53)
(cid:53)
(cid:19)(cid:17)(cid:25)
(cid:19)(cid:17)(cid:24)
(cid:92)
(cid:81)(cid:86)(cid:76)(cid:87) (cid:19)(cid:17)(cid:23)
(cid:72)
(cid:39) (cid:19)(cid:17)(cid:22)
(cid:19)(cid:17)(cid:21)
(cid:19)(cid:17)(cid:20)
(cid:19)(cid:17)(cid:19)
(cid:20)(cid:23) (cid:20)(cid:25) (cid:20)(cid:27) (cid:21)(cid:19) (cid:21)(cid:21) (cid:21)(cid:23) (cid:21)(cid:25) (cid:21)(cid:27)
(cid:68)(cid:16)(cid:43)(cid:72)(cid:79)(cid:76)(cid:91)(cid:3)(cid:11)(cid:8)(cid:3)(cid:76)(cid:81)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:83)(cid:85)(cid:82)(cid:87)(cid:72)(cid:76)(cid:81)(cid:12)
B (cid:43)(cid:76)(cid:86)(cid:87)(cid:82)(cid:74)(cid:85)(cid:68)(cid:80)(cid:3)(cid:82)(cid:73)(cid:3)(cid:137)(cid:3)(cid:86)(cid:75)(cid:72)(cid:72)(cid:87)
(cid:49)(cid:82)(cid:85)(cid:80)(cid:68)(cid:79)(cid:3)
(cid:20)(cid:26) (cid:20)(cid:27) (cid:20)(cid:28) (cid:21)(cid:19) (cid:21)(cid:20) (cid:21)(cid:21)
(cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:20) (cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:22) (cid:40)(cid:55)(cid:53)
(cid:22)(cid:17)(cid:19) (cid:49)(cid:53)
(cid:53)
(cid:21)(cid:17)(cid:24)
(cid:21)(cid:17)(cid:19)
(cid:92)
(cid:72)(cid:81)(cid:86)(cid:76)(cid:87) (cid:20)(cid:17)(cid:24)
(cid:39)
(cid:20)(cid:17)(cid:19)
(cid:19)(cid:17)(cid:24)
(cid:19)(cid:17)(cid:19)
(cid:20)(cid:26) (cid:20)(cid:27) (cid:20)(cid:28) (cid:21)(cid:19) (cid:21)(cid:20) (cid:21)(cid:21)
(cid:137)(cid:3)(cid:86)(cid:75)(cid:72)(cid:72)(cid:87)(cid:3)(cid:11)(cid:8)(cid:3)(cid:76)(cid:81)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:83)(cid:85)(cid:82)(cid:87)(cid:72)(cid:76)(cid:81)(cid:12)
C (cid:43)(cid:76)(cid:86)(cid:87)(cid:82)(cid:74)(cid:85)(cid:68)(cid:80)(cid:3)(cid:82)(cid:73)(cid:3)(cid:38)(cid:82)(cid:76)(cid:79)
(cid:49)(cid:82)(cid:85)(cid:80)(cid:68)(cid:79)(cid:3)
(cid:24)(cid:20) (cid:24)(cid:23) (cid:24)(cid:26) (cid:25)(cid:19) (cid:25)(cid:22) (cid:25)(cid:25) (cid:25)(cid:28)
(cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:20) (cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:22) (cid:40)(cid:55)(cid:53)
(cid:19)(cid:17)(cid:28) (cid:49)(cid:53)
(cid:53)
(cid:19)(cid:17)(cid:27)
(cid:19)(cid:17)(cid:26)
(cid:19)(cid:17)(cid:25)
(cid:92)
(cid:81)(cid:86)(cid:76)(cid:87) (cid:19)(cid:17)(cid:24)
(cid:72)
(cid:39) (cid:19)(cid:17)(cid:23)
(cid:19)(cid:17)(cid:22)
(cid:19)(cid:17)(cid:21)
(cid:19)(cid:17)(cid:20)
(cid:19)(cid:17)(cid:19)
(cid:24)(cid:20) (cid:24)(cid:23) (cid:24)(cid:26) (cid:25)(cid:19) (cid:25)(cid:22) (cid:25)(cid:25) (cid:25)(cid:28)
(cid:38)(cid:82)(cid:76)(cid:79)(cid:3)(cid:11)(cid:8)(cid:3)(cid:76)(cid:81)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:83)(cid:85)(cid:82)(cid:87)(cid:72)(cid:76)(cid:81)(cid:12)
Figure1StatisticalanalysisinsecondStructurecomposition(%)distributionoftheNS5Asequences.Plot:Differenceinhelix(A),sheet
(B)andcoil(C)percentageintheNS5Aproteinbetweengenotype1and3/therapyoutcome.X-axisdescribesthepercentageofeachsecond
structuretypeintotalprotein;y-axismeansdensity(numberofsequenceswithcorrespondpercentage/totalnumberofsequences)ETR-Endof
therapyresponder,NR-nonresponder,R-responder.
The results from NS5A domain I suggested that this varied among transcription regulation, growth factor,
region is related to the functional category of energy immune response, transcription and none of these cate-
metabolism, with enzymatic activity from unknown gories. To calculate the chi-square, categories below 5
class. For domain I, prediction of gene ontology category sequences were excluded. Significant difference was
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A (cid:55)(cid:72)(cid:86)(cid:87)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:40)(cid:84)(cid:88)(cid:68)(cid:79)(cid:3)(cid:57)(cid:68)(cid:85)(cid:76)(cid:68)(cid:81)(cid:70)(cid:72)(cid:86)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:302)(cid:68) (cid:16)(cid:43)(cid:72)(cid:79)(cid:76)(cid:91)(cid:3)(cid:76)(cid:81)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:22)
(cid:37)(cid:68)(cid:85)(cid:87)(cid:79)(cid:72)(cid:87)(cid:87)(cid:10)(cid:86)(cid:3)(cid:55)(cid:72)(cid:86)(cid:87)
(cid:55)(cid:72)(cid:86)(cid:87)(cid:3)(cid:54)(cid:87)(cid:68)(cid:87)(cid:76)(cid:86)(cid:87)(cid:76)(cid:70) (cid:27)(cid:26)(cid:17)(cid:19)(cid:24)
(cid:49)(cid:53) (cid:51)(cid:16)(cid:57)(cid:68)(cid:79)(cid:88)(cid:72) (cid:19)(cid:17)(cid:19)(cid:19)(cid:19)
(cid:47)(cid:72)(cid:89)(cid:72)(cid:81)(cid:72)(cid:10)(cid:86)(cid:3)(cid:55)(cid:72)(cid:86)(cid:87)
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(cid:51)(cid:16)(cid:57)(cid:68)(cid:79)(cid:88)(cid:72) (cid:19)(cid:17)(cid:19)(cid:19)(cid:22)
(cid:80)(cid:72)
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(cid:50)
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(cid:28)(cid:24)(cid:8)(cid:3)(cid:37)(cid:82)(cid:81)(cid:73)(cid:72)(cid:85)(cid:85)(cid:82)(cid:81)(cid:76)(cid:3)(cid:38)(cid:82)(cid:81)(cid:73)(cid:76)(cid:71)(cid:72)(cid:81)(cid:70)(cid:72)(cid:3)(cid:44)(cid:81)(cid:87)(cid:72)(cid:85)(cid:89)(cid:68)(cid:79)(cid:86)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:54)(cid:87)(cid:39)(cid:72)(cid:89)(cid:86)
B (cid:55)(cid:72)(cid:86)(cid:87)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:40)(cid:84)(cid:88)(cid:68)(cid:79)(cid:3)(cid:57)(cid:68)(cid:85)(cid:76)(cid:68)(cid:81)(cid:70)(cid:72)(cid:86)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:137)(cid:3)(cid:86)(cid:75)(cid:72)(cid:72)(cid:87)(cid:3)(cid:76)(cid:81)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:22)
(cid:37)(cid:68)(cid:85)(cid:87)(cid:79)(cid:72)(cid:87)(cid:87)(cid:10)(cid:86)(cid:3)(cid:55)(cid:72)(cid:86)(cid:87)
(cid:55)(cid:72)(cid:86)(cid:87)(cid:3)(cid:54)(cid:87)(cid:68)(cid:87)(cid:76)(cid:86)(cid:87)(cid:76)(cid:70) (cid:20)(cid:25)(cid:17)(cid:22)(cid:23)
(cid:49)(cid:53) (cid:51)(cid:16)(cid:57)(cid:68)(cid:79)(cid:88)(cid:72) (cid:19)(cid:17)(cid:19)(cid:19)(cid:19)
(cid:47)(cid:72)(cid:89)(cid:72)(cid:81)(cid:72)(cid:10)(cid:86)(cid:3)(cid:55)(cid:72)(cid:86)(cid:87)
(cid:55)(cid:72)(cid:86)(cid:87)(cid:3)(cid:54)(cid:87)(cid:68)(cid:87)(cid:76)(cid:86)(cid:87)(cid:76)(cid:70) (cid:23)(cid:17)(cid:23)(cid:19)
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(cid:80)(cid:72)
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(cid:28)(cid:24)(cid:8)(cid:3)(cid:37)(cid:82)(cid:81)(cid:73)(cid:72)(cid:85)(cid:85)(cid:82)(cid:81)(cid:76)(cid:3)(cid:38)(cid:82)(cid:81)(cid:73)(cid:76)(cid:71)(cid:72)(cid:81)(cid:70)(cid:72)(cid:3)(cid:44)(cid:81)(cid:87)(cid:72)(cid:85)(cid:89)(cid:68)(cid:79)(cid:86)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:54)(cid:87)(cid:39)(cid:72)(cid:89)(cid:86)
C (cid:55)(cid:72)(cid:86)(cid:87)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:40)(cid:84)(cid:88)(cid:68)(cid:79)(cid:3)(cid:57)(cid:68)(cid:85)(cid:76)(cid:68)(cid:81)(cid:70)(cid:72)(cid:86)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:82)(cid:76)(cid:79)(cid:3)(cid:76)(cid:81)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:42)(cid:72)(cid:81)(cid:82)(cid:87)(cid:92)(cid:83)(cid:72)(cid:3)(cid:22)
(cid:37)(cid:68)(cid:85)(cid:87)(cid:79)(cid:72)(cid:87)(cid:87)(cid:10)(cid:86)(cid:3)(cid:55)(cid:72)(cid:86)(cid:87)
(cid:55)(cid:72)(cid:86)(cid:87)(cid:3)(cid:54)(cid:87)(cid:68)(cid:87)(cid:76)(cid:86)(cid:87)(cid:76)(cid:70) (cid:20)(cid:19)(cid:27)(cid:17)(cid:22)(cid:25)
(cid:49)(cid:53) (cid:51)(cid:16)(cid:57)(cid:68)(cid:79)(cid:88)(cid:72) (cid:19)(cid:17)(cid:19)(cid:19)(cid:19)
(cid:47)(cid:72)(cid:89)(cid:72)(cid:81)(cid:72)(cid:10)(cid:86)(cid:3)(cid:55)(cid:72)(cid:86)(cid:87)
(cid:55)(cid:72)(cid:86)(cid:87)(cid:3)(cid:54)(cid:87)(cid:68)(cid:87)(cid:76)(cid:86)(cid:87)(cid:76)(cid:70) (cid:20)(cid:20)(cid:17)(cid:20)(cid:24)
(cid:51)(cid:16)(cid:57)(cid:68)(cid:79)(cid:88)(cid:72) (cid:19)(cid:17)(cid:19)(cid:19)(cid:19)
(cid:80)(cid:72)
(cid:70)(cid:82) (cid:53)
(cid:88)(cid:87)
(cid:50)
(cid:40)(cid:55)(cid:53)
(cid:19)(cid:17)(cid:24) (cid:20)(cid:17)(cid:19) (cid:20)(cid:17)(cid:24) (cid:21)(cid:17)(cid:19) (cid:21)(cid:17)(cid:24)
(cid:28)(cid:24)(cid:8)(cid:3)(cid:37)(cid:82)(cid:81)(cid:73)(cid:72)(cid:85)(cid:85)(cid:82)(cid:81)(cid:76)(cid:3)(cid:38)(cid:82)(cid:81)(cid:73)(cid:76)(cid:71)(cid:72)(cid:81)(cid:70)(cid:72)(cid:3)(cid:44)(cid:81)(cid:87)(cid:72)(cid:85)(cid:89)(cid:68)(cid:79)(cid:86)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:54)(cid:87)(cid:39)(cid:72)(cid:89)(cid:86)
Figure2Statisticalanalysisinsecondstructurecomposition(%)varianceoftheNS5Agenotype3sequences.Plot:Statisticalresultsfor
helix(A),sheet(B)andcoil(C)varianceingenotype3.Squareintheleftshowsthecalculatedvaluesforeachtest.Notethevariancedifferent
distributionineachresponsegroup.StDev-standarddeviation.
observed between genotypes but not between therapy between therapy outcome groups (except between non-
response groups, with statistical power of 0.999. responders–NR and end of therapy responders- ETR
Prediction for domain II sequences varied in func- groups) and genotype. Enzymatic function prediction
tional category, enzymatic function and gene ontology and gene ontology (GO) category prediction had signifi-
category. Resulted functional category includes energy cant difference between genotypes, with statistical power
metabolism and translation, with statistical difference of 0.801 and 1, respectively.
Yamasakietal.VirologyJournal2012,9:14 Page5of10
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Table 1Statistical results(t-test) forcomparison between IFN therapy. Viral kinetics was greatly different. In gen-
genotype 1and 3secondary structurecomposition otype 1 infected patients, IFN effectiveness, free virion
Genotype No.ofsequences(n) Mean StDev SEMean p-value clearance rate and cell death rate were lower than geno-
a-helix type 2 HCV hosts. In contrast, percentage of individuals
1 109 18.06 0.52 0.05 0.00 that reached an undetectable level during 14 days of
3 143 17.51 0.41 0.03 therapy was higher in genotype 2 infected individuals
b-sheet [23]. Our group also found in vivo indication of diver-
gences in NS5A quasispecies composition and muta-
1 109 19.58 0.27 0.03 0.00
tional profiles between genotypes 1 and 3 in baseline
3 143 18.61 0.33 0.03
specimens [24,25]. Using the sequences derived from
Coil
these studies, we showed that there is indication of
1 109 62.35 0.52 0.05 0.00
structural and functional differences between NS5A-1a/
3 143 63.88 0.38 0.03
b and NS5A-3 (Table 3). In addition, at least for geno-
StDev:StandardDeviation,SE:StandardError
type 3, there were differences in variance of structure
between the different responses groups (Figure 2). This
For domain III analysis, all the sequences were pre- divergence is not observed when we compare the
dicted to be similar to proteins related to transport and sequences extracted from patients with the same therapy
binding, with nonenzyme function. The gene ontology response. Possibly resulting from the structural differ-
category varied among growth factor, immune response, ences, the functional prediction profile was also different
stress response, hormone, voltage-gated ion channel and between genotypes.
unknown. These differences were significant different Invivo and derived in silico results pointto a relation-
between end of therapy responders (ETR) sequences ship between NS5A and genotypic IFN response rate.
and the other two outcome groups (non-responders– However,invitroresearchesresultsarestillcontroversial.
NR, responders–R) and between genotypes. Statistical In 2008, it was reported that cells infected by 2a-NS5A-
power values were 0.6 and 1, respectively. containingrepliconspresentedlowerdegreeofIFNantag-
onism than the counterpart containing 1b-NS5A. The
Discussion same study also found suggestions that the V3 domain
Infection with genotype 1 results in lower therapy suc- andtheC-terminalregionoftheNS5AarerelatedtoIFN
cess rates than other genotypes [22]. No previous study differential reaction [26]. Other study in 2010 did not
was found connecting NS5A amino acids composition reachedsimilarconclusions. Inthiscase,cellsexpressing
or secondary structure type (Figure 1, 2 and Table 1) to 1band2aNS5Aproteinpresentedanalogouscapabilityof
this difference in response rate. The present study sug- IFN responses inhibition and IL-8 expression induction.
gests that these two characteristics present genotypic The author suggests that other viral factors and/or host
differences. These differences could affect NS5A func- factors may be involved in the genotypic difference of
tions, by modifying its interactions with other viral and HCV[27].Posterior,itwasalsodescribedthatHCVwith
host proteins, or its stability. Consequently, these differ- recombinantNS5Afromdifferentgenotypespresentsdif-
ences could affect viral resistance, replication and other ferentsensibilitytoNS5Ainhibitors,butnotforIFN[28].
properties linked to NS5A that differ among genotypes. Thesehypothesisdoessupportourgroupresults,sinceit
Indeed, some observations of the viral genotype speci- seemsthatNS5Ahasstillnumerousunknowndirectand
fic features are reported between genotype 1 and 2. indirect interactions with host and viral proteins, conse-
Viral dynamics was the first property detected, in a quence from the high structural flexibility observed in
study that collected viral load data in patients receiving domainsIIandIIIoftheprotein[15,21].
Table 2Conservation ofpatterns/motifs predictedon NS5A,described inpercentage ofsequences
Motif/patternname Presence ConservedPositions No.ofsitesinsamesequence
N-glycosylation 95,65% 69,268(90%) Twouptosix
cAMP/cGMP-dependentphosphorylation 95,65% 357(90,14%) Oneuptotwo
PKCphosphorylation 100% 71,238(98,84%) Fiveuptoeight
CKIIphosphorylation 100% - Fouruptoeight
Tyrosinekinasephosphorylation 99,99% 122or123(99,99%) Fouruptoeight
N-myristoylation 100% - Fouruptoeight
Cellattachmentsequence 100% 48(100%) 1
Yamasakietal.VirologyJournal2012,9:14 Page6of10
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Figure3ResultsofMEMSAT3andProsite.SummaryforNS5AreferenceAF009606.TM:transmembraneregion,CRS:cytoplasmicretention
site,PKR-bd:PKRbindingsite,NLS:nuclearlocalizationsite,V3:variableregion3.Lines-purple:n-glycosylationsites,blue:phosphorylationsites,
green:n-myristoylationsites,orange:RGDsite.
High structural flexibility is the key of the multifunc- shows a high percentage of coils in NS5A, which results
tionality in promiscuous proteins [29]. The NS5A pro- in the high structural flexibility of the NS5A. In addition
tein, as other promiscuous proteins, presents an with ProtFun predictions, we observed that NS5A may
intrinsic disorder. Recent studies showed that the have genotypic differences in performed functions, vary-
domain III can be unfold or partially fold in helix [21], ing also between the domains, with a great statistical
another feature of promiscuous proteins, which can pre- difference (p < .001) and power (> .95) (Table 3).
sent different conformations depending on the interac- Despite of the essential functions for HCV infection,
tions made [29]. Our secondary structure prediction NS5A from different genotypes may have different
Table 3Statistical resultsfor comparison between therapy outcome andgenotype inProtFun analysis
Region Feature Comparison Chi-square p-value Statpower
NS5A Functionalcategory Outcome 18.832 0.004 0.711
RandNR 10.697 0.013 0.617
RandETR 15.279 0.002 0.627
NRandETR 5.103 0.164 0.676
Genotype 218.730 0.000 1
DomainI GO* Outcome 1.370 0.504 0.694
Genotype 51.316 0.000 0.999
DomainII Functionalcategory Outcome 27.769 0.000 0.861
RandNR 15.903 0.000 0.881
RandETR 16.776 0.000 0.581
NRandETR 0.241 0.623 0.663
Genotype 21.755 0.000 0.780
Non/enzyme Genotype 22.711 0.000 0.801
GO Outcome 1.792 0.408 0.665
Genotype 116.636 0.000 1
DomainIII GO* Outcome 24.680 0.000 0.572
RandNR 5.488 0.064 0.587
RandETR 11.307 0.010 0.616
ETRandNR 12.374 0.006 0.609
Genotype 54.217 0.000 0.998
*values<05wereexcludedforthestatisticalcalculation
Yamasakietal.VirologyJournal2012,9:14 Page7of10
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secondary functions. These functions may lead directly myristoylation in the NS5A protein. However, we sug-
or indirectly to the different SVR rate in genotypes. gest that this process is important in structural/func-
Also, it would explain why NS5A from different geno- tional stabilization of NS5A. If experimental data
types have different behaviour when the mutational and demonstrated that these modifications are present in
quasispecies profile are analysed [24,25]. NS5A, these sites could be possible targets for new anti-
Prosite analysis demonstrated the presence of several viral agents.
potential co-and post-translational modification sites Interestingly, a cell attachment site (RGD) was present
that are well conserved in the sequence, including N- in all sequences between residues 48 and 52. This region
glycosylation sites (Figure 3 and Table 2). Carbohydrate is inside the trasmembrane region predicted by MEM-
binding can confer a different function on a protein. For SAT3. RGD is a sequence for interaction with integrins,
example, it can lead to the addition of epitopes that proteins located on the cell surface that act on cell-cell
facilitate the recognition of other proteins [30,31]. There and cell-extracellular matrix interactions [44]. Although
are no studies that describe glycosylation of NS5A, but the intracellular functions of RGD require further inves-
in some viruses, glycosylated proteins can be essential tigation, studies concerning proprotein convertase 1
for viral assembly [32]. Helenius (1994) demonstrated showed that this sequence is essential for correct folding
that glycosylation promoted an increase in solubility, in the endoplasmatic reticulum and transport to secre-
and possibly in interactions with chaperones on the tory glands [45].
endoplasmatic reticulum, thereby affecting folding and Micelles with cyclic RGD peptides transfected into
stabilization of the protein. Proteins without this modifi- HeLa cells tend to congregate in the perinuclear region
cation could assemble in a non-reversable form or exit [46]. Therefore, the RGD sequence in NS5A genotypes
the endoplasmatic reticulum [33]. N- and O-linked gly- 1 and 3 could have a role in (1) folding and intracellular
cosylation are also described in non-reticulum compart- transport and/or (2) nuclear and perinuclear localiza-
ments, such as nucleus and cytosol [34]. Since this tion. The NS5A protein has a functional nuclear loca-
discover, several cytosolic proteins involved in the pro- tion signal (NLS) at its carboxy terminal [47]. The
cess of adding carbohydrate to proteins were character- complete protein form is predominantly located in the
ized [35,36]. Proteins which undergo these unusual cytoplasm and/or in the perinuclear region [47,48].
glycosylation processes are linked with several functions, However, forms in which the NS5A amino terminal
including nuclear membrane structure and transcription region (residues 1-31) has been deleted predominate in
factors [37]. the nucleus [49]. These deleted forms occur naturally
Following this hypothesis, NS5A glycosylation may be during infection, resulting in cell caspase activity [50,51].
essential for maintaining its functional structure, as The function of these nuclear forms requires further
these modifications sequence appear to be conserved. In study but they have been shown to be transcriptional
addition, this modification may play an important role regulators [52-54]. Furthermore, these forms can trans-
in nuclear localization of NS5A mutants. port other proteins complexed with NS5A. The c-Raf
Potential phosphorylation sites were identified in protein interacts with the NS5A carboxy terminal and is
NS5A. This modification has been experimentally detected in the cell nucleus with these deleted forms of
described and is important for interaction with core the NS5A protein [54].
proteins and for viral assembly [38]. Phosphorylation is Regarding these studies, we suggest that the factors
a reversible modification process, and may be key to the participating in NS5A nuclear localization are (1) dele-
multifunctionality of NS5A. Several proteins were identi- tion of the amino terminal region, which inhibits the
fied as playing a role in NS5A phosphorylation including NLS region; (2) presence of the NLS without mutations;
AKT, p70s6K, MEK, MKK1, CKI, CKIIe and Syk [39-41] (3) possible interactions between RGD and proteins
but we found no study has described phosphorylation by related to transport through the nuclear envelope (4)
PKC or cAMP-/cGMP-dependent protein kinase. These possible glycosylation of NS5A similar to other nuclear
proteins are still candidates for this process, as details functional proteins.
concerning NS5A phosphorylation have yet to be fully It is important to highlight that if other proteins are
elucidated. transported to the nucleus with c-Raf, nuclear NS5A
Possible myristoylation sites with qualitative conserva- could be important to the regulation and modulation of
tion were recognized in NS5A. Covalent myristate bind- cell processes.
ing is not reversible and alters the protein’s
hydrophobicity. In viral proteins such as Arenavirus and Conclusions
Arterivirus, this modification is related to functions such This research presents new insights regarding HCV
as protein cell localization and protein-protein interac- NS5A genotypes 1 and 3. In addition, it demonstrates
tions [42,43]. There are no studies describing the importance of applying bioinformatic tools to the
Yamasakietal.VirologyJournal2012,9:14 Page8of10
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study of proteins that are difficult to investigate by other Table 4Characteristics ofthe study population and
experimental procedures. There was no relationship number ofNS5A different sequences
between the response to therapy and primary structure, Patient# Genotype Therapyoutcome No.ofsequences
but for genotype 3 secondary structure variances were RF03 1A ETR 12
different between the three outcome groups. In addition, RF37 1A ETR 11
there is evidence that the primary/secondary structure RF42 1B ETR 14
differs among genotypes and that this could be impor- RF45 1A ETR 13
tant during the infection process. Functional prediction RF16 1A NR 13
also indicated that NS5A may have functional difference RF22 1B NR 5
between genotypes. Altogether, structural and functional RF39 1B NR 3
properties show that the two genotypes behaviour dur- RF44 1B NR 11
ing infection have differences. The acquired data can be RF05 1B R 12
compared with future experimental data regarding the RF35 1A R 10
NS5A protein and may help in developing new antiviral RF40 1A R 5
strategies, considering the genotypic differences present RF20 3 ETR 14
in Hepatitis C virus. RF31 3 ETR 10
RF109 3 ETR 13
Materials and methods
RF119 3 ETR 12
Sequence bank RF07 3 NR 12
The sequence bank included 345 NS5A complete RF60 3 NR 8
sequences that were obtained from previous studies by RF75 3 NR 12
our group [24,25] and nine reference sequences from RF145 3 NR 10
Genbank. These sequences were extracted from 23 Bra- RF80 3 R 14
zilian patients infected with HCV genotypes 1a, 1b or 3. RF15 3 R 10
The accession numbers in Genbank are RF18 3 R 15
EU309511aEU309673 for genotype 1; EU826174 to RF59 3 R 13
EU826233 and from EU826249 to EU826352 for geno- TOTAL - - 252
type 3. These samples comprised patients who had a
sustained virological response (SVR), non response (NR)
or end of treatment response (ETR) after conventional is a pattern data bank, based on scientific publications
therapy based on Interferon (genotype 3) or Peginter- or research describing the function of determined pro-
feron (genotype 1) plus Ribavirin. Details of the study tein groups [59].
population are presented in Table 4. Redundant amino
acid sequences were excluded, using the software Prediction of functional features
LOCQSPEC 1.0 [55], resulting in 252 different Sequences were submitted to ProtFun 2.2 Server. The
sequences of complete NS5A. method is based on sequence derived protein features
such as predicted post translational modifications, pro-
Amino acids and secondary structure analysis tein sorting signals and physical/chemical properties
The percentage of each amino acid type was calculated calculated from amino acid composition. This allows
and secondary structures investigated in all complete prediction of functionality for proteins which no
NS5A sequences from the sequence bank. These calcu- homology can be found [60,61]. Acquired data were
lations were performed using the PROF program [56], organized in tables (not shown) to posterior statistical
using the Predict Protein Server http://www.predictpro- analysis.
tein.org[57].
Statistical analysis
Transmembrane region prediction In order to establish if there were differences between
Prediction of transmembrane regions was developed by the prediction results between the response groups or
the MEMSAT3 program [58]http://bioinf.cs.ucl.ac.uk/ genotypes, test of homogeneity (chi-square test), t-test
psipred/. All NS5A sequences from the sequence bank and equal variance test was calculated using the soft-
were analyzed using this program. ware MiniTab® 15 (Minitab Inc., USA). Values under 5
(five) were excluded from statistical calculation, since
Prediction of sites these results could be deviation from the sample. Statis-
All sequences were analyzed using the PROSITE pro- tical power calculation was performed using online soft-
gram [59], from the Predict Protein Server [57]. Prosite ware Russ Lenth’s power and sample size page [62].
Yamasakietal.VirologyJournal2012,9:14 Page9of10
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Acknowledgements 17. TellinghuisenTL,MarcotrigianoJ,GorbalenyaAE,RiceCM:TheNS5A
ThisworkwassupportedbygrantsfromFAPESP,CNPqandCAPES. proteinofhepatitisCvirusisazincmetalloprotein.JBiolChem2004,
279:48576-48587.
Authordetails 18. MoradpourD,BrassV,PeninF:Functionfollowsform:thestructureofthe
1DepartmentofBiology,SaoPauloStateUniversity–UNESP,SaoJosedoRio N-terminaldomainofHCVNS5A.Hepatology2005,42:732-735.
Preto,SP,Brazil.2DepartmentofClinicalMedicine,SaoPauloUniversity–USP, 19. BrassV,BieckE,MontserretR,WolkB,HellingsJA,BlumHE,PeninF,
MedicineCollege,SaoPaulo,SP,Brazil.3ViralImmunologyLaboratory, MoradpourD:Anamino-terminalamphipathicalpha-helixmediates
ButantanInstitute,SaoPaulo,SP,Brazil.4RuaCristovaoColombo,2265 membraneassociationofthehepatitisCvirusnonstructuralprotein5A.
LaboratoriodeEstudosGenomicos,JardimNazareth,CEP:15054-000,Sao JBiolChem2002,277:8130-8139.
JosedoRioPreto,SP,Brazil. 20. LiuS,AnsariIH,DasSC,PattnaikAK:Insertionanddeletionanalyses
identifyregionsofnon-structuralprotein5AofHepatitisCvirusthatare
Authors’contributions dispensableforviralgenomereplication.JGenVirol2006,87:323-327.
LHTYcarriedoutallexperiments,acquisitionofdata,analysisand 21. VerdegemD,BadilloA,WieruszeskiJM,LandrieuI,LeroyA,
interpretationofdata,anddraftingthemanuscript;HAAhelpedwiththe BartenschlagerR,PeninF,LippensG,HanoulleX:Domain3ofNS5A
acquisitionofdataandinterpretationofresults;ACGJ,CBandIMVGCM proteinfromthehepatitisCvirushasintrinsic{alpha}-helicalpropensity
participatedinthestudyandthewritingofthemanuscript,PRconceived andisasubstrateofCyclophilinA.JBiolChem2011.
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