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Virology, Molecular Biology, and Serology of Hepatitis C Virus
Virology, Molecular Biology, and Serology of Hepatitis C Virus Daniel W. Bradley
EPATITIS C VIRUS (HCV) has reeently heen cloned and completely or partialIy sequenced by several different laboratories. I -3 HCV is the major eause of parenteralIy transmitted nonA, non-B hepatitis (PT-NANBH) worldwide. 4 ,5 Although transfusions alone aecount for a smalI proportion of aIl cases of welI-documented HCV, it is a virus of immense interest beeause it is assoeiated with a high frequency of chronie infection and later-occurring morbidity in a significant number of infeeted individuals. For example, studies condueted at the United States Centers for Disease ControI (M. Alter, personał communieation, July 1989) indicate that there are an estimated 170,000 HCV infections per year, of which 127,000 are asymptomatic. However, at least 50% of alI HCV infections lead to persistent disease, including 17,000 eases of chronic active hepatitis (CAH) with or without concurrent eirrhosis. Approximately 45% to 50% of alI cases of HCV have risk factors that involve overt or potential parenteral exposure to blood, such as transfusions (3%), intravenous (IV) drug abuse (approximately 38%), hemodialysis, or employment in a health care profession. However, nearly 45% of aIl eases of PTNANBH in the US have no obvious risk factors for aequiring HCV infection (including parenteral exposure), leaving unanswered the question of virus transmission via as yet unidentified routes of exposure. Worldwide, the prevalence of antibodies to HCV (anti-CI00-3/NS4) ranges between 0.2% to 1.7% (about 0.5% in the US), which suggests that HCV is a readily transmitted virus with a re1atively low frequency of clinicalIy apparent disease, as previously noted. HCV has been implieated as one of the major causative agents of primary hepatocelIular earcinoma in Japan6 and has also been shown to be strongly associated with eases of "eryptogenie" cirrhosis. Although HCV has yet to be visualized by electron microscopy (EM) or immune EM (lEM), physicochemical characterization of the virus 7 - 1O and moleeular analysis of the viral genorne strongly indicate that it is an enveloped pestilike/ f1avilike virus with a diameter of less than 50 nm. Isopycnic banding studies of HCV in suerose gradients l l have also shown that it has a buoyant den-
H
Transfusion Medicine Reviews, Vat VI, Na 2 (April), 1992: pp 93-102
sity of approximately 1.10 gm/cm3 , a finding that is consistent with the dedueed pestiviruslike organization of the putative struetural region of the genome (subsequently discussed). HCV was first cloned from a large-volume, chronie-phase chimpanzee plasma pool shown to have a titer of at least l X 106 ehimpanzee infectious doses (CID)/mL. 12 Using an established immunosereening procedure for (plasma) cDNA inserted into a lambda gt ll expression veetor,13 an immunoreaetive clone, identified as S-l-l, was shown to be virus-specific when used to express antigen for the deteetion of anti-HCV (5-1-1) in sera from humans and experimentalIy infected chimpanzees. 14 The faetor VIII-derived HCV was subsequently shown to contain an approximately 9.4-kilobase (kb) single-stranded, positive-sense RNA molecule with one single open reading frame (ORF) that eneoded for a poIyprotein composed of structural and nonstruetural proteins. 2 TabIe l deseribes the moIecuIar properties of HCV and compares them to those of typical f1aviviruses, pestiviruses, and alphaviruses. MOLECULAR VIROLOGY
Initial analysis of the HCV nucleotide (nt) sequenee (published in European Patent 88310922.5, 11/18/88, 1988) showed that the dedueed amino acid sequenee and hydropathic profile eouId be aligned with the 3' -nonstructural region of Dengue2 (DEN2) virus based on the eharacteristic, highly hydrophobic NS2a1b and NS4a1b regions. The ability to align this partial HCV sequenee with the 3'-nonstructural region of either flavivirus or pestivirus genomes suggests that this segment of the HCV genorne also encodes nonstructural proteins. The 3' end of the f1avivirus (and pestivirus) genome encodes NS5, the putative RNA-dependent
From the Virology Section, Hepatitis Branch, National Center for lnfectious Diseases, Centers For Disease Control, Atlanta, GA. This is a US government work. There are no restrictions on its use. Copyright © 1992 by W.B. Saunders Company 0887-7963/92/0602-0002$3.00/0 93
DANIEL W. BRADLEY
94 Table 1. Molecular Characteristics of HCV and Other Related Enveloped Viruses
Genome
Sense Size (kb) Poly-A Subgenomic RNAs ORFs Genomie Organization
Hepatitis C
Flavivirus
Pestivirus
Alphavirus
ssRNA
ssRNA
ssRNA
ssRNA
+
+
+
+
-9.4 ?
10 to 11
12 to 13
11 to 12
1 5'-S 3'-NS
+ + 5'-S 3'-NS
1 5'-S 3'-NS
>1 5'-NS 3'-S
Abbreviations: ss, single-stranded; ORF, open reading trame; S, struetural; NS, nonstruetural. * poly-A: tiek-borne eneephalitis virus.
RNA polymerase (RDRP). This conclusion is based on the presence of the consensus GDD (GlyAsp-Asp) sequence and the spacing of surrounding residues exhibited by other RNA viruses. These consensus sequences are also found in the same order and relative location in the genomes of HCV and the pestiviruses bovine viral diarrhea virus (BVDV) and hog cholera virus (HogCV), suggesting that a replicase function is encoded at the 3' end of these genomes. Preceding the putative RDRP is the extremely hydrophobic NS4a1b region that, although identified in flavivirus-infected cells, has no defined function as yet. The analogous sequence in HCV is also extremely hydrophobic, but is only marginally hydrophobic in the pestiviruses. During HCV infection this region has been shown to elicit antibodies that can be detected by a recombinant protein, CIOO-3 (derived from the putative NS4 region of HCV); this recombinant protein provided the basis for the first commercially available assay to detect anti-HCV (CIOO-3) in potentially infectious donor blood. 15 Upstream of NS4a1b is the NS3 protein that, at its NH3 -terminal end, has consensus sequences found in serine proteases, including those from many RNA viruses. Support for an NS3 protease function comes from recent biochemical evidence that NS3 acts as a protease to cleave at dibasic residues between itself and NS2b during processing of the polyprotein. However, additional data are needed to determine whether NS3 is the viral protease responsible for eleavage at dibasic residues throughout the polyprotein. The presence of these same consensus sequences in the same relative location in HCV and the pestiviruses would suggest a simi1ar function will be observed with
these viruses. Immediately downstream (toward the 3' end) of the putative protease sequences, but still within NS3, are consensus sequences indicative of the proposed superfamily 2 helicase proteins. The function of these proteins in positivesense RNA viruses is not totally elear, but they are probably necessary for unwinding of the doublestranded RNA intermediates, such as replicative form (RF), during replication of the viral genome. As with the consensus polymerase and protease regions, these helicase sequences are found in the same relative location in HCV and the pestiviruses, again emphasizing their organizational and functional relatedness. Furthermore, the simi1arity of the putative HCV NS3 and NSS regions to analogous regions within the genomes of certain plant viruses (polyviruses and carmoviruses, respectively) may indicate that these regions arose from ancestral helicase/protease and polymerase genes that were absolutely essential for the replication of positive-sense, single-stranded RNA viruses. The rerrtaining upstream nonstructural segments of the flavivirus genome include NS2a1b that, like NS4a1b, are extreme1y hydrophobic in character and have only recently been identified in flavivirus-infected cells. No functionis current1y known for the former proteins, but it has been postulated that NS2a may be a cis-acting protease required for cleavage at the NSl (or E2 in the case of HCV)I NS2a junction. As with the flaviviruses, HCV and the related pestiviruses share this hydrophobic segment and, as expected, the spacing between NS2 and NS4 is similar for each of these viruses. However, the region upstream from NS2a1b differs significant1y between HCV and either flaviviruses or pestiviruses. In flaviviruses, this region includes
95
VIROLOGY OF HCV
El has been shown by radioimmunoprecipitation studies to be linked via disulfide bridges to gp33 (E2). Thus, in contrast to the flaviviruses, this region of the pestivirus genome seems to encode only structural proteins and not any NS l-like proteins. Recent reports indicate that the analogous region of the HCV genome is significantly shorter than the respective regions of the flaviviruses and pestiviruses. In vitro processing analysis l6 and eukaryotic (vaccinia virus) expression of recombinant HCV clones (T. Miyamura, personal communication, September 1990) indicate that the 5' end encodes for a minimum of three proteins, two of which are heavily glycosylated. They include: (1) a very basic, unglycosylated, hydrophilic 18- to 22-kd protein encoded by sequence at the extreme 5' end of the HCV genome (and tentatively considered to be the viral capsid protein); (2) a hydrophobic 33-kd glycoprotein (18-kd "backbone"); and (3) a 70- to 72-kd glycoprotein (38-kd "backbone"). Based on the general similarities of HCV to portions of both flavivirus and pestivirus genomes, including the presence of characteristic consensus sequences and the spacing between the hydrophobic NS2 and NS4 regions, investigators at the Na-
sequence encoding for: (1) a capsid protein; (2) a premembrane protein that is subsequently cleaved to yield the mature membrane (M) protein; (3) an envelope protein (E) that is the major target for neutralizing antibodies; and (4) NSl, a nonstructural protein found on the infected cell surface (and in circulation) that can protect against infection with the virus. Figure 1 summarizes the current concept of the HCV genome, including its organizational and functional elements, as weB as structural and nonstructural regions selected to produce recombinant DNA expressed proteins for use in diagnostic assays (discussed subsequent1y). A target polypeptide for use in a candidate vaccine is indicated in the lower left portion of the figure. COMPARATIVE VIROLOGY AND CLASSIFICATION
Recent molecular cloning and genome analyses have shown that the pestivirus proteins encoded by the extreme 5' region (Fig 2) are: (1) the highly basic putative capsid protein, p20; (2) gp62 that is subsequent1y cleaved to gp44/48 and gp33 (possibly analogous to the flavivirus preM/M cleavage); and (3) gp55 that has been shown with hog cholera virus to be the structural protein El and the target ofneutralizing monoclonal antibodies. In addition,
500
AA
I
1500
1_ _1_ _
1 5'
1000 gp33
2000
gp70
r------r----------
~E2(NS1)I NS2
@@@
I
NS3
3000 C_O_O~H 3'
,NS4
I
NS5
----;::1~C~2=O=O;}~trt:~~=-::::;I-'-----~
I C33c I STRUCTURAL
I
3010
1_------lU
1
1
NH2
2500
D5·1·1 (tirst clone identitied) I C100·3 I NON-STRUCTURAL
"Target" protein for recombinant vaccine? Fig 1. location of expressed proteins used in diagnostic assavs.
96
DANIEL W. BRADLEY
HCV
862 AA
~==20==:*:ł;8=:łł~r---2-1-p20
BVDV
gp33
tł ł t
29
I
p20
27 gp48
II ł
,
I
ł
I
I
20
42
32
gp25
gp53
NS2A
1330 AA
1 13
39
2_4-- - l
r--4--,-*.&...---o...ł ---------.--..*-*-------, . ~ 5_4 ---,-
C preM M Fig 2.
NS2A
1320 AA
******
DEN2
gp70
E
NS1
NS2A
Struetural relationships between the HCV and related enveloped viruses: a pestivirus (BVDVI and a łłavivirus (DEN 21.
tional Center for Infectious Diseases believe that HCV should be included within the family Flaviviridae as a separate genus along with another genus encompassing the pestiviruses BVDV, HogCV, and border disease virus (BDV) of sheep. Further molecular characteńzation and biochemical studies will be required, however, to substantiate the current notion that the structural gene region of HCV more closely resembles that of the pestiviruses rather than that of the f1aviviruses. CONSERVED AND HYPERVARIABLE REGIONS OF THE HCV GENOME
Recent studies have shown that the genome of HCV contains both variable and conserved domains, that is, specific regions of the genome where sequence changes are more or less frequently observed, respectively (Fig 3). The most conserved region of the HCV genome in all isolates sequenced to date is found at the extreme S' end (left-hand side) of the genome, including the S' untranslated or noncoding region (NCR). Comparison of a Japanese HCV isolate at the nt and
deduced amino acid (aa) sequence levels with the prototypie factor VIII (US) isolate shows that the putative 22-kd capsid protein of both isolates shares a high degree of homology (90.5% at the nt level, and 97.4% at the aa level).17 This region is of diagnostic importance because tests for antibodies to the capsid protein or nt sequences specific for the S' NCR/capsid region are most likely to detect individuals infected with different isolates ofHCV. With regard to variable domains, a moderately variable region is found between amino acids 215 and 255 within the putative El (envelope/matńx) protein, in which more than 60% of the nt changes are in· the third codonic position resułting in more than 90% silent mutations (Fig 4).18 By a contrast, a hypervariable domain (HV), found between aa 386 and aa 411 within E2/NS 1, accounts for approximately 50% of all aa changes observed within this generał region of the genome (Fig 4).18 One other study has shown that the sequence within the HV region of HCV can change (mutate) over a period of years in a persistent1y infected individ-
97
VIROLOGY OF HCV
500
AA
1500
2500
3010
IL...--_I_ _I_ _I_--&-I_---.L..-I__IJ 1
1000 gp33
5'
2000
gp70
3000 3'
NS5 L..:; NS4 NS3 ~E2{NS1}1 NS2 ___________________---JI'
Kd
I@
@~38
Region II
Region I
Icons~rved I j..-Re lon-l
HV Region STRUCTURAL ....------ł NON·STRUCTURAL
{Carboxy Terminus}
(Amino Terminus) HCV·FVIII 9,379 nt 3011 aa ORF ] HCV-Jap1 9,416 nt 3010 aa ORF Fig 3.
o
Yo homology - 85 {aa}
Conserved and hypervariable (HV) domains of HCV genorne.
ual. 19 HCV isolates in two plasmas eolleeted from a patient in 1977 and again in 1990 (ehronic phase of disease) were partially sequenced and shown to differ by 2.5% overall at the nt level (123 of 4923 nt); base substitutions in the two isolates were generally unevenly distributed throughout the genome, although a relatively large number of nt changes (44 of 960 nt) were found within the E2/ NS l region corresponding to the HV region deseribed previously. One domain of 39 nucleotides was found to differ in sequence by 28.2% between the two isolates. By contrast, only 2 of 276 nt (0.7%) contained within the 5' NCR were found to be different between the two isolates of HCV. Evidence for the existence of at least two serotypes of HCV derives from studies that have shown serological and/or sequenee-speeific differences between geographically distinct isolates. 20 Table 2 summarizes the results of one study (A. Nomoto, personal communication, May 1991) that clearly indicate that two groups of HCV, referred to here as groups I and II, can be differentiated on the basis of unique antibody responses in HCVinfected individuals. For example, recombinant DNA expressed protein from the NS4 region of group II HCV was not detected by antibody from individuals infected with group I HCV. Likewise, polymerase chain reaction (PCR) studies have also shown that the NS4 region of groups I and II iso-
lates are sufficiently divergent in sequence that PCR primers used for group II virus do not amplify a homologous sequence from the group I isolate (Table 3). Other studies conducted at the Centers for Disease Control further suggest that some isolates of HCV may even contain nonviral inserts (RNA) because PCR analysis of the NS3 region of one attenuated strain of HCV (shown to be avirulent in two chimpanzees) demonstrated a much larger than expected eDNA/PCR product on agarose gels. If confirmed, these studies would suggest that HCV, like BVDV, may occasionally incorporate host message RNA within well-defined regions of the viral genorne that can confer phenotypieally different properties to the "chimeric" virus. This finding mayaIso account for differences in the apparent "virulence" af some HCV isolates and recrudescence and/or course of disease observed in some individuals. DIAGNOSTIC ASSAYS FOR VIRUS-SPECIFIC ANTIBODY AND NUCLEIC ACID
HCV infeetion, once a diagnosis of "exclusion" based on the absenee of markers of aeute infeetion with hepatitis A virus (HAV) and aeute or chronic infeetion with hepatitis B virus (HBV) in clinieally ill patients, can now be specifieally detected by using serodiagnostic assays for virus-
DANIEL W. BRADlEY
98
Deduced Amino Acid
o
o
....
O
AA
o o
o
O C\l
O
o o
'Ot
CO)
o o co
10
II---+---I----ł---+--
o o,....
I
I-
1. 40 aa in E1 modo variable 2. 94% nt seq. homology: a) 80% T1 ; 20% TV b) 60% ot changes in 3rd pos'n. c) >90% silent mutations Fig 4.
HV (Region V) 28 aa 1. 8% ot nt (78/963) 2. Accounts tor - 50% aa changes (40-63% range)
Variable regions in genome of HCV.
specific antibodies, as previously discussed. Firstgeneration enzyme immunoassays (ElA) relied on the use of a recombinant DNA-expressed fusion protein as a source of antigen. This fusion protein, Cl00-3, comprised of a fragment of a superoxide dismutase (SOD) protein fused to a portion of the HCV NS4 protein, was shown to detect antibody Table 2. Evidence for a Second Serotype of HCV Assay
o oo
T
411 255 386 Region V (HV Region)
Region IV (aa129-437)
o o m
-I
Region IV
215
o o co
Result
Antibody or Nucleic Acid (PGR)
Group
Group
I
II
Anti-C (Capsid; C22c) Anti-NS3 (group I antigen)* Anti-NS4 (group I antigen; C100-3) Anti-NS4 (group II antigen; clone "E")t PCR (5' UTR-Capsid) PCR (NS4, group I) PCR (NS4, group II)
Pos Pos
Pos Pos
Pos
Pos
Neg Pos Pos Neg
Pos Pos Pos Pos
* Group II NS3 antigen not tested. tA. Nomoto: personal communication (May 1991).
in approximately 50% of acutely infected patients and more than 70% of chronically infected patients (range: 60% to 95%).21-24 Second-generation EIAs based on the use of multiple (three) recombinant proteins derived from both the structural and nonstructural regions of the HCV genorne (Fig l), have shown improved sensitivity with regard to the diagnosis of acute infection and have shortened the time to the initial detection of virus-specific antibodies by 3 to 12 weeks in those individuals who did not demonstrate seroconversion during the acute phase of disease. Another ElA format for the detection of virus-specific antibodies has recently been described. 25 This assay makes use of synthetic peptide antigens representing three immunodorninant regions of the structural and nonstructural proteins of HCV, namely, capsid (C22), NS3, and NS4. When used to screen pane1s of human and chimpanzee sera, this assay was found to detect antibody 4 to 10 weeks earlier than the original ElA that used the C100-3 (NS4) recombinant protein. Although the "window" period between the acute phase of disease and appearance of
VIROLOGY OF HCV
virus-specifie antibodies has been shortened by both types of second-generation assays, a significant proportion of individuals (25% to 40%) are still found to be negative for HCV-specific antibody. It is anticipated that other more sensitive serologieal assays based on the use of novel antigens and formats will further shorten this window in acutely infected patients. PCR methodologies, although experimental in nature and cumbersorne to perform, may mature to the level of clinically useful tests and provide a reliable means of detecting virus-specific genorne sequences in patient sera or donor plasma. 26-28 Reverse transcription (RT) ofHCV RNA followed by amplifieation of the cDNA by PCR or "nested" PCR (the latter employs the use of pairs of external and internal primers complementary to the HCV cDNA) has been shown to be highly sensitive and specific for the detection of HCV when used under controlled conditions. 26 For example, the RT-PCR procedure can detect as little as 0.1 cm in chronic-phase plasma (A. Weiner, personal communication, June 1990). In cases in whieh acute HCV infection is suspected, but antibody results are still negative, ie, the window period, the RTPCR method can be used to confirm infection with HCV. The results of one studl7 also suggest that the detection of HCV viral sequences in donor blood by PCR is a better predietor of infectivity than the presence of anti-C100-3 (anti-NS4) alone. PCR-based assays may also prove to be useful adjunctive tests to monitor the course of disease in chronically infected patients. PCR methods have already been used to determine the relative abundance of HCV RNA in patients who have been treated with a-interferon (J. Hoofnagle, personal communieation, September 1990); a similar use of PCR can be anticipated for patients who may undergo alternative antiviral therapy, including treatment with synthetic organie molecules incorporating antihelicase and/or antiviral protease activities (protease and helicase functions are encoded by the NS3 region of the HCV genome). PERSPECTIVES IN VACCINE DEVELOPMENT
In HogCV, gp55 or the analogous BVDV gp53 seems to be of major importance for neutralization of pestiviruses because neutralizing antibodies are directed toward this gene produet. As previously tnentioned, it has recent1y been shown that HogCV gp55 or BVDV gp53 forms disulfide-linked het-
99
erodimers with a putative transmembrane protein, HogCV gp33 or BVDV gp25, respectively (Fig 2 illustrates BVDV). Although live attenuated virus vaccines for HogCV have been used, some countries have placed restrictions on their use because infection with virulent virus cannot be readily differentiated from immunization with tissue culturederived, attenuated HogCV. Rumenupf et al recently developed a series of recombinant vaccinia virus/HogCV constructs, one of whieh expressed all four structural proteins; the latter construct was successfully used to immunize pigs against a lethal challenge with wild-type HogCV. 29 Similar vaccinia virus/HCV constructs have been commercially prepared (as proprietary candidate vaccines) and recently used to immunize chimpanzees against infection with 100 chimpanzee infectious doses of a factor VIII HCV isolate. The status (success) of this trial is current1y unknown; however, the strong similarity between HCV and HogCV within the putative structural gene region of their respective genomes would suggest that this strategem may eventually lead to the development of an effective recombinant vaccine for HCV, provided proper glycosylation, cleavage of the polyprotein, and folding ofthe individual viral proteins occurs. However, if the presumption (previously discussed) that the "NSI" region of HCV encodes for a structural protein of HCV (E2) is incorrect, then additional approaches involving recombinants of HCV with vaccinia or other vectors and other regions of the genorne will have to be explored. Recent vaccinia virus expression studies by Shimotohno et al (personal communication, May 1991) seem to support the notion that three structural proteins (capsid, El, and E2) are encoded within the HCV genorne covering a region from the extreme 5' end of the ORF to the carboxy terminus of NS 1 (putative E2). Cleavages of the expressed polyprotein seem to occur between aa 191 and 192 (amino terminus of El) and aa 384 and 385 (amino terminus of E2); the carboxy terminus of E2 seems to resuIt from a cleavage between aa 737 and 738. These cleavages have been verified by mierosequencing of the amino termini of the respective proteins. Other expression studies conducted by Saitoet al (personal communication) suggest that HCV El contains approximately 190 aa and is glycosylated to yield gp35; E2 (NSl) yields a glycosylated protein with a molecular weight of 70 to 72
100
kd. It is elear that definitive biochemical studies of authentie viral proteins, ineluding microsequencing of gel-separated proteins recovered from hightitered, HCV-infected chimpanzee liver tissue are needed to confirm and elarify the above results. Other studies that might further define the exact nature of the structural proteins of HCV could involve in vitro translation of the entire ORF of HCV with production of bona fide virus particIes and/or transfection of cells with full-Iength cONA with synthesis of complete virus partieles. Although much of what is described herein is speculative or incomplete in nature, there remain other possible impediments to the development of a recombinant or tissue culture-derived HCV vaccine. There is nowelear evidence for the existence of a hypervariable (HV) region within the amino terminus of the putative HCV E2 (NS 1) protein, as previously described (Fig 3). This finding has lead to the concern that antibodies directed against one variant may not necessarily protect against infection by another variant if the HV region is involved in virus neutralization. Although chimpanzee cross-challenge studies conducted in the early 1980s indicated that reinfection by HCV does not readily occur, it is equally important to note that animals infected with HCV often developed persistent infection, thereby confounding (and probably negating the value of) these results because of the probabIe effects of viral interference. Clearly, more work needs to be focused on the importance of the HV region of HCV and its potential for compounding the problems of vaccine development. Another potentially confounding factor to be cdnsidered in vaccine development would be the presence of different serotypes of HCV. We and other investigators (A. Nomoto, et al, personal communication, May 1990) have recent1y acquired experimental evidence for the existence of serologically distinct groups of HCV that can be distinguished from the prototypie factor VIII isolate on the basis of virulence in primates and/or the results of serological and nucleic acid (PCR) tests (Table 2). In spite of the potential difficulties in developing a vaccine we believe that the unexpectedly high worldwide prevalence of HCV (average, 0.6%), and the related long-term sequellae (cirrhosis, liver cancer, aplastic anemia, etc) will demand that more resources be focused on efforts to: (l) further
DANIEL W. BRADLEY
delineate the molecular biology of HCV; (2) propagate HCV in tissue culture; (3) define the mechanisms of pathogenesis, including persistence of infection; and (4) develop asafe, cost-effective vaccine based on either cell culture-derived virus or a recombinant DNA construct. PATHOGENETIC EVENTS OF HCV REPlICATION INVIVO
Recent studies condueted in two ehimpanzees that had been experimentally infeeted with patient "H" plasma showed that HCV sequenees could be detected as early as 3 days after inoculation; HCV RNA (as eONA) was found to be positive through the peak of alanine aminotransferase (ALT) aetivity.30 In these same animals, anti-ClOO-3 beeame detectable 13 and 32 weeks after inoculation, respectively, or 5 and 25 weeks after the respective peaks of ALT activity. These findings indicated a very early replicative phase of HCV in whieh no virus-specifie antibody could be detected before, or just after, the peak of ALT aetivity. These findings imply that some blood donors who are in the incubation phase of disease could be potentially infectious, underseoring the need to eonsider deteetion of virus-specifie nueleic acid sequenees rather than antibody. In another study, long-term continued observation follow-up of five human patients and four chimpanzees infected with HCV 31 again showed that viremia oeeurred shortly after transfusion/infection (1 to 5 weeks) and that antiClOO-3 appeared 12 to 30 weeks after exposure to HCV. This same study also showed that the disappearance of HCV from serum was eorrelated with resolution of disease. Recent studies eonducted at the US Centers for Oisease Control32 ,33 have clearly shown that early viremia is biphasic in experimentally infected chimpanzees and that HCV-speeifie antigen ean be deteeted in hepatoeyte eytoplasm by an immunofluorescent antibody probe up to several weeks before the first elevated ALT value. As observed earlier, anti-ClOO-3 responses were often delayed or even absent in HCV-infeeted ehimpanzees. The use of a modified second-generation assay for antibodies to a eombination of HCV-speeifie proteins representing portions of the capsid, NS3, and NS4 domains showed that the antibody "window" eould be shortened but that viremia still preeeded
101
VIROLOGY OF HCV
the appearance of antibody. Furthermore, no particular pattem of antibody response(s) could be correlated with either resolution of disease or progression to chronicity. Although some investigators have speculated that development of persistent HCV infection may be associated with the introduction of HV region neutralization "escape" mutants, no data have yet been presented to support this notion. Further stud-
ies of the HV region of the HCV genome, as wen as other regions of interest, may provide critical insight into the mechanism(s) underlying persistence of infection. It should also be noted that some variants of HCV may be more or less virulent than others, as previously discussed. How do such variants .arise and what are the important genetic factors (mutations, deletions, insertions, etc) that confer these unusual phenotypic properties?
REFERENCES 1. Kato N, Hijikata M, Ootsuyama Y, et al: Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Nad Acad Sci USA 87:9524-9528, 1990 2. Choo Q-L, Richman K, Han J, et al: Genetic organization and diversity of the hepatitis C virus. Proc Nad Acad Sci USA 88:2451-2455, 1991 3. Takamazawa A, Mori C, Fuke I, et al: Structure and organization of the hepatitis C virus genorne isolated from human carriers. J ViroI65:1105-1113, 1991 4. Alter H: The hepatitis C virus and its relationship to the clinical spectrum of NANB hepatitis. J Gastroenterol Hepatol 1:78-94, 1990 (Suppl) 5. Choo Q-L, Weiner A, Overby L, et al: Hepatitis C virus: The major causative agent of viral non-A, non-B hepatitis. Br Med Buli 46:423-441, 1990 6. Saito I, Miyamura T, Ohbayashi A, et al: Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. PNAS 87:6547-6549, 1990 7. Bradley D, Maynard J, Popper H, et al: Posttransfusion non-A, non-B hepatitis: Physicochemieal properties of two distinct agents. J Infect Dis 148:254-265, 1983 8. Bradley D, McCaustland K, Cook EH, et al: Posllransfusion non-A, non-B hepatitis in chimpanzees: Physicochemical evidence that the tabule forming agent is a smali, eveloped virus. Gastroentology 88:773-779, 1985 9. He, L-F, Alling D, Popkin T, et al: Determining the size of non-A, non-B hepatitis virus by fiItration. J Infect Dis 156:636-640, 1987 10. Yuasa T, Ishikawa G, Manabe S, et al: The particIe size of hepatitis C virus estimated by filtration through microporous regenerated cellulose fibre. J Gen Virol 72:2021-2024, 1991 11. Bradley D, McCaustland K, Krawczynski K, et al: Hepatitis C virus: Buoyant density of the factor VIII-derived isolate in sucrose. J Med Virol 34:206-208, 1991 12. Bradley D, Krawczynski K, Beach M, et al: Non-A, non-B hepatitis: Toward the discovery of hepatitis C and E viruses. Semin Liver Dis 11:128-146, 1991 13. Young R, Davis R: Efficient isolation of genes by using mtibody probes. PNAS 80:1194-1198, 1983 14. Choo Q-L, Kuo G, Weiner A, et al: Isolation of a cDNA :Ione derived from a blood-borne non-A, non-B viral hepatitis ~enome. Science 244:359-362, 1989
15. Kuo G, Choo Q-L, Alter J, et al: An assay for circulating antibodies to a major etiologie agent of human non-A, non-B hepatitis. Science 244:362-364, 1989 16. Hijikata M, Kato N, Ootsuymama Y, et al: Gene mapping of the putative structural region of the hepatitis C virus genorne by in vitro processing analysis. PNAS 88:5547-5551, 1991 17. Takeuchi K, Kubo Y, Boonmar S, et al: Nucleotide sequence of core and envelope genes of the hepatitis C virus genorne derived direcdy from human healthy carriers. Nucleic Acids Res 18:4626, 1990 18. Weiner A, Brauer M, Rosenblatt J, et al: Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NSl proteins and the pestivirus envelope glycoproteins. Virology 180:842-848, 1991 19. Ogata N, AIter H, Miller R, et al: Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. PNAS 88:3392-3396, 1991 20. Takeuchi K, Boonmar S, Kubo Y, et al: Hepatitis C viral cDNA c10nes isolated from a healthy carrier donor implicated in post-transfusion non-A, non-B hepatitis. Gene 91:287291, 1990 21. Alter H, PurcelI R, Shih J, et al: Detection of antibody to hepatitis C virus in prospectively folIowed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 321:1494-1500, 1989 22. Miyamura T, Saito I, Katayama T, et al: Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis. PNAS 87:983-987, 1989 23. Boonmar S, Pojanagaroon B, Watanabe Y, et al: Prevalence of hepatitis C virus antibody among healthy blood donors and non-A, non-B hepatitis patients in Thailand. Jpn J Med Sci Biol 43:29-36, 1990 24. Esteban J, Gonzalez A, Hernandez J, et al: Evaluation of antibodies to hepatitis C virus in a study of transfusionassociated hepatitis. N Engl J Med 323:1107-1112,1990 25. Hosein B, Fang C, Popovsky M, et al: Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein. PNAS 88:3647-3651, 1991 26. Weiner A, Kuo G, Bradley D, et al: Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet 335:1-3, 1990 27. Garson J, Tedder R, Briggs M, et al: Detection of hep-
102
atitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet 335:1419-1422, 1990 28. Ulrich P, Romeo J, Lane P, et al: Detection, semiquantitation, and genetic variation in hepatitis C virus sequences amplified from the plasma of blood donors with e1evated alanine aminotransferase. J Clin lnvest 86:1609-1614, 1990 29. Rumenapf T, Stark R, Meyers G, et al: Structural proteins of hog cholera virus expressed by vaccinia virus: Further characterization and induction of protective immunity. J Virol 65:589-597, 1991
DANIEL W. BRADLEY
30. Shimizu Y, Weiner A, Rosenblatt J, et al: Earlyevents in hepatitis C virus infection in chimpanzees. PNAS 87:64416444, 1990 31. Farci P, Alter H, Wong D, et al: A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 325:98-104, 1991 32. Krawczynski K, Beach M, Bradley D, et al: Hepatitis C virus antigen in hepatocytes: lmmunomorphologic detection and identification. (submitted for publication) 33. Beach M, Meeks E, Krawczynski K, et al: (manuscript in preparation)
EPATITIS C VIRUS (HCV) has reeently heen cloned and completely or partialIy sequenced by several different laboratories. I -3 HCV is the major eause of parenteralIy transmitted nonA, non-B hepatitis (PT-NANBH) worldwide. 4 ,5 Although transfusions alone aecount for a smalI proportion of aIl cases of welI-documented HCV, it is a virus of immense interest beeause it is assoeiated with a high frequency of chronie infection and later-occurring morbidity in a significant number of infeeted individuals. For example, studies condueted at the United States Centers for Disease ControI (M. Alter, personał communieation, July 1989) indicate that there are an estimated 170,000 HCV infections per year, of which 127,000 are asymptomatic. However, at least 50% of alI HCV infections lead to persistent disease, including 17,000 eases of chronic active hepatitis (CAH) with or without concurrent eirrhosis. Approximately 45% to 50% of alI cases of HCV have risk factors that involve overt or potential parenteral exposure to blood, such as transfusions (3%), intravenous (IV) drug abuse (approximately 38%), hemodialysis, or employment in a health care profession. However, nearly 45% of aIl eases of PTNANBH in the US have no obvious risk factors for aequiring HCV infection (including parenteral exposure), leaving unanswered the question of virus transmission via as yet unidentified routes of exposure. Worldwide, the prevalence of antibodies to HCV (anti-CI00-3/NS4) ranges between 0.2% to 1.7% (about 0.5% in the US), which suggests that HCV is a readily transmitted virus with a re1atively low frequency of clinicalIy apparent disease, as previously noted. HCV has been implieated as one of the major causative agents of primary hepatocelIular earcinoma in Japan6 and has also been shown to be strongly associated with eases of "eryptogenie" cirrhosis. Although HCV has yet to be visualized by electron microscopy (EM) or immune EM (lEM), physicochemical characterization of the virus 7 - 1O and moleeular analysis of the viral genorne strongly indicate that it is an enveloped pestilike/ f1avilike virus with a diameter of less than 50 nm. Isopycnic banding studies of HCV in suerose gradients l l have also shown that it has a buoyant den-
H
Transfusion Medicine Reviews, Vat VI, Na 2 (April), 1992: pp 93-102
sity of approximately 1.10 gm/cm3 , a finding that is consistent with the dedueed pestiviruslike organization of the putative struetural region of the genome (subsequently discussed). HCV was first cloned from a large-volume, chronie-phase chimpanzee plasma pool shown to have a titer of at least l X 106 ehimpanzee infectious doses (CID)/mL. 12 Using an established immunosereening procedure for (plasma) cDNA inserted into a lambda gt ll expression veetor,13 an immunoreaetive clone, identified as S-l-l, was shown to be virus-specific when used to express antigen for the deteetion of anti-HCV (5-1-1) in sera from humans and experimentalIy infected chimpanzees. 14 The faetor VIII-derived HCV was subsequently shown to contain an approximately 9.4-kilobase (kb) single-stranded, positive-sense RNA molecule with one single open reading frame (ORF) that eneoded for a poIyprotein composed of structural and nonstruetural proteins. 2 TabIe l deseribes the moIecuIar properties of HCV and compares them to those of typical f1aviviruses, pestiviruses, and alphaviruses. MOLECULAR VIROLOGY
Initial analysis of the HCV nucleotide (nt) sequenee (published in European Patent 88310922.5, 11/18/88, 1988) showed that the dedueed amino acid sequenee and hydropathic profile eouId be aligned with the 3' -nonstructural region of Dengue2 (DEN2) virus based on the eharacteristic, highly hydrophobic NS2a1b and NS4a1b regions. The ability to align this partial HCV sequenee with the 3'-nonstructural region of either flavivirus or pestivirus genomes suggests that this segment of the HCV genorne also encodes nonstructural proteins. The 3' end of the f1avivirus (and pestivirus) genome encodes NS5, the putative RNA-dependent
From the Virology Section, Hepatitis Branch, National Center for lnfectious Diseases, Centers For Disease Control, Atlanta, GA. This is a US government work. There are no restrictions on its use. Copyright © 1992 by W.B. Saunders Company 0887-7963/92/0602-0002$3.00/0 93
DANIEL W. BRADLEY
94 Table 1. Molecular Characteristics of HCV and Other Related Enveloped Viruses
Genome
Sense Size (kb) Poly-A Subgenomic RNAs ORFs Genomie Organization
Hepatitis C
Flavivirus
Pestivirus
Alphavirus
ssRNA
ssRNA
ssRNA
ssRNA
+
+
+
+
-9.4 ?
10 to 11
12 to 13
11 to 12
1 5'-S 3'-NS
+ + 5'-S 3'-NS
1 5'-S 3'-NS
>1 5'-NS 3'-S
Abbreviations: ss, single-stranded; ORF, open reading trame; S, struetural; NS, nonstruetural. * poly-A: tiek-borne eneephalitis virus.
RNA polymerase (RDRP). This conclusion is based on the presence of the consensus GDD (GlyAsp-Asp) sequence and the spacing of surrounding residues exhibited by other RNA viruses. These consensus sequences are also found in the same order and relative location in the genomes of HCV and the pestiviruses bovine viral diarrhea virus (BVDV) and hog cholera virus (HogCV), suggesting that a replicase function is encoded at the 3' end of these genomes. Preceding the putative RDRP is the extremely hydrophobic NS4a1b region that, although identified in flavivirus-infected cells, has no defined function as yet. The analogous sequence in HCV is also extremely hydrophobic, but is only marginally hydrophobic in the pestiviruses. During HCV infection this region has been shown to elicit antibodies that can be detected by a recombinant protein, CIOO-3 (derived from the putative NS4 region of HCV); this recombinant protein provided the basis for the first commercially available assay to detect anti-HCV (CIOO-3) in potentially infectious donor blood. 15 Upstream of NS4a1b is the NS3 protein that, at its NH3 -terminal end, has consensus sequences found in serine proteases, including those from many RNA viruses. Support for an NS3 protease function comes from recent biochemical evidence that NS3 acts as a protease to cleave at dibasic residues between itself and NS2b during processing of the polyprotein. However, additional data are needed to determine whether NS3 is the viral protease responsible for eleavage at dibasic residues throughout the polyprotein. The presence of these same consensus sequences in the same relative location in HCV and the pestiviruses would suggest a simi1ar function will be observed with
these viruses. Immediately downstream (toward the 3' end) of the putative protease sequences, but still within NS3, are consensus sequences indicative of the proposed superfamily 2 helicase proteins. The function of these proteins in positivesense RNA viruses is not totally elear, but they are probably necessary for unwinding of the doublestranded RNA intermediates, such as replicative form (RF), during replication of the viral genome. As with the consensus polymerase and protease regions, these helicase sequences are found in the same relative location in HCV and the pestiviruses, again emphasizing their organizational and functional relatedness. Furthermore, the simi1arity of the putative HCV NS3 and NSS regions to analogous regions within the genomes of certain plant viruses (polyviruses and carmoviruses, respectively) may indicate that these regions arose from ancestral helicase/protease and polymerase genes that were absolutely essential for the replication of positive-sense, single-stranded RNA viruses. The rerrtaining upstream nonstructural segments of the flavivirus genome include NS2a1b that, like NS4a1b, are extreme1y hydrophobic in character and have only recently been identified in flavivirus-infected cells. No functionis current1y known for the former proteins, but it has been postulated that NS2a may be a cis-acting protease required for cleavage at the NSl (or E2 in the case of HCV)I NS2a junction. As with the flaviviruses, HCV and the related pestiviruses share this hydrophobic segment and, as expected, the spacing between NS2 and NS4 is similar for each of these viruses. However, the region upstream from NS2a1b differs significant1y between HCV and either flaviviruses or pestiviruses. In flaviviruses, this region includes
95
VIROLOGY OF HCV
El has been shown by radioimmunoprecipitation studies to be linked via disulfide bridges to gp33 (E2). Thus, in contrast to the flaviviruses, this region of the pestivirus genome seems to encode only structural proteins and not any NS l-like proteins. Recent reports indicate that the analogous region of the HCV genome is significantly shorter than the respective regions of the flaviviruses and pestiviruses. In vitro processing analysis l6 and eukaryotic (vaccinia virus) expression of recombinant HCV clones (T. Miyamura, personal communication, September 1990) indicate that the 5' end encodes for a minimum of three proteins, two of which are heavily glycosylated. They include: (1) a very basic, unglycosylated, hydrophilic 18- to 22-kd protein encoded by sequence at the extreme 5' end of the HCV genome (and tentatively considered to be the viral capsid protein); (2) a hydrophobic 33-kd glycoprotein (18-kd "backbone"); and (3) a 70- to 72-kd glycoprotein (38-kd "backbone"). Based on the general similarities of HCV to portions of both flavivirus and pestivirus genomes, including the presence of characteristic consensus sequences and the spacing between the hydrophobic NS2 and NS4 regions, investigators at the Na-
sequence encoding for: (1) a capsid protein; (2) a premembrane protein that is subsequently cleaved to yield the mature membrane (M) protein; (3) an envelope protein (E) that is the major target for neutralizing antibodies; and (4) NSl, a nonstructural protein found on the infected cell surface (and in circulation) that can protect against infection with the virus. Figure 1 summarizes the current concept of the HCV genome, including its organizational and functional elements, as weB as structural and nonstructural regions selected to produce recombinant DNA expressed proteins for use in diagnostic assays (discussed subsequent1y). A target polypeptide for use in a candidate vaccine is indicated in the lower left portion of the figure. COMPARATIVE VIROLOGY AND CLASSIFICATION
Recent molecular cloning and genome analyses have shown that the pestivirus proteins encoded by the extreme 5' region (Fig 2) are: (1) the highly basic putative capsid protein, p20; (2) gp62 that is subsequent1y cleaved to gp44/48 and gp33 (possibly analogous to the flavivirus preM/M cleavage); and (3) gp55 that has been shown with hog cholera virus to be the structural protein El and the target ofneutralizing monoclonal antibodies. In addition,
500
AA
I
1500
1_ _1_ _
1 5'
1000 gp33
2000
gp70
r------r----------
~E2(NS1)I NS2
@@@
I
NS3
3000 C_O_O~H 3'
,NS4
I
NS5
----;::1~C~2=O=O;}~trt:~~=-::::;I-'-----~
I C33c I STRUCTURAL
I
3010
1_------lU
1
1
NH2
2500
D5·1·1 (tirst clone identitied) I C100·3 I NON-STRUCTURAL
"Target" protein for recombinant vaccine? Fig 1. location of expressed proteins used in diagnostic assavs.
96
DANIEL W. BRADLEY
HCV
862 AA
~==20==:*:ł;8=:łł~r---2-1-p20
BVDV
gp33
tł ł t
29
I
p20
27 gp48
II ł
,
I
ł
I
I
20
42
32
gp25
gp53
NS2A
1330 AA
1 13
39
2_4-- - l
r--4--,-*.&...---o...ł ---------.--..*-*-------, . ~ 5_4 ---,-
C preM M Fig 2.
NS2A
1320 AA
******
DEN2
gp70
E
NS1
NS2A
Struetural relationships between the HCV and related enveloped viruses: a pestivirus (BVDVI and a łłavivirus (DEN 21.
tional Center for Infectious Diseases believe that HCV should be included within the family Flaviviridae as a separate genus along with another genus encompassing the pestiviruses BVDV, HogCV, and border disease virus (BDV) of sheep. Further molecular characteńzation and biochemical studies will be required, however, to substantiate the current notion that the structural gene region of HCV more closely resembles that of the pestiviruses rather than that of the f1aviviruses. CONSERVED AND HYPERVARIABLE REGIONS OF THE HCV GENOME
Recent studies have shown that the genome of HCV contains both variable and conserved domains, that is, specific regions of the genome where sequence changes are more or less frequently observed, respectively (Fig 3). The most conserved region of the HCV genome in all isolates sequenced to date is found at the extreme S' end (left-hand side) of the genome, including the S' untranslated or noncoding region (NCR). Comparison of a Japanese HCV isolate at the nt and
deduced amino acid (aa) sequence levels with the prototypie factor VIII (US) isolate shows that the putative 22-kd capsid protein of both isolates shares a high degree of homology (90.5% at the nt level, and 97.4% at the aa level).17 This region is of diagnostic importance because tests for antibodies to the capsid protein or nt sequences specific for the S' NCR/capsid region are most likely to detect individuals infected with different isolates ofHCV. With regard to variable domains, a moderately variable region is found between amino acids 215 and 255 within the putative El (envelope/matńx) protein, in which more than 60% of the nt changes are in· the third codonic position resułting in more than 90% silent mutations (Fig 4).18 By a contrast, a hypervariable domain (HV), found between aa 386 and aa 411 within E2/NS 1, accounts for approximately 50% of all aa changes observed within this generał region of the genome (Fig 4).18 One other study has shown that the sequence within the HV region of HCV can change (mutate) over a period of years in a persistent1y infected individ-
97
VIROLOGY OF HCV
500
AA
1500
2500
3010
IL...--_I_ _I_ _I_--&-I_---.L..-I__IJ 1
1000 gp33
5'
2000
gp70
3000 3'
NS5 L..:; NS4 NS3 ~E2{NS1}1 NS2 ___________________---JI'
Kd
I@
@~38
Region II
Region I
Icons~rved I j..-Re lon-l
HV Region STRUCTURAL ....------ł NON·STRUCTURAL
{Carboxy Terminus}
(Amino Terminus) HCV·FVIII 9,379 nt 3011 aa ORF ] HCV-Jap1 9,416 nt 3010 aa ORF Fig 3.
o
Yo homology - 85 {aa}
Conserved and hypervariable (HV) domains of HCV genorne.
ual. 19 HCV isolates in two plasmas eolleeted from a patient in 1977 and again in 1990 (ehronic phase of disease) were partially sequenced and shown to differ by 2.5% overall at the nt level (123 of 4923 nt); base substitutions in the two isolates were generally unevenly distributed throughout the genome, although a relatively large number of nt changes (44 of 960 nt) were found within the E2/ NS l region corresponding to the HV region deseribed previously. One domain of 39 nucleotides was found to differ in sequence by 28.2% between the two isolates. By contrast, only 2 of 276 nt (0.7%) contained within the 5' NCR were found to be different between the two isolates of HCV. Evidence for the existence of at least two serotypes of HCV derives from studies that have shown serological and/or sequenee-speeific differences between geographically distinct isolates. 20 Table 2 summarizes the results of one study (A. Nomoto, personal communication, May 1991) that clearly indicate that two groups of HCV, referred to here as groups I and II, can be differentiated on the basis of unique antibody responses in HCVinfected individuals. For example, recombinant DNA expressed protein from the NS4 region of group II HCV was not detected by antibody from individuals infected with group I HCV. Likewise, polymerase chain reaction (PCR) studies have also shown that the NS4 region of groups I and II iso-
lates are sufficiently divergent in sequence that PCR primers used for group II virus do not amplify a homologous sequence from the group I isolate (Table 3). Other studies conducted at the Centers for Disease Control further suggest that some isolates of HCV may even contain nonviral inserts (RNA) because PCR analysis of the NS3 region of one attenuated strain of HCV (shown to be avirulent in two chimpanzees) demonstrated a much larger than expected eDNA/PCR product on agarose gels. If confirmed, these studies would suggest that HCV, like BVDV, may occasionally incorporate host message RNA within well-defined regions of the viral genorne that can confer phenotypieally different properties to the "chimeric" virus. This finding mayaIso account for differences in the apparent "virulence" af some HCV isolates and recrudescence and/or course of disease observed in some individuals. DIAGNOSTIC ASSAYS FOR VIRUS-SPECIFIC ANTIBODY AND NUCLEIC ACID
HCV infeetion, once a diagnosis of "exclusion" based on the absenee of markers of aeute infeetion with hepatitis A virus (HAV) and aeute or chronic infeetion with hepatitis B virus (HBV) in clinieally ill patients, can now be specifieally detected by using serodiagnostic assays for virus-
DANIEL W. BRADlEY
98
Deduced Amino Acid
o
o
....
O
AA
o o
o
O C\l
O
o o
'Ot
CO)
o o co
10
II---+---I----ł---+--
o o,....
I
I-
1. 40 aa in E1 modo variable 2. 94% nt seq. homology: a) 80% T1 ; 20% TV b) 60% ot changes in 3rd pos'n. c) >90% silent mutations Fig 4.
HV (Region V) 28 aa 1. 8% ot nt (78/963) 2. Accounts tor - 50% aa changes (40-63% range)
Variable regions in genome of HCV.
specific antibodies, as previously discussed. Firstgeneration enzyme immunoassays (ElA) relied on the use of a recombinant DNA-expressed fusion protein as a source of antigen. This fusion protein, Cl00-3, comprised of a fragment of a superoxide dismutase (SOD) protein fused to a portion of the HCV NS4 protein, was shown to detect antibody Table 2. Evidence for a Second Serotype of HCV Assay
o oo
T
411 255 386 Region V (HV Region)
Region IV (aa129-437)
o o m
-I
Region IV
215
o o co
Result
Antibody or Nucleic Acid (PGR)
Group
Group
I
II
Anti-C (Capsid; C22c) Anti-NS3 (group I antigen)* Anti-NS4 (group I antigen; C100-3) Anti-NS4 (group II antigen; clone "E")t PCR (5' UTR-Capsid) PCR (NS4, group I) PCR (NS4, group II)
Pos Pos
Pos Pos
Pos
Pos
Neg Pos Pos Neg
Pos Pos Pos Pos
* Group II NS3 antigen not tested. tA. Nomoto: personal communication (May 1991).
in approximately 50% of acutely infected patients and more than 70% of chronically infected patients (range: 60% to 95%).21-24 Second-generation EIAs based on the use of multiple (three) recombinant proteins derived from both the structural and nonstructural regions of the HCV genorne (Fig l), have shown improved sensitivity with regard to the diagnosis of acute infection and have shortened the time to the initial detection of virus-specific antibodies by 3 to 12 weeks in those individuals who did not demonstrate seroconversion during the acute phase of disease. Another ElA format for the detection of virus-specific antibodies has recently been described. 25 This assay makes use of synthetic peptide antigens representing three immunodorninant regions of the structural and nonstructural proteins of HCV, namely, capsid (C22), NS3, and NS4. When used to screen pane1s of human and chimpanzee sera, this assay was found to detect antibody 4 to 10 weeks earlier than the original ElA that used the C100-3 (NS4) recombinant protein. Although the "window" period between the acute phase of disease and appearance of
VIROLOGY OF HCV
virus-specifie antibodies has been shortened by both types of second-generation assays, a significant proportion of individuals (25% to 40%) are still found to be negative for HCV-specific antibody. It is anticipated that other more sensitive serologieal assays based on the use of novel antigens and formats will further shorten this window in acutely infected patients. PCR methodologies, although experimental in nature and cumbersorne to perform, may mature to the level of clinically useful tests and provide a reliable means of detecting virus-specific genorne sequences in patient sera or donor plasma. 26-28 Reverse transcription (RT) ofHCV RNA followed by amplifieation of the cDNA by PCR or "nested" PCR (the latter employs the use of pairs of external and internal primers complementary to the HCV cDNA) has been shown to be highly sensitive and specific for the detection of HCV when used under controlled conditions. 26 For example, the RT-PCR procedure can detect as little as 0.1 cm in chronic-phase plasma (A. Weiner, personal communication, June 1990). In cases in whieh acute HCV infection is suspected, but antibody results are still negative, ie, the window period, the RTPCR method can be used to confirm infection with HCV. The results of one studl7 also suggest that the detection of HCV viral sequences in donor blood by PCR is a better predietor of infectivity than the presence of anti-C100-3 (anti-NS4) alone. PCR-based assays may also prove to be useful adjunctive tests to monitor the course of disease in chronically infected patients. PCR methods have already been used to determine the relative abundance of HCV RNA in patients who have been treated with a-interferon (J. Hoofnagle, personal communieation, September 1990); a similar use of PCR can be anticipated for patients who may undergo alternative antiviral therapy, including treatment with synthetic organie molecules incorporating antihelicase and/or antiviral protease activities (protease and helicase functions are encoded by the NS3 region of the HCV genome). PERSPECTIVES IN VACCINE DEVELOPMENT
In HogCV, gp55 or the analogous BVDV gp53 seems to be of major importance for neutralization of pestiviruses because neutralizing antibodies are directed toward this gene produet. As previously tnentioned, it has recent1y been shown that HogCV gp55 or BVDV gp53 forms disulfide-linked het-
99
erodimers with a putative transmembrane protein, HogCV gp33 or BVDV gp25, respectively (Fig 2 illustrates BVDV). Although live attenuated virus vaccines for HogCV have been used, some countries have placed restrictions on their use because infection with virulent virus cannot be readily differentiated from immunization with tissue culturederived, attenuated HogCV. Rumenupf et al recently developed a series of recombinant vaccinia virus/HogCV constructs, one of whieh expressed all four structural proteins; the latter construct was successfully used to immunize pigs against a lethal challenge with wild-type HogCV. 29 Similar vaccinia virus/HCV constructs have been commercially prepared (as proprietary candidate vaccines) and recently used to immunize chimpanzees against infection with 100 chimpanzee infectious doses of a factor VIII HCV isolate. The status (success) of this trial is current1y unknown; however, the strong similarity between HCV and HogCV within the putative structural gene region of their respective genomes would suggest that this strategem may eventually lead to the development of an effective recombinant vaccine for HCV, provided proper glycosylation, cleavage of the polyprotein, and folding ofthe individual viral proteins occurs. However, if the presumption (previously discussed) that the "NSI" region of HCV encodes for a structural protein of HCV (E2) is incorrect, then additional approaches involving recombinants of HCV with vaccinia or other vectors and other regions of the genorne will have to be explored. Recent vaccinia virus expression studies by Shimotohno et al (personal communication, May 1991) seem to support the notion that three structural proteins (capsid, El, and E2) are encoded within the HCV genorne covering a region from the extreme 5' end of the ORF to the carboxy terminus of NS 1 (putative E2). Cleavages of the expressed polyprotein seem to occur between aa 191 and 192 (amino terminus of El) and aa 384 and 385 (amino terminus of E2); the carboxy terminus of E2 seems to resuIt from a cleavage between aa 737 and 738. These cleavages have been verified by mierosequencing of the amino termini of the respective proteins. Other expression studies conducted by Saitoet al (personal communication) suggest that HCV El contains approximately 190 aa and is glycosylated to yield gp35; E2 (NSl) yields a glycosylated protein with a molecular weight of 70 to 72
100
kd. It is elear that definitive biochemical studies of authentie viral proteins, ineluding microsequencing of gel-separated proteins recovered from hightitered, HCV-infected chimpanzee liver tissue are needed to confirm and elarify the above results. Other studies that might further define the exact nature of the structural proteins of HCV could involve in vitro translation of the entire ORF of HCV with production of bona fide virus particIes and/or transfection of cells with full-Iength cONA with synthesis of complete virus partieles. Although much of what is described herein is speculative or incomplete in nature, there remain other possible impediments to the development of a recombinant or tissue culture-derived HCV vaccine. There is nowelear evidence for the existence of a hypervariable (HV) region within the amino terminus of the putative HCV E2 (NS 1) protein, as previously described (Fig 3). This finding has lead to the concern that antibodies directed against one variant may not necessarily protect against infection by another variant if the HV region is involved in virus neutralization. Although chimpanzee cross-challenge studies conducted in the early 1980s indicated that reinfection by HCV does not readily occur, it is equally important to note that animals infected with HCV often developed persistent infection, thereby confounding (and probably negating the value of) these results because of the probabIe effects of viral interference. Clearly, more work needs to be focused on the importance of the HV region of HCV and its potential for compounding the problems of vaccine development. Another potentially confounding factor to be cdnsidered in vaccine development would be the presence of different serotypes of HCV. We and other investigators (A. Nomoto, et al, personal communication, May 1990) have recent1y acquired experimental evidence for the existence of serologically distinct groups of HCV that can be distinguished from the prototypie factor VIII isolate on the basis of virulence in primates and/or the results of serological and nucleic acid (PCR) tests (Table 2). In spite of the potential difficulties in developing a vaccine we believe that the unexpectedly high worldwide prevalence of HCV (average, 0.6%), and the related long-term sequellae (cirrhosis, liver cancer, aplastic anemia, etc) will demand that more resources be focused on efforts to: (l) further
DANIEL W. BRADLEY
delineate the molecular biology of HCV; (2) propagate HCV in tissue culture; (3) define the mechanisms of pathogenesis, including persistence of infection; and (4) develop asafe, cost-effective vaccine based on either cell culture-derived virus or a recombinant DNA construct. PATHOGENETIC EVENTS OF HCV REPlICATION INVIVO
Recent studies condueted in two ehimpanzees that had been experimentally infeeted with patient "H" plasma showed that HCV sequenees could be detected as early as 3 days after inoculation; HCV RNA (as eONA) was found to be positive through the peak of alanine aminotransferase (ALT) aetivity.30 In these same animals, anti-ClOO-3 beeame detectable 13 and 32 weeks after inoculation, respectively, or 5 and 25 weeks after the respective peaks of ALT activity. These findings indicated a very early replicative phase of HCV in whieh no virus-specifie antibody could be detected before, or just after, the peak of ALT aetivity. These findings imply that some blood donors who are in the incubation phase of disease could be potentially infectious, underseoring the need to eonsider deteetion of virus-specifie nueleic acid sequenees rather than antibody. In another study, long-term continued observation follow-up of five human patients and four chimpanzees infected with HCV 31 again showed that viremia oeeurred shortly after transfusion/infection (1 to 5 weeks) and that antiClOO-3 appeared 12 to 30 weeks after exposure to HCV. This same study also showed that the disappearance of HCV from serum was eorrelated with resolution of disease. Recent studies eonducted at the US Centers for Oisease Control32 ,33 have clearly shown that early viremia is biphasic in experimentally infected chimpanzees and that HCV-speeifie antigen ean be deteeted in hepatoeyte eytoplasm by an immunofluorescent antibody probe up to several weeks before the first elevated ALT value. As observed earlier, anti-ClOO-3 responses were often delayed or even absent in HCV-infeeted ehimpanzees. The use of a modified second-generation assay for antibodies to a eombination of HCV-speeifie proteins representing portions of the capsid, NS3, and NS4 domains showed that the antibody "window" eould be shortened but that viremia still preeeded
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VIROLOGY OF HCV
the appearance of antibody. Furthermore, no particular pattem of antibody response(s) could be correlated with either resolution of disease or progression to chronicity. Although some investigators have speculated that development of persistent HCV infection may be associated with the introduction of HV region neutralization "escape" mutants, no data have yet been presented to support this notion. Further stud-
ies of the HV region of the HCV genome, as wen as other regions of interest, may provide critical insight into the mechanism(s) underlying persistence of infection. It should also be noted that some variants of HCV may be more or less virulent than others, as previously discussed. How do such variants .arise and what are the important genetic factors (mutations, deletions, insertions, etc) that confer these unusual phenotypic properties?
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30. Shimizu Y, Weiner A, Rosenblatt J, et al: Earlyevents in hepatitis C virus infection in chimpanzees. PNAS 87:64416444, 1990 31. Farci P, Alter H, Wong D, et al: A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 325:98-104, 1991 32. Krawczynski K, Beach M, Bradley D, et al: Hepatitis C virus antigen in hepatocytes: lmmunomorphologic detection and identification. (submitted for publication) 33. Beach M, Meeks E, Krawczynski K, et al: (manuscript in preparation)