The effects of genetic diversity of HCV on the success of antiviral therapy are complex for many reasons. First, two drugs are used, both drugs are likely to have more than one effect, and peginterferon at least does not act directly on any of the HCV proteins. Second, viral genetic variability is only one factor in response; host genetics and immune function are also important. Third, even closely related HCV subtypes such as 1a and 1b differ by ~10% to ~12% at the amino acid level; hence, isolates from different genotypes or subtypes may not be affected the same way at the molecular level by the drugs even when overall response rates to therapy are similar. Finally, all HCV isolates from treated individuals are wild-type strains that established chronic infections; hence, stark differences between isolates are not expected in parameters essential for viral replication or immune evasion. Rather, differences in susceptibility to therapy between isolates from different individuals are likely to be due to quantitative differences in viral functions, with some isolates being somewhat more sensitive than others. Therefore, variations at simple genetic motifs are unlikely to correlate highly with response. Consistent with this prediction, we found only three positions at which variation showed ≥60% correlation with response to therapy but found many diversity differences that were highly correlated with day 28 virological response.
The diversity data summarized in Table led to three conclusions. First, there were strong associations between response to therapy and genetic diversity of the consensus sequences from infected individuals for a few viral genes but little to no association for the other genes. Higher variation was found by multiple analytical methods in sequences from the marked compared to poor responders in genotype 1a NS3 and NS5A and in genotype 1b core and NS3. Associations with response to therapy were also seen with E1, E2, and NS2, depending on the analytical method used. Second, the patterns of association between genetic diversity and response to therapy were not the same for genotypes 1a and 1b. This implies that the two genotypes may be affected by therapy through somewhat different molecular mechanisms. Finally, diversity differences correlating with the race of the patient were seen only for genotype 1b.
Summary of HCV genetic diversity differences by response and race
The diversity differences between infected individuals correlating with response to therapy may be due to either allelic variation leading to differential sensitivity to therapy and/or to differential accumulation of escape mutations due to differing immune pressures in the marked and poor responders. Immune escape mutations can reduce viral fitness (10
), and our data do not preclude such effects in this cohort. However, genetic variability was also significantly higher for the marked compared to poor responders in sequences that are very unlikely to be targets of cellular or humoral immune selective pressures (Fig. ). These include nonepitope regions in core, NS3, and NS5A where the correlation between genetic diversity and response to therapy was strongest. Therefore, selection of immune escape variants cannot explain all of the correlation of pretreatment viral genetic variations with response to therapy. This implies that the higher diversity in the marked samples was due to preexisting allelic variation directly associated with response to therapy. It is well accepted that HCV isolates can differ in sensitivity to alpha interferon-based therapy at the level of the major genotypes (e.g., genotypes 2 and 3 are more sensitive than genotype 1 [18
]). However, the idea that interferon-sensitive and -resistant isolates exist within the major genotypes is controversial. The strong association of viral diversity with early response to therapy within both genotypes 1a and 1b reported here indicates that even within the major genotypes, viral genetic variability leads to differential sensitivity to peginterferon and ribavirin therapy.
A possible confounding issue concerning the relevance of pretherapy HCV sequences to the outcome of therapy is that HCV replicates as a quasispecies and hence can evolve rapidly in response to the selective pressures induced by antiviral therapy. Viral evolution during the first month of therapy could skew our results by misclassifying sensitive HCV sequences into the poor-responder group or resistant sequences into the marked-responder group. Misclassifying resistant sequences into the marked-responder group was not a major problem in this study because 28 of the 31 marked responders achieved SVR; hence, the large majority of HCV quasispecies variants in the marked responders were sensitive to therapy. It is possible that some of the pretreatment sequences in the poor-responder group were sensitive to the drugs and were rapidly supplanted by resistant strains during therapy. To evaluate the degree of viral evolution during therapy, we are sequencing the full ORFs at 24 weeks posttherapy from the 1a patients who did not achieve SVR. Posttherapy sequence data have been obtained for the full NS3 gene and for NS5A amino acids 1 to 328 (of 448) from all 24 1a non-SVR patients for whom a follow-up week 24 blood sample is available. In NS3 the median numbers of variations relative to the reference sequence were 18 prior to therapy and 19 after therapy. When the pre- and posttherapy NS3 sequences from the same patient were compared, the median number of amino acid changes per patient was only 1. In the first 328 amino acids of NS5A, the median number of total variations relative to the reference sequence in both the pre- and posttherapy sequences was 14, and the median number of amino acid changes between the pre- and posttherapy sequences from the same patient was again just 1. Therefore, the pre- and posttherapy sequences were very similar in NS3 and NS5A, the two genotype 1a genes in which high pretherapy diversity correlated with suppression of viremia. This indicates that viral evolution during therapy does not alter the observation that higher sequence diversity correlates with successful suppression of HCV during therapy.
Alpha interferon causes the large majority of the decline in viral titers by day 28 during combination therapy (12
); hence, our marked- and poor-response categories were defined primarily by the response to peginterferon. Alpha interferon is a key player in the innate immune system, and HCV has evolved strategies to inhibit its effects (reviewed in references 22
, and 60
). Therefore, the variable sensitivity to peginterferon of HCV isolates from different individuals is likely due to variations in the sequences and functions of viral proteins that dampen host antiviral responses. The strongest correlations between response to therapy and viral variation were in NS3 and NS5A for genotype 1a and in core and NS3 for 1b. Importantly, all of these genes can actively block the action of alpha interferon in vitro (reviewed in reference 19
). Therefore, the genetic data reported here provide strong support for a role for core, NS3, and NS5A (and, to a lesser extent, E2) in antagonizing the type 1 interferon response induced pharmacologically during treatment of HCV infections in humans.
Core residues R70 and M91 have been associated with response to therapy (2
), but few other studies have examined the role of diversity in the core in the outcome of therapy. We observed a similar association of R70 with marked response for genotype 1b but not 1a; however, M91 was highly dominant in both the marked- and poor-responder sequences. E2 has been more thoroughly studied, because it has a PKR-phosphorylation homology domain (PePHD) which inhibits PKR in vitro (65
). Increased diversity in the PePHD has been correlated with response to therapy in studies of patients infected with genotype 3 (58
) but not in other studies that included genotypes 1 to 3 (1
). We found that by some measures, E2 from marked responders was more diverse than E2 from poor responders for genotype 1a, and this difference approached significance for genotype 1b. However, these differences clustered in the N-terminal third of E2, and there were almost no variations among the isolates in the PePHD for either genotype 1a or 1b.
We observed a strong correlation between HCV diversity in NS3 between infected individuals and response to therapy, and to our knowledge variation in NS3 has not previously been associated with response to alpha interferon-based therapy in humans. The NS3 protease can block induction of type 1 interferon in response in vitro by cleaving signal transduction molecules downstream of the double-stranded RNA sensors Rig-I and TLR3 (reviewed in reference 30
). In our sequences, diversity differences in NS3 correlating with response to therapy mapped exclusively to the helicase domain rather than the protease domain (Fig. ). This implies that variation in NS3 correlating with response to therapy may affect the helicase activity rather than the protease activity or that the helicase domain may modulate the activity of the protease domain allosterically; interdomain communication within NS3 has been reported previously (32
). These results also raise the possibility that variability in the viral replication rate modulated by variation in helicase activity may correlate with sensitivity to antiviral therapy and/or that the helicase may limit accumulation of double-stranded RNA that can trigger innate immune responses.
We found significant differences between viral sequences from marked and poor responders in NS5A, but only in genotype 1a. The functions of NS5A during HCV infection are unknown, but NS5A can attenuate the type 1 interferon response in vitro (19
). Variation within a short region of NS5A termed the ISDR (15
) has been associated with interferon sensitivity during antiviral therapy in some populations (47
). We observed higher variability in the ISDR of marked responders relative to the poor responders, but only in genotype 1a CA patients (Fig. ). The role of genetic variation in NS5A outside the ISDR or the overlapping PKR binding site in response to therapy has been investigated in relatively few studies (14
). These studies found greater diversity in responders to alpha interferon-based therapy, especially in the carboxy-terminal half of the protein which includes the V3 hypervariable region (28
). The Virahep-C samples were more diverse in the marked responders primarily in the carboxy-terminal half of the protein, including the V3 region (data not shown), so our results agree with previous studies of the full NS5A gene. Because most of the variability associated with response in NS5A in our samples was outside the ISDR/PKR binding site, variation in the ISDR may have modulated sensitivity to therapy in some patients, but this is not the only contribution that diversity in NS5A could have made to response of HCV to therapy.
Two recent studies examined diversity differences in E2 and NS5A between CA and AA patients (31
). In E2, sequences from CA patients were more diverse than those from AA patients for both genotype 1a and genotype 1b. This diversity was very significant in genotype 1b, with the majority of the difference clustering in the middle third of the protein. In NS5A, higher viral V3 variability was found for CA than for AA patients, and this increased variability correlated with response to therapy (36
). We found a trend to higher diversity in the CA compared to the AA sequences throughout the viral ORF. For genotype 1a, these differences did not achieve statistical significance for any gene, but for 1b significant differences in E1, NS2, and NS5B were found by at least three analytical methods. The association between diversity and race for NS2 is novel, but the implications of this association are not clear because the role(s) of NS2 in HCV biology is not well known.
This study is the most comprehensive analysis to date of HCV genetic variation in the AA population. Our data do not support the idea that unusual genotype 1a HCV strains infecting AA patients exist, but there may be minor strain differences for genotype 1b between the AA and CA populations that could contribute to the poor response of AA patients to therapy. However, this equivocal conclusion does not preclude a role for viral diversity contributing to the unusually poor response of AA patients to therapy. We selected equal numbers of marked, intermediate, and poor responders in the AA and CA populations for sequence analysis, despite significantly lower response rates to therapy for the Virahep-C AA patients (28% versus 52% SVR rates) (9
). The genetic similarity of HCV strains in the AA and CA patients across the three response classes and the intermingling of the AA and CA sequences in phylogenetic analyses (data not shown) indicate that very similar HCV strains circulate in the two racial groups; hence, the higher proportion of poor responders in the AA population implies that a higher proportion of relatively resistant HCV variants may circulate in the AA population, even for genotype 1a. Therefore, viral sequence variation may contribute to the unusually low response of AA patients to therapy, but the mechanism would be circulation in the AA population of a higher proportion of the same resistant strains found in the CA population rather than circulation of novel resistant strains that are absent from the CA population. Genotype 1b is more common in the AA population than in the CA population (53
), so there is a precedent for differential circulation of HCV strains in the two racial groups.
The goal of antiviral therapy for HCV is SVR, the clearance of detectable viral RNA from serum for at least 6 months posttherapy. Large declines in viral titers by day 28 (such as occurred in the marked responders) correlate well with SVR (17
), with a major confounding factor being the failure or inability of the patients to take sufficient drug amounts during the demanding 48-week treatment regimen. A complete analysis of how viral genetic diversity is related to SVR and other clinical variables will be presented elsewhere, but we have performed an initial characterization of how pretreatment HCV genetic diversity correlates with SVR. The pretherapy sequences were stratified as SVR or non-SVR, and then the total and unique numbers of variations relative to the population consensus reference sequence, the total Shannon's entropy values, and the mean genetic distances in the two groups were compared. For both genotype 1a and genotype 1b, the polyproteins of the SVR sequences were significantly more diverse than those of non-SVR samples at P
≤ 0.01. For genotype 1a, NS3 and NS5A were significantly more diverse in the SVR samples by at least three of the four analytical methods. For genotype 1b, core was significantly more diverse in SVR patients by all four measures, and E2, NS2, and NS3 were more diverse by three of the four measures. Therefore, our basic observation that pretherapy sequence diversity was higher in the day 28 marked responders than in the poor responders for a few select viral genes is also true when the sequences are analyzed with respect to the ultimate goal of therapy, SVR.
Taking the data together, the focal distribution of viral genetic diversity at the patient level correlating with response to therapy and the inability of immune selection to account for all of this diversity lend strong support to the hypothesis that HCV genetic variation modulates efficacy of antiviral therapy in human patients. Because most of the reduction in viral levels at day 28 was due to peginterferon therapy, these data imply that the genes for which diversity differences correlate with early response to therapy function at least in part to blunt the type 1 interferon response in humans. This is consistent with the in vitro immune evasion activities reported for core, NS3, and NS5A (19
). The consistently higher diversity in isolates from the marked responders (for whom the virus failed to counteract the drugs) compared to the poor responders (for whom the drugs were ineffective) implies that the functions of the proteins which counteract the effects of therapy can be impaired by amino acid differences at multiple locations. In essence, diversity would correlate with clearance because there are only a few ways to optimize activity of the viral proteins but many ways to interfere with their function.