Abstract
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Hepatitis B Virus genotypic differences map structurally close to NRTI resistance hot spots
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Stefan G. Sarafianos, Ph.D., 471d Christopher S. Bond Life Sciences Center, 1201 Rollins St., Columbia, MO 65211, USA, Tel: +1 (573) 882-9357, Fax: +1 (573) 884 - 9676, Abstract
Despite the availability of a Hepatitis B Virus (HBV) vaccine, there are approximately 350 million people that are chronically infected with this virus that can cause liver cirrhosis and hepatocellular carcinoma. Currently, most approved anti-HBV drugs are nucleoside RT inhibitors (NRTIs) that target the viral enzyme reverse transcriptase (RT or P gene product). They suppress viral replication very efficiently but require long-term therapies, which invariably lead to the development of drug resistant viral strains with drug resistance mutations at the P gene. Because the reading frames of the P and S (surface antigen) genes partially overlap, selection of NRTI-resistance mutations may impart changes on the surface structural landscape of the virus. Conversely, genotypic differences on viral surface residues may also change the amino acid composition of the P gene and in terms affect HBV RT properties such as susceptibility to NRTIs. Interestingly, several studies have shown that patients infected with HBV from various genotypes respond differently to NRTI therapies. Here, we built a three-dimensional homology model of the catalytic core of HBV RT using HIV-1 RT as a template. We then mapped on the molecular model the residues that vary among various HBV genotypes. Surprisingly, the genotypic variability residues are generally in the vicinity of residues that are involved in NRTI resistance. Our results suggest that emergence of NRTI resistance mutations in HBV RT may be constrained by structural interactions with residues that vary among different genotypes.
1. INTRODUCTION
Hepatitis B is a life-threatening disease that affects about 350 million people who are chronically infected with the Hepatitis B virus. HBV is the prototype member of the Hepadnaviridae family of DNA viruses, which includes viruses that cause liver infections in humans and animals. The highest prevalence of HBV infection is in Africa and Asia. The virus is transmitted through blood and infected bodily fluids including mother-to-child transmission. Most adults that come in contact with the virus recover after an acute phase and develop immunity against the virus. However, most of the infants and children are not able to clear the infection and are chronically infected. The virus itself does not kill the infected cells, but the immune response that is elicited upon HBV infection can cause dangerous symptoms. These symptoms include liver inflammation and jaundice that may lead to chronic hepatitis. Chronically infected HBV patients are more susceptible to hepatocellular damage, which may eventually lead to liver cirrhosis and hepatocellular carcinoma.
Hepatitis B prevention involves vaccination of infants which induces protective antibodies in more than 95% of infants and children. Currently, there are seven FDA approved drugs for the treatment of chronic HBV infection; two formulations of interferon alpha (IFNα) and five nucleos(t)ide analogs: lamivudine (LVD), adefovir (ADV), entecavir (ETV), telbivudine (LdT), and tenofovir (TDF). Despite the availability of a successful vaccine and several antiviral drugs, HBV remains a serious global health problem that causes an estimated 600,000 deaths per year (World Health Organization).
The HBV genome is circular partially double-stranded DNA of about 3,200 bases for the full-length strand. It has a compact organization with four partially overlapping open reading frames (ORFs) that encode for seven known proteins. For example, the S ORF encodes the surface antigen, or HBsAg, which overlaps with the P ORF, which encodes the viral polymerase or reverse transcriptase (RT). Therefore, changes in the surface protein may affect the polymerase, and vice versa. This is important because most approved anti-HBV drugs target the viral RT and the development of drug resistance RT mutations may lead to treatment failure.
Based on differences in the whole genomic sequences there are ten major HBV genotypes worldwide. Several studies have associated genotypic differences with differences in the clinical outcome and response to anti-HBV therapy (1, 3, 4, 6, 11, 12). Here, we have built a molecular model of HBV RT and used it to show that genotypic differences on the surface of HBV viruses may result in changes in the RT gene that may affect NRTI-related drug resistance. Interestingly, many residues that vary among different HBV genotypes are proximal to RT residues that are mutated during NRTI drug treatment, suggesting a possible correlation.
2. EXPERIMENTAL
We used the Stanford HIV Drug Resistance Database to identify residues of HBV RT that vary among different genotypes and compiled a list of these residues in Table 2. A three-dimensional homology model of the HBV RT was built using the sequence alignment-based three-dimensional structure-generating program MODELLER-4 (8) and the coordinates of HIV RT as a template structure (2, 5, 9, 10). Details of the model building are described in (2). Based on this model we analyzed the list of the genotypically variable residues and identified those that are in proximity to drug resistance residues in the primary and/or tertiary structures. The figures of the molecular models were built using PyMOL (www.pymol.org).
Table 2
Residues in red are genotypically variable in treatment-naïve patients and may have an effect on NRTI drug resistance because of three dimensional proximity to residues that are mutated in response to NRTI treatment (shown in green). The table is based on the HBV RT Stanford Drug Resistance Database (http://hivdb.stanford.edu/HBV/DB/cgi-bin/MutPrevByGenotypeRxHBV.cgi) (7).
| RT Residue | Changes in various HBV genotypes | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| A | B | C | D | E | F | G | H | I | |
| 55 | R | R | H | R | R | R | R | R | R |
| 80 | L | L | L | L | L | L | L | L | L |
| 84 | V | V | V | V | V | V | V | V | V |
| 85 | S | S | S | S | S | S | S | S | S |
| 91 | I | L | I | L | L | L | I | L | L |
| 169 | I | I | I | I | I | I | I | I | I |
| 180 | L | L | L | L | L | L | L | L | L |
| 181 | A | A | A | A | A | A | A | A | A |
| 184 | T | T | T | T | T | T | T | T | T |
| 204 | M | M | M | M | M | M | M | M | M |
| 214 | V | V | V | V | V | V | V | V | V |
| 215 | Q | Q | Q | Q | Q | Q | Q | Q | Q |
| 217 | R | L | L | L | L | L | L | L | L |
| 221 | Y | Y | F | F | Y | Y | Y | Y | Y |
| 222 | T | A | T | T | T | T | T | T | T |
| 223 | A | A | S | A | S | A | A | A | A |
| 224 | V | V | I | V | V | V | V | V | V |
| 233 | I | I | I | I | I | I | I | I | I |
| 236 | N | N | N | N | N | N | N | N | N |
| 237 | P | P | P | P | P | T | P | T | P |
| 238 | N | H | N | N | N | S | N | A | N |
| 242 | R | R | R | R | R | R | R | W | R |
| 248 | N | N | N | H | N | H | N | H | N |
| 250 | M | M | M | M | M | M | M | M | M |
| 253 | I | V | V | V | V | V | V | I | V |
| 267 | Q | Q | L | Q | M | H | Q | H | Q |
| 291 | V | V | V | V | V | V | T | V | S |
| 317 | A | A | S | S | S | A | A | A | A |
| 322 | T | T | T | T | T | V | T | V | T |
3. RESULTS AND DISCUSSION
The genotypic polymorphisms of HBV polymerase were identified using sequences retrieved from the HBV RT Stanford database (http://hivdb.stanford.edu/HBV/DB/cgi-bin/MutPrevByGenotypeRxHBV.cgi) (7). We used structural analysis and molecular modeling tools to display the positions of the drug resistance mutations in the HBV RT model that we built as described above. HBV RT mutations that cause resistance to various NRTIs are listed in Table 1. In Figure 1 it is shown that the drug-resistance residues are generally located in close proximity in primary and/or tertiary structure to residues that vary among different genotypes. Table 2 summarizes these residues of RT with the genotypic variations in treatment-naïve patients and the drug resistance mutations in treatment-experienced patients. Specifically, residues that are involved in NRTI resistance and residues that vary among different genotypes are clustered in four regions of RT. The side chains of these residues are shown in Figure 1.
HBV RT homology model was built as described in Methods and in Das et al. (2). Positions that vary among different genotypes and are in proximity to NRTI-resistance residues are shown in magenta in all panels. In panels A, B, C, and D residues involved in resistance to LVD, ADV, ETV, and TDF are shown in yellow, cyan, brown, and blue, respectively. These resistance residues may be at interacting distances from residues that vary among different genotypes. In panel B residues 237 and 238, which are both ADV-resistance mutations and genotypic variants, are shown in green. In panels A, C, and D, the position of residue 204 is depicted with a star and other residues are numbered accordingly. Circles indicate the general location of clusters I–IV. In panel B, cluster I includes residues 84 and 85 and cluster II includes residue 233.
Table 1
Drug resistance mutations in HBV RT in response to NRTI treatments.
| NRTI | RT Mutations |
|---|---|
| LVD | L80V/I, I169T, V173L, L180M, A181T, T184S, M204V/I/S, Q215S |
| ADV | L80V/I, V84M, S85A, A181V/T, V214A, Q215S, I233V, N236T, P237H, N238H |
| ETV | I169T, V173L, L180M, T184G, S202I, M204V, M250V |
| TDF | L180M, A181V, A194T, M204V/I, V214A, Q215S |
| LdT | L180V/I, M204V/I |
Cluster I contains residues 236–238, 221–224, and 84–85
Residues 236–238 (Figure 1B)
RT residues 236, 237 and 238 are important for two reasons: first, they are ADV resistance sites (N236T, P237H, and N238H) (Table 1) and also vary among different genotypes in treatment-naïve patients (residues 237, 238). Specifically, residue 237 is proline (P) in all genotypes, except in F and H, where it is a more flexible threonine (T) (Table 2). Similarly, residue 238 is an asparagine (N) in all genotypes except for genotype B where it is a histidine (H), genotype F where it is a serine (S), and genotype H where it is an alanine (A). In addition, residue 242 is an arginine (R) in all genotypes except for genotype H where it is a tryptophan (W).
Residues 221–224
Residues 221, 222, 223, and 224 vary among different genotypes and are in the vicinity of residue 236. Specifically, residue 221 is a phenylalanine (F) in genotypes C and D and a tyrosine (Y) in all other genotypes. Based on our molecular model the hydroxyl group of Y221 forms a hydrogen bond with the amide side chain of residue 236. This interaction would not be present in F221 (genotypes C and D). Because residue 221 has the potential to interact with residues from clusters I and III, we discuss it as part of both clusters. Residue 222 is an alanine (A) in genotype B and threonine (T) in all other genotypes. Residue 223 is usually a serine (S) in genotypes C and E, and an alanine (A) in the other genotypes. Residue 224 is usually an isoleucine (I) in genotype C, and valine (V) in all the other genotypes. In our model in Figure 1 for clarity purposes residues 222, 223, and 224 are not shown.
Residues 84–85
The drug resistance mutations V84M and S85A cause ADV resistance and are close to residues 237 and 238 that vary among different genotypes and also proximal to the I233V ADV-resistance position, which is close to residue 91 that varies among different genotypes (233 and 91 are in cluster II).
Cluster II contains NRTI drug resistance sites 169, 180, 184, 233, and genotypically variable residue 91
Residue 169 (Figure 1A and C)
Mutation of this residue to a threonine (I169T) causes resistance to ETV and LVD (Table 1). Residue 169 is proximal to residue 91, which is an isoleucine (I) in genotypes A, C, and G and a leucine (L) in all other genotypes.
Residues 180, 181, and 184 (Figure 1A, C and D)
Residues 180, 181 and 184 are involved in drug resistance of multiple NRTIs. Specifically, the L180M mutation causes resistance to LVD, ETV and TDF, the A181V/T mutation causes resistance to LVD, ADV and TDF, and T184G/L/S mutation causes resistance to LVD and ETV. These residues are close to each other and also close to residue 91 which varies among different genotypes and interacts with ETV resistance residue 169.
Cluster III contains LVD- and ADV-resistance residue 80, and genotypically variable residues 217, 242, 221 and 214–215
Residue 80 (Figure 1A and B)
The L80V/I mutation causes resistance to LVD and ADV. It is proximal to three residues that vary among different genotypes: a) residue 217, which is a leucine (L) in all genotypes except for genotype A where it is an arginine (R); b) residue 242, which is an arginine (R) in all genotypes except for genotype H where it is a tryptophan (W); c) residue 221, which was also discussed in Cluster I.
Residue 214 and 215 (not shown)
The V214A mutation causes resistance to ADV and TDF and the Q215S mutation causes resistance to LVD, ADV and TDF. Both 214 and 215 are close to residue 217 which is a leucine (L) in all genotypes except for genotype A where it is an arginine (R). Since residue 217 interacts with resistance mutation L80V/1, we propose that differences in the genotype in residues of Cluster II may have an effect on the role of residue 80 in resistance to LVD and ADV.
Cluster IV contains ETV-resistance residue 250 and genotypically variable residues 248, 253, and 267
Residue 250 (Figure 1C)
The M250V mutation causes resistance to ETV. This residue is part of the “primer grip”, a structural element that helps position the primer strand at the polymerase active site, and is also proximal to residues that vary among different genotypes: a) residue 248 is an asparagine (N) in most genotypes except for genotypes D, F, and H where it is typically a histidine (H); b) residue 253 is usually an isoleucine (I) in genotype A and H and a valine (V) in all other genotypes; c) residue 267 is part of the thumb subdomain and close to the DNA primer grip which interacts with the nucleic acid (thumb subdomain, Figure 1); it is a glutamine (Q) in genotypes A, B, D, G, and I, a leucine (L) in genotype C, a methionine (M) in genotype E, and a histidine (H) in genotypes F and H. The general proximity of residues 248, 253, and 267 to 250 opens the possibility that their interactions are different among different genotypes, thereby having the potential to affect the resistance function of 250 in a distinct ways.
Moreover, we identified genotypically variable residues that do not directly interact with drug resistance residues, but may still have an indirect effect on NRTI resistance as they are close to the DNA binding cleft and may affect nucleic acid binding. Specifically, residue 55 is part of the palm subdomain of RT and close to the template strand of the DNA substrate (not shown) and is an arginine (R) in most genotypes, except in C where it is a histidine (H). In addition, other thumb residues may affect nucleic acid binding: a) residue 291, which is a valine (V) in all genotypes except G and I where it is a threonine (T) or serine (S); b) residue 317, which is usually an alanine (A) except for genotypes C, D, and E where it is a serine (S), and c) residue 322, which is usually a threonine (T) except for genotypes F and H where it is a valine (V).
4. CONCLUSION
Despite the development of a successful vaccine, hundreds of millions of people are still affected by HBV. Although a growing number of NRTIs has helped improve the treatment of HBV infections, NRTI resistance remains a major public health problem. Design of efficient HBV regimens will require a better understanding of the effect of HBV genotypic variability on NRTI resistance. The molecular basis of this relationship is not known. Our study provides the first glimpses of possible interactions between residues that vary in different genotypes and residues that are involved in NRTI resistance. Future biological studies will address the effect of these residues and their interactions in viral fitness, efficiency of replication, and drug resistance.
Acknowledgments
This work was supported by the Ministry of Knowledge and Economy, Bilateral International Collaborative Research and Development Program, Republic of Korea, National Institutes of Health Grants AI076119, AI094715 and AI079801.
Abbreviations
| HBV | Hepatitis B Virus |
| RT | Reverse Transcriptase |
| N(t)RTI | Nucleos(t)ide Reverse Transcriptase Inhibitor |
| LVD | Lamivudine |
| ADV | Adefovir Dipivoxil |
| ETV | Entecavir |
| LdT | Telbivudine |
| TDF | Tenofovir Disoproxil Fumarate |
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Funding
Funders who supported this work.
NIAID NIH HHS (9)
Grant ID: R01 AI076119
Grant ID: R01 AI100890
Grant ID: R21 AI079801
Grant ID: R21 AI094715-02
Grant ID: R01 AI076119-04
Grant ID: R33 AI079801
Grant ID: R33 AI079801-04
Grant ID: R37 AI076119
Grant ID: R21 AI094715
