KR101723581B1 - A method for predicting replication and diagnosing multi-drug resistant hepatitis b virus - Google Patents

Abstract

The present invention relates to a method for predicting and diagnosing replication of a multidrug-resistant hepatitis B virus.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for predicting and diagnosing a drug-resistant hepatitis B virus

The present invention relates to a method for predicting and diagnosing replication of a multidrug-resistant hepatitis B virus.

Chronic infection by hepatitis B virus (HBV), including inflammation, cirrhosis and liver cancer, is a major cause of the global disease. WHO estimates that approximately 350 to 400 million people are affected by chronic hepatitis B infection (Lee WM. N Engl J Med 1997; 337: 1733-45). Antiviral drugs for CHB relieve liver disease and inhibit progression to liver cancer.

Recently, several kinds of nucleic acid derivatives (nucleos (t), including laminovudine (LMV), telbivudine (LdT), adefovir (ADV), entecavir (ETV), cevudine (CLV), and tenofovir ) ide analogues NAs are used to treat CHB patients (Zoulim F, Locarnini S. Gastroenterology 2009; 137: 1593-608 e1-2 .; Kim KH, Kim ND, Seong BL Molecules 2010; 15: 5878- Marcellin P, Chang T T, Lim SG et al. al . N Engl J Med 2003; 348: 808-16; Chang TT, Gish RG, de Man R et al . N Engl J Med 2006; 354: 1001-10).

All clinically available HBV drugs have a common target for the inhibition of viral reverse transcriptase (RT), so all anti-HBV drugs are reported to exhibit virus resistance resulting from a specific mutation in the RT domain (Zoulim F, Locarnini S. Gastroenterology 20092009; 137: 1593-608 e1-2 .; Locarnini S, Mason WS. Cellular and virological mechanisms of HBV drug resistance. J Hepatol 2006; 44: 422-31; Ghany M, Liang TJ. Gastroenterology 2007; 132: 1574-85; Yim HJ, Hwang SG. Clin Mol Hepatol 2013; 19: 195-209).

LMV is a potent inhibitor of HBV replication, but the resistance rate of LMV has been reported to expand to 23% and 80% of HBV carriers after 1 and 5 years of treatment, respectively (Zoulim F, Locarnini S. Gastroenterology 20092009; 137: 1593-608 (e1-2).

The major mutation in the resistance of LMV is rtM204I / V of the YMDD motif. Since YMDD mutants are mostly replication-defective (Melegari M, Scaglioni PP, Wands JR. Hepatology 1998; 27: 628-33),

This mutation often involves a second mutation that compensates for the replication-deficit of the first resistant mutation to the anti-HBV drug. A mutation involving rtL180M, rtL80I / V, and rtV173L was known as a second mutation, which increases viral replication of the replication-deficient rtM204I / V mutant (Zoulim F, Locarnini S. Gastroenterology 20092009; 137: 1593-608 (e1-2)

ETV is the strongest of the currently used anti-HBV drugs with very low tolerance (Zoulim F, Locarnini S. Gastroenterology 20092009; 137: 1593-608 e1-2;). Mutations associated with this drug resistance include rtI169T, rtL180M, rtS184S / A / I / L / G / C / M, M204I / V, rtS202G / I, and rtM250I / V do. The second mutation was not yet known.

CLV has a profile similar to resistance to LMV.

Genotypic analysis revealed that rtN236T and / or rtA181T / V mutations confer ADV resistance.

Therefore, the occurrence of secondary mutations in drug-resistant HBV polymerase genes is associated with treatment failure, so identification of this mutation will have a significant impact on the treatment strategy for CHB patients.

[Prior Patent Literature]

Korean Patent Publication No. 2001-0075192

The present invention has been made in view of the above needs, and an object of the present invention is to identify a mutation associated with the occurrence of a second mutation in a polymerase gene of drug resistant HBV.

In order to accomplish the above object, the present invention provides a method for detecting hepatitis B virus infection comprising the steps of: a) extracting hepatitis B virus (HBV) DNA from a sample;

b) amplifying the reverse transcriptase (RT) gene of said HBV by PCR, and determining mutation by sequencing,

And determining that the replication activity of HBV is increased when the amino acid of the 269th residue of the reverse transcriptase (RT) of HBV is replaced with isoleucine in the leucine as a result of the sequencing.

In one embodiment of the present invention, the reverse transcriptase (RT) of the HBV is preferably composed of the amino acid sequence of SEQ ID NO: 1,

Preferably, the HBV is drug resistant HBV, but is not limited thereto.

The present invention also provides a composition for predicting HBV replication activity comprising a mutant in which the amino acid at position 269 of the reverse transcriptase (RT) of HBV is substituted with isoleucine in leucine as an active ingredient.

The present invention also relates to a method for detecting hepatitis B virus infection, comprising the steps of: a) extracting drug-resistant hepatitis B virus (HBV) DNA from a sample;

b) amplifying the reverse transcriptase (RT) gene of said HBV by PCR, and determining mutation by sequencing,

As a result of the sequencing, when the amino acid of the 269th residue of the reverse transcriptase (RT) of HBV is replaced with isoleucine in leucine, the HBV has strong replication activity and is resistant to lamivudine (LMV), entecavir (ETV) (CLV) of the HBV.

The present invention also provides a composition for determining the resistance and intractability of a hepatitis B virus multidrug drug comprising a mutant in which the amino acid at residue 269 of the reverse transcriptase (RT) of HBV is substituted with isoleucine in leucine as an active ingredient.

In one embodiment of the invention, the agent is preferably but not limited to lamivudine (LMV), entecavir (ETV), and cevadine (CLV).

Hereinafter, the present invention will be described.

In the present invention, the effect of each mutation on replication activity and drug resistance was examined using mutation analysis and drug susceptibility assay.

The present inventors have confirmed that the substitution in rtL269 is associated with strong replication of multiple drug resistant HBV. Molecular modeling studies have also shown that rtL269I substitution affects replication. Finally, clinical validity of rtL269I replacement was investigated.

Hereinafter, the present invention will be described in detail.

RT  Domain has five ( quintuple ) Mutant CHB  Multiple drug resistance isolated from patient HBV  Characterization of mutants

The present inventors have already reported (Kwon SY, Park YK, Ahn SH et al . J Virol 2010; 84: 4494-503). A multidrug resistant HBV mutant (clone 50-2) was further analyzed for its replication activity and drug sensitivity (Fig. 1).

The Southern blot results showed that clone 50-2 was twice as high as the wild-type WT 1.2mer (Fig. 1a). To confirm that the drug resistance of the clones in vitro drug sensitivity assays were performed using anti-HBV drugs including LMV, CLV, ETV, ADV, and TDF. WT HBV was used as a control; it appeared to be sensitive to all test agents (FIG. 1B). Clones with rtM129L + V173L + M204I + L269I + H337N mutations show complete resistance to LMV, CLV, and ETV; Sensitive to ADV and TDF (Fig. 1C).

This analysis confirmed that the five rtM129L + V173L + M204I + L269I + H337N mutants had strong replication activity and were multidrug resistant HBV.

rtL269I  Substitution WT  And drug resistance HBV  Increase replication of everyone

Since the YMDD mutants are mostly replication-defective, a two-fold increase in the replication activity of the five mutants (with the YMDD mutation) relative to the WT results in the effect of each mutation on the replication activity.

A series of artificial replication -competent 1.2mer HBV replicons reflecting the five mutants were constructed as shown in FIG. 2A. First, the effect of rtM129L, rtV173L, and rtH337N mutations on the replication of rtM204I YMDD mutants was tested. Southern blot analysis of the four mutants (rtM204I, rtV173L + M204I, rtM129L + V173L + M204I, and rtM129L + V173L + M204I + H337N) was performed to determine the cloning activity of each clone compared to WT (FIG. The replication level of the rtM204I mutant was scarcely detected (less than 10% of WT). The second mutation of the LMV-resistant YMDD mutant, rtV173L, enabled the replication of the rtM204I mutant. However, rtM129L and rtH337N did not affect the replication of the YMDD mutant (Fig. 2b).

Although the rtM129L + V173L + M204I + H337N mutation increased replication, replication levels remained less than 10% of the five mutants. Thus, mutants containing different rtL269I were constructed (Figure 2a) and the effect of rtL269I on the replication of all artificial mutants (Figure 2c) was determined. Surprisingly, rtL269I substitutions showed 2 to 7-fold higher replication activity in both WT and YMDD-mutant skeletal HBV constructs (Fig. 2d). This indicates that the effect of rtL269I for replication higher in the mutant skeletons than WT, the order is rtM129L + V173L + M204I + H337N> rtV173L + M204I>rtM204I> WT ( about 7-fold, respectively vs vs times 4- 3- Pear vs 2-fold).

Taken together, these data indicate that the substitution at rtL269 is associated with strong replication of multiple drug resistant HBV and WT.

rtL269I Of multidrug resistance YMDD HBV  Identified as a second mutation in the mutant

The effect of rtL269I substitution on drug resistance was investigated. Because pentavalent mutants showed resistance to LMV and ETV (FIG. 1c), the effect of rtL269I on LMV (FIG. 3) and ETV (FIG. 4) resistance was determined using all artificial mutants.

rtL269I single substitution was sensitive to LMV and ETV treatment similar to WT HBV. When the rtL269I mutation was added to the YMDD mutant, the relative level of replication (no drug versus LMV or ETV treatment) of all artificial mutants was significantly increased despite LMV (Figures 3a and 3b) or ETV treatment (Figures 4a and 4b) It did not change. This data indicates that rtL269I substitution does not affect the degree of LMV and ETV resistance.

To investigate the role of rtL269I on drug resistance, the effect of rtL269I on the replication of all artificial mutants treated with LMV (Figure 3c) or ETV (Figure 4c) was determined. Similar to the results shown in Figure 2c, rtL269I substitution increased replication by about 2.5 to 5 fold (only when treated with LMV or ETV) only in YMDD mutant skeletal HBV.

Taken together, these results indicate that the rtL269I substitution is a secondary mutation for the multidrug resistant YMDD HBV mutant.

rtL269I  Substitution molecule modelling

To determine the molecular basis for the increased activity of HBV polymerase (expressing rtL269I substitution) in WT or rtM204I YMDD drug resistant mutants, the three-dimensional structure of HBV polymerase was modeled through comparative modeling studies (Figure 5a) . rtL269 is distant from the catalytic site of polymerase (~ 27 Å); Ile is not believed to directly affect the substrate binding of the catalyst. On the other hand, since rtL269 is near the template binding residue (rtW284), a small difference between Leu and Ile will affect the binding affinity to the template's nucleoside ( N-3 ) affinity (Figures 5b and 5c).

Compared to rtL269, the rtL269I substitution has a long side chain occupying the same space with the adjacent residue rtR289 (bold arrow in Figure 5c). To avoid the space constraints caused, rtR289 causes a morphological change to move away from the bulky side of rtI269, which makes the nearest rtW284 closer to the template strand (dashed arrow in Fig. Thus, this molecular modeling study shows that rtL269I substitution causes morphological changes in both the RT site and the template strand.

Based on these biochemical results, rtL269I substitution is located in a template strand that forms a more effective complex with the incoming dNTP substrate for the polymerase, suggesting that increased activity of rtI269-HBV polymerase results in both WT and YMDD mutants.

CHB In the patient group rtL269I  Appropriateness of substitution

Based on the in vitro results of rtL269I replacement strongly restoring the weakened HBV polymerase activity of the YMDD mutant, treatment failure or 'sub-optimal' treatment of 'treatment-naive' patients with chronic HBV infection and LMV and CLV, Patients who experienced a response were analyzed. This enables the hypothesis that the rtL269I mutation is associated with persistent viral replication despite antiviral therapy.

First, none of the treatment-naive patients treated with LMV showed rtL269I displacement (Table 1). Of the four patients treated with CLV, one patient (patient 22) showed rtL269I mutation prior to treatment. We analyzed patient 22 who showed a serum HBV DNA level> 60 IU / mL for at least 6 months during LMV or CLV unilateral therapy. 16 patients showed the appearance of a YMDD mutation during a follow-up time of 96 weeks (patients 1 to 18) or 12 to 17 weeks (patients 19 to 22). In conclusion, among 16 patients, 7 (43.8%) showed rtL269I substitution in addition to the YMDD mutation (Table 1).

Despite the history of multiple antiviral therapies, so-called 'inconvenient' cases of persistent viral replication were also analyzed. These cases showed ADV and / or ETV resistance in the background mutant environment. This is because continuous antiviral therapy (switch to ADV or ETV 1.0 mg) is generally administered as rescue therapy in the case of LMV resistance instead of combination therapy. Sequential analysis confirmed that almost all patients (13 in 14 patients with incontinence) showed rtL269I displacement during the follow-up type (Table 2). Thus, this clinical data suggests that the appearance of rtL269I substitution accumulates during therapy because of more complex genotypic resistance and is associated with persistent viral replication during antiviral therapy. This result also supports the in vivo suitability of rtL269I displacement as a secondary mutation in multidrug resistant HBV.

Number gender age HBeAg HBV DNA (IU / mL) Treatment history Mutation profile rtL269I Before Treatment During treatment One M 47 + 5.4 x 10 ⁶ LMV N / A - 2 F 38 + 2.4 x 10 ³ LMV rtL180M, rtM204V, rtL269I - + 3 M 45 + 5.1 x 10 ³ LMV rtL180M, rtM204V, rtL269I - + 4 M 28 + 4.9 x 10 ⁶ LMV rtL180M, rtM204V, rtL269I - + 5 M 32 + 9.1 x 10 ⁶ LMV → ETV 1.0 mg N / A - 6 M 53 + 2.4 x 10 ⁴ LMV rtM204I - - 7 F 54 + 3.8 x 10 ⁵ LMV → ETV 1.0 mg N / A - 8 M 55 + 3.3 x 10 ³ LMV rtL180M, rtM204V / I - - 9 M 28 + 5.6 x 10 ⁴ LMV N / A - 10 F 47 + 2.4 x 10 ² LMV rtL180M, rtM204V - - 11 M 46 + 7.5 x 10 ⁵ LMV rtM204I, rtL269I - + 12 F 36 + 5.1 x 10 ⁶ LMV rtM204I - - 13 F 59 + 4.2 x 10 ⁶ LMV → ETV 1.0 mg rtL180M, rtM204V / I, rtL269I - + 14 F 46 + 8.0 x 10 ⁴ LMV → ETV 1.0 mg N / A - 15 F 41 + 1.6 x 10 ³ LMV N / A - 16 F 25 + 9.8 x 10 ⁴ LMV rtL180M, rtM204V - - 17 M 47 + 9.6 x 10 ¹ LMV rtL180M, rtM204V - - 18 M 31 + 1.1x10 ⁵ LMV rtL180M, rtM204V - - 19 F 48 + 1.1x10 ⁷ CLV rtM204I - - 20 M 39 + 2.6 x ^{10 8} CLV rtM204I, rtL269I - + 21 M 29 + 5.5x10 ⁶ CLV rtM204I - - 22 F 52 + 8.0x10 ⁵ CLV rtM204I, rtL269I + +

Table 1 shows the mutation profiles of patients exhibiting a suboptimal response during LMV or CLV therapy

Number gender age HBeAg HBV DNA (IU / mL) Treatment history Mutation profile rtL269I One M 51 + 7.1 x 10 ³ LMV → ADV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtA181T, rtT184L, rtM204V, rtL269I + 2 M 52 + 1.0 x 10 ³ LMV → ADV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtA181T, rtT184L, rtS202G, rtM204V / I, rtL269I + 3 M 37 - 1.8 x 10 ⁵ LMV → ADV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtM204V, rtN236T, rtM250V, rtL269I + 4 M 36 + 5.9 x 10 ⁵ LMV → ADV → ETV 1.0 mg rtL180M, rtA181V, rtM204V, rtL269I + 5 M 58 + 1.1 x 10 ³ LMV → ADV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtT184L, rtM204V / I, rtN236T, rtL269I + 6 M 52 + 1.9 x 10 ⁵ LMV → ADV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtA181V, rtM204V, rtM250V, rtL269I + 7 M 48 + 4.2 x 10 ⁵ LMV → ADV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtA181T, rt184L, rtM204V, rtN236T, rtL269I + 8 M 58 + 6.4 x 10 ⁴ LMV → ADV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtA181T, rtM204V, rtN236T, rtM250V, rtL269I + 9 M 52 + 1.93 x 10 ⁷ LMV → LMV + ADV → ETV 1.0 mg rtL180M, rt184L, rtM204V, rtL269I + 10 M 53 + 5.20 x 10 ² ADV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtT184L / I, rtS202G, rtM204V, rtL269I + 11 F 67 + 5.73 x 10 ⁴ LMV → ETV 1.0 mg rtM204V, rtL269I + 12 * M 48 + 8.11 x 10 ⁴ LMV → ETV 1.0 mg rtL180M, rtM204V - 13 F 60 + 5.54 x 10 ⁵ LMV → ETV 1.0 mg → ETV 1.0 mg + ADV rtL180M, rtT184L, rtM204V, rtL269I + 14 M 45 + 1.78 x 10 ⁶ LMV → ETV 1.0 mg rtL180M, rtS202G, rtM204V, rtL269I +

Table 2 shows the mutation profile of the patient showing the intact CHB,

* Only one out of 14 challenged CHB cases showed rtL269I mutation.

As can be seen from the present invention, substitution in rtL269 has been shown to be associated with strong replication of multidrug resistant HBV, and molecular modeling studies have shown that rtL269I substitution affects replication.

Figure 1 shows the characteristics of multidrug-resistant HBV isolated from chronic HBV infected patients expressing five mutations rtM129L + V173L + M204I + L269I + H337N. HBV 1.2mer Construct Plasmid (2 mg) was transfected into Huh7 hepatoma cells cultured in 6 well plates (harvesting after 72 h of transfection). (a) Southern blot analysis of HBV DNA levels. (b) In vitro drug susceptibility assay of WT HBV 1.2 mer using LMV, CLV, ETV, ADV and TDF. Cells were treated with each drug for 3 days and the level of replication compared to that of WT (no treatment). (c) In vitro drug susceptibility assays (LMV, CLV, ETV, ADV, and TDF) of mutant HBV 1.2 mers (rtM129L + V173L + M204I + L269I + H337N) from CHB patients. Relative HBV replication levels were quantified using a Phosphorimager. The standard deviation of three independent experiments was measured (***, P <0.001).
Figure 2 shows that rtL269I replacement increases replication of WT and drug resistant HBV. (a) Figure of each HBV mutant construct used in the present invention. (bc) Huh7 cells (70% to 80% confluence) cultured in 6 well plates were transfected with HBV plasmid. HBV DNA levels were analyzed by Southern blot. (d) The relative replication activity of HBV mutants was determined by Southern blot analysis. Relative HBV replication levels were quantified using a Phosphorimager. The standard deviation of three independent experiments was measured (**, P <0.01; ***, P <0.001).
Figure 3 shows the effect of rtL269I substitution on resistance to LMV. (a) All HBV 1.2mer DNA was transfected into Huh7 cells and treated with LMV for 3 days. Intracellular HBV DNA was prepared and DNA levels were determined by Southern blot analysis. Representative results are shown. (b) Relative replicative levels of each HBV mutant (no drug treated vs. LMV treated) were calculated based on the results of Figures 2C and 3A. (c) The relative replication activity of HBV mutants treated with LMV was determined by Southern blot and quantified with a phosphorimager (*, P < 0.05). The relative replica level of each HBV construct was expressed as an average of at least three independent experiments.
Figure 4 shows the effect of rtL269I displacement on resistance to ETV. (a) All HBV 1.2mer DNAs were transfected into Huh7 cells and treated with ETV for 3 days. Intracellular HBV DNA was prepared and DNA levels were determined by Southern blot analysis. Representative results are shown. (b) Relative replicative levels of each HBV mutant (no drug treatment vs. ETV treatment) were calculated based on the results of Figures 2C and 4A. (c) The relative replication activity of ETV-treated HBV mutants was determined by Southern blot and quantified with a Phosphorimager (*, P <0.05; **, P <0.01; ***, P <0.001). The relative replica level of each HBV construct was expressed as an average of at least three independent experiments.
Figure 5 (a) shows the tertiary structure of the modeled HBV polymerase-DNA-TTP complex and (b) the amplified structure around rtL269. N-3 starts at the 3'-end just above the substrate binding site and is the third nucleotide in the template strand. (c) When Leu is mutated to Ile, the rtL269I residue experiences spatial collision with adjacent rtR289 (block arrow), which causes a morphological change in the substrate binding residue, rtW284 (dashed arrow) Causing morphological changes in Tide N-3.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  1: Positioning On mutation  by HBV RT  Mutant Reflexion  build

The HBV RT domain of wild-type WT and the mutant clone with the rtM129L + V173L + M204I + L269I + H337N mutation was obtained from the patient serum and cloned into the HBV 1.2mer replicon using pGEM-4z vector (Promega Corporation, Madison, Wis., USA) (Kwon SY, Park YK, Ahn SH et al . J Virol 2010; 84 : 4494-503).

All artificial mutant clones containing the rtL269I, rtM204I, rtM204I + L269I, rtV173L + M204I, rtV173L + M204I + L269I, rtM129L + V173L + M204I, and rtM129L + V173L + M204I + H337N mutants were all located using WT HBV 1.2mer Specific mutagenesis (Ahn SH, Park YK, Park ES et &lt; RTI ID = 0.0 &gt; al . J Virol 2014; 88 : 6805-18).

All mutant clones were identified by sequencing.

Example  2: cell culture, Transfection  And drug treatment

Huh7 human hepatoma cells, the 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) supplemented a Dulbecco's modified Eagle medium cultured at (Gibco, Grand Island, NY, USA) 37 ℃ in 5% CO ₂ environment in Respectively. These cells were transfected with 2 μg HBV WT and mutant 1.2mer replicons using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

LMV, CLV and ETV were purchased from GlaxoSmithKline (Brentford, United Kingdom), Bukwang Pharm (Seoul, South Korea), and Moravek (Brea, CA, USA). ADV and TDF were purchased from Gilead Science (Foster City, CA, USA). All drugs were treated for 4 days with daily replacement with fresh medium containing each drug. The final treatment concentrations of the drugs were 20 μM for LMV, CLV, ADV, and TDF, and 1 μM for ETV (Kwon SY, Park YK, Ahn SH et al . J Virol 2010; 84 : 4494-503).

Example  3: Drug sensitivity and replication Assay

Cloned HBV DNA was extracted from intracellular core particles and analyzed by Southern blot for the determination of drug sensitivity (Kim JH, Park YK, Park ES et al. al . World J Gastroenterol 2014; 20 : 5708-20).

In summary, cells were transfected with HBV replicon and treated with the indicated drug for 4 days. The cells were disrupted with HEPES buffer containing 1% NP-40. The cell lysate was treated with DNase I (Clontech / Takara Bio, Mountain View, CA, USA) and mung bean nuclease (Clontech / Takara Bio) for 15 min at 37 ° C to remove the transfected plasmid DNA. The intracellular core particles were precipitated with a polyethylene glycol (PEG) solution and the HBV DNA was obtained by treating the capsid protein with proteinase K (Roche Applied Science, Indianapolis, IN, USA) at 37 ° C for 2 hours. After extraction of HBV DNA with phenol and ethanol precipitation, total DNA was separated on 0.8% agarose gel and transferred onto a Hybond-N + membrane (GE Healthcare, Buckinghamshire, UK). HBV DNA was detected using purified and lambda probes labeled with [? - ³² P] dCTP (500 mCi / mmol; Perkin-Elmer Inc., Waltham, Mass., USA). To quantify the replication activity of HBV, the membranes were analyzed using a Phosphorimager and Multi-Gauge v3.2 software (Fujifilm, Tokyo, Japan).

Example  4: molecule modelling

A comparative model of HBV polymerase-DNA-TTP complexes was constructed by sequencing the sequences between HBV polymerase and HIV-1 RT (Das K, Xiong X, Yang H et al . J Virol 2001; 75 : 4771-9), and the crystal structure of the HIV-1 RT-DNA-dNTP ternary complex (Protein Data Bank accession No. 1 RTD) (Huang H, Chopra R, Verdine GL et al . Science 1998; (Maestro, version 7.5, Schrodinger, LLC (2006), New York, NY, USA) based on the Maestro software (Maestro, 282 : 1669-75) Additional HBV polymerase models were constructed by rtL269I replacement. Two Mg ^{2 +} ions, thymidine triphosphate, and substrate-adopts the double-stranded primer from the HIV-1 RT-DNA-dNTP composite structure (1RTD), were placed in the same location in the modeled structure HBV polymerase.

The modeled HBV polymerase-DNA-TTP complexes were equilibrated by molecular mechanics simulation and energy minimization. Modeling studies were performed using a MacroModel (Maestro, version 7.5; Schrodinger, LLC) running on a Linux platform. The complexes were minimized in the presence of the GB / SA continuum water model using the OPLS_2005 force field until no significant migration was observed in the atomic axis prior to molecular role simulation. Conjugate gradient and Polak-Ribiere 1st derivative method were used for energy minimization. Molecular dynamics simulations of the HBV polymerase-DNA-TTP complex were heated from 0 to 300 K in 5 ps using OPLS_2005 in the presence of the GB / SA continuum water model and an additional 10 ps at 300 K Lt; / RTI &gt; Production dynamics simulations were performed for 500 ps from 300 K to 1.5 fs. A shake algorithm was used to limit covalent bonding to hydrogen atoms.

Example  5: HBV RT  Patients and genotypes of genes

A total of 36 chronic HBV carriers registered at Severance Hospital and Konkuk University Hospital were examined. Serological markers for HBsAg and HBeAg were determined as enzyme-linked immunoassays (Behringwerke, Marburg, Germany). HBV DNA levels in all patients were determined by PCR using the Roche COBAS Amplicor system (Roche Applied Diagnostics). All serum samples were reviewed and approved by the Institutional Review Board (IRB).

HBV DNA was extracted from serum samples using DNA Blood Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. The HBV RT gene was amplified by PCR and the mutation was determined by direct sequencing (Kwon SY, Park YK, Ahn SH et al . J Virol 2010; 84 : 4494-503).

Statistical analysis of the present invention was performed using Student t- test. Values of P < 0.05 were considered statistically significant.

<110> Konkuk University Industrial Cooperation Corp <120> A METHOD FOR PREDICTING REPLICATION AND DIAGNOSING MULTI-DRUG          RESISTANT HEPATITIS B VIRUS <130> HY150184 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 344 <212> PRT <213> Hepatitis B virus <400> 1 Glu Asp Trp Gly Pro Cys Thr Glu His Gly Glu His Asn Ile Arg Ile   1 5 10 15 Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys              20 25 30 Asn Pro His Asn Thr Thr Glu Ser Arg Leu Val Val Asp Phe Ser Gln          35 40 45 Phe Ser Arg Gly Ser Thr His Val Ser Trp Pro Lys Phe Ala Val Pro      50 55 60 Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu  65 70 75 80 Ser Leu Asp Val Ser Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala                  85 90 95 Ala Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Pro Arg Tyr Val             100 105 110 Ala Arg Leu Ser Ser Thr Ser Arg Asn Ile Asn Tyr Gln His Gly Thr         115 120 125 Met Gln Asp Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu     130 135 140 Leu Leu Leu Tyr Lys Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His 145 150 155 160 Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser                 165 170 175 Pro Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg             180 185 190 Arg Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val         195 200 205 Leu Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ser Ile     210 215 220 Thr Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr 225 230 235 240 Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Ser                 245 250 255 Trp Gly Ala Leu Pro Gln Glu His Ile Val Gln Lys Leu Lys Gln Cys             260 265 270 Phe Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln         275 280 285 Arg Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly     290 295 300 Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala 305 310 315 320 Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu                 325 330 335 His Leu Tyr Pro Val Ala Arg Gln             340

Claims (11)

a) extracting hepatitis B virus (HBV) DNA from the sample;
b) amplifying the reverse transcriptase (RT) gene of said HBV by PCR, and determining mutation by sequencing,
(LMV) or entecavir (ETV), comprising determining that the replication activity of HBV is increased when the amino acid of the 269th residue of the reverse transcriptase (RT) of HBV is replaced with isoleucine in the result of the sequencing Methods for predicting replication activity in HBV with drug resistance. The method according to claim 1, wherein the reverse transcriptase (RT) of the HBV comprises an amino acid sequence as set forth in SEQ ID NO: 1. Prediction of replication activity in HBV having drug resistance to lamivudine (LMV) or entecavir (ETV) Way. delete delete delete delete delete delete delete delete delete

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