Structural Changes in Human Immunodeficiency Virus Type 1 Mutant Integrase Proteins

Shin,Cha-Gyun 中央大學校 遺傳工學硏究所 1993 遺傳工學硏究論集 Vol.6 No.1

본 연구에서는 인간면역결핍 바이러스(HIV-1)의 유전자가 숙주세포의 유전자에 끼어들어 갈 때 작용하는 바이러스 효소(integrase)의 돌연변이 단백질의 구조변화에 대한 연구를 함으로써, 돌연변이 integrase를 갖는 바이러스의 복제와 형태가 비정상적인 것으로 판명된 앞선 연구결과에 대한 원인을 돌연변이 integrase 단백질구조 측면에서 이해하고자 하였다. 돌연변이 integrase 단백질을 in vitro transcription과 translation을 이용하여 생성하기 위하여, 돌연변이 proviral DNA로부터 PCR를 이용하여 돌연변이 integrase유전자를 expression vector에 cloning하였고, 이를 이용하여 돌연변이 integrase 단백질들을 생성하였다. 또한 이미 바이러스 integrase 단백질과 잘 결합하는 것으로 알려진 항체를 이용하여, immunoprecipitation하였을때, precipitation되는 정도가 비슷하여 돌연변이 integrase 단백질이 외형상으로는 wild type과 비슷한것으로 여겨지나, trypsin digestion 형태에서 S147I 돌연변이는 wild type과 달라서, 이 point mutation은 integrase단백질의 부분적인 구조를 바꾼 것으로 사료되며, 이러한 변화가 돌연변이바이러스의 복제불능을 유발할 수 있을 것으로 추측된다. In a previous work (Shin et al., 1993) several human immunodeficiency virus type 1 mutant viruses which have point mutation at integrase region showed defective viral replication, abortive infection or abnormal virion morphology. To understand these results in aspects of protein conformation, some of mutant integrase DNAs (wild type, D116A, S147I, and E152A) were cloned into expression vector by using PCR-amplification. Wild type and mutant integrase proteins were prepared through in vitro transcription and translation, respectively. All integrase proteins were immunoprecipitated by rabbit anti-integrase antiserum in same level, indicating that there is no dramatic changes in structure of all mutant proteins. However, partial digestion of the proteins with trypsin showed that cleavage pattern of S147I mutant protein was different from that of the wild type, suggesting that point mutation in the mutant protein may induce conformational change in local region and be further responsible for defective viral replication.

면역결핍 바이러스의 효소 integrase의 endonucleolytic activity

신차균 中央大學校 遺傳工學硏究所 1995 遺傳工學硏究論集 Vol.8 No.1

면역결핍바이러스의 integrase 단백질은 바이러스 DNA 유전자를 감염세포의 유전자에 공유결합으로 끼워넣어 바이러스 유전자의 복제와 바이러스의 생성을 가능하게하는 바이러스 증식에 필요불가결한 효소이다. 본연구에서는 박테리아에서 overexpression으로 생산한 integrase 단백질의 endonucleolytic 활성의 발현 조건을 조사하였다. 방사능으로 표식된 이중나선의 oligonucleotide 기질 0.1 pmol를 integrase 30 pmol로 처리하였을 때, 90분 정도의 반응시간에서 대부분의 기질이 선택적으로 절단되었다. 또한 이러한 절단반응에 조효소로써 Mg++이온과 Mn++이온이 필요한가를 각 이온의 여러 농도에서 조사해보았을 때, Mn++이온의 존재는 endonucleolytic 활성을 위하여는 꼭 필요하며, 최고 20 mM까지 효소활성이 나타나게하였다. 그러나, Mg++이온은 조사된 모든 농도에 있어서 효소활성이 관측되지 않았다. 또한 방사능으로 표식되지않은 경쟁 oligonucleotide를 사용하여 효소활성의 저해를 조사하였을 때, 1 ug이상의 oligonucleotide는 효과적으로 반응을 억제하였다. The integrase protein mediates insertion of viral DNA into cellular DNA that is essential for viral replication and virion production. Using the human immunodeficiency virus type-1(HIV-1) integrase protein prepared from the overexpressed bacteria, basic conditions for evaluation of the endonucleolytic activity were studied in vitro. Short duplex oligonucleotides corresponding to the ends of linear viral DNA were used as substrates. Endonucleolytic cleavages in a reaction containing 0.1 pmol oligonucleotide as substrate and 30 pmol integrase were almost completed in 90 min. Mn++ was absolutely required for the endonucleolytic activity of the HIV-1 integrase while Mg++ was not. The endonucleolytic activities were observed in the presence of 0.2 to 20 mM Mn++. However, the endonucleolytic activity was not detected in any concentrations of Mg++ in the absence of Mn++. Furthermore, the endonucleolytic activity was fairly inhibited in the presence of competitive oligonucleotides. T

Characterization of the Functional Domains of Human Foamy Virus Integrase Using Chimeric Integrases

신차균,Hak Sung Lee,Seung Yi Kang 한국분자세포생물학회 2005 Molecules and cells Vol.19 No.2

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous Nterminal and C-terminal domains.

In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

Yoon, B.,Kim, I.,Nam, J.A.,Chang, H.I.,Ha, C.H. Academic Press 2016 Biochemical and biophysical research communication Vol.473 No.1

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system.

인간면역결핍 바이러스의 효소 integrase의 disintegration 활성

신차균 中央大學校 遺傳工學硏究所 1997 遺傳工學硏究論集 Vol.10 No.1

면역결핍바이러스의 integrase 단백질은 바이러스 DNA 유전자를 감염세포의 유전자에 공유 결합으로 끼워넣어 바이러스 유전자의 복제와 바이러스의 생성을 가능하게하는 바이러스 증식에 필요불가결한 효소이다. 본연구에서는 박테리아에서 생산한 면역결핍바이러스 integrase의 disintegration 활성의 발현 조건을 조사하였다. Oligonucleotide를 이용하여 Y-구조의 기질을 제작하고, integrase와 90 분간 반응시켰을 때, 기질의 20-30 % 정도만이 생성물로 전환되었다. 또한 조효소로 망간과 마그네슘의 적정농도를 조사해보았을 때, disintegration 활성을 위하여 망간을 꼭 필요로 하며, 적정농도는 5-10 mM이었다. 이런 결과는 disintegration활성이 endonucleolytic 활성에 비하여 조효소로 고농도의 망간을 필요로 하며, 반응을 진행시키는 활성의 정도가 상대적으로 약한 것으로 사료된다. Integration of a viral DNA into the host chromosome is a process essential for the complete life cycle of retrovirus, and is mediated by a viral protein, integrase. Using the human immunodeficiency virus type-1 (HIV-1) integrase isolated from the overexpressed bacteria, characteristics of the disintegration activity were studied in vitro. Only 20-30% of the disintegration substrate were converted into the product in 90 min, indicating that the disintegration activity of the HIV integrase is relatively weaker than the endonucleolytic activity of that. As a cofactor, Mn++ ion was required absolutely, while Mg++ ion was not. The optimal concentration of Mn++ ion is 5 to 10mM.

인간 포미바이러스 인테그라제의 생화학적 특성

강승이,오수아,이학성,한성태,서진욱,신차균 대한약학회 2004 약학회지 Vol.48 No.1

A bacterial expression vector for the human foamy virus (HFV) integrase was constructed and expressed in Escherichia coli. By two-step purification using a nickel-chelated column and a SP-sepharose chromatography; the HFV into-grase protein of 43 kDa was purified to near homogeneity, and used to investigate biochemical characteristics of the enzymatic activities, such as endonucleolytic and disintegration activities. Oligonucleotide substrates were specifically and efficiently cleaved by the purifed HFV integrase in the presence of Mn $^{+2}$, but not in the presence of Mg $^{+2}$, indicating that the HFV integrase is not able to use Mg $^{+2}$ as a cofactor Endonucleolytic reaction was almost completed in 60 min at 37 $^{\circ}C$. In addition, the maximum enzymatic activities were observed at 5 mM Mn $^{+2}$ in the buffer of which pH was from 7.0 to 9.0. The endonucleolytic activities were dose-dependently blocked in the addition of baicalein or chicolic acid which is a well-known inhibitor of human immunodeficiency virus integrase.

An analysis of the arm-type site binding domain of bacteriophage .lambda. integrase

Cho, Eun-Hee The Microbiological Society of Korea 1995 The journal of microbiology Vol.33 No.2

The 356 amino acid long lambda integrase protein of bacteriophage .lambda. constains two autonomous DNA binding domains with distinct sequence specificities. The amino terminal domain of integrase is implicated to bind to the arm-type sequences and the carboxyl domain interacts with the coretype sequencess. As a first step to understand the molecular mechanism of the integrase-DNA interaction at the arm-type site, the int(am)94 gene carrying an amber mutation at the 94th codon of the int was cloned under the control of the P$\_$tac/ promoter and the lacI$\_$q/ gene. The Int(am)94 mutant protein of amino terminal 93 amino acid residues can be produced at high level from a suppressor free strain harboring the plasmid pInt(am)94. The arm-type binding activity of Int(am)94 were measured in vivo and in vitro. A comparison of the arm-type binding properties of the wild-type integrase and the truncated Int(am)94 mutant indicated that the truncated fragment containing 93 amino acid residues carry all the determinants for DNA binding at the arm-type sites.

면역결핍바이러스 인테그라제 효소활성을 복구하는 돌연변이

김도진,오유택,신차균 中央大學校 遺傳工學硏究所 1998 遺傳工學硏究論集 Vol.11 No.1

면역결핍바이러스의 integrase는 바이러스 DNA 유전자를 감염세포의 유전자에 끼워 넣어 바이러스 유전자 복제와 바이러스 증식을 가능하게 하는 중요한 효소이다. integrase는 central 지역에 보존적인 DD(35)E motif가 존재하며, 이 지역은 효소의 3가지 활성에 필요하다. 면역결핍바이러스의 integrase는 이 외의 아미노산 돌연변이에서도 활성이 사라짐을 확인할 수 있다. 본 연구에서는 endonucleolytic cleavage 활성과 integration 활성이 사라진 V79E 돌연변이와 endonucleolytic cleavage 활성만 사라진 N120E 돌연변이를 제작, 활성을 확인하고, 두 지역이 모두 변한 돌연변이를 제작하여 활성을 확인한 결과, endonucleolytic cleavage 활성은 여전히 사라지나 V79E에서 소실된 integration 활성이 복원된 것을 확인하였다. The retroviral integrase (IN) mediates a sequence of DNA cleavage and joining reactions in integration of viral DNA copy into host chromosome in the retroviral life cycle. Several residues including valine 79 (V79) in the human immunodeficiency virus type-1 (HIV-1) IN have been known to be important for enzymatic activities. An HIV-IN mutant with V79 mutation was defective for endonucleolytic activity and integration activity but displayed disintegration activity. In order to understand roles of this residue. A second site mutant of N120E in the V79E backbone was constructed. The mutant IN was purified by using a nickel-chelated column. The biochemical properties of the mutant INs were characterized in terms of enzymatic activities. A V79E/N120E mutant was showed to restore the integration activity which was not observed in a V79E mutant. Therefore, it is suggested that the second site mutation of N120E in HIV-1 IN is partially compensatory for structural defectiveness created by the first mutation.

단백질 융합 시스템을 이용한 Bacteriophage Lambda Integrase의 발현 및 정제

이나영,유승구 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.6

The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E.coli TG1 strain crrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni^++-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.

람다파지 Integrase의 이차 구조 예측

유정아,조은희 조선대학교 생명과학연구소 1997 생명과학 연구 Vol.5 No.-

람다 파지의 Integrase(Int) 단백질은 356 아미노산 잔기로 이루어져 있으며 type Ⅰtopoisomerase의 활성을 이용하여 부위특이 재조합 과정을 수행하는 재조합 효소이다. 람다 파지 Int 단백질의 이차 구조를 컴퓨터를 이용하여 예측하였다. 예측된 결과를 x-ray 결정으로부터 이미 그 구조가 알려진 Int 단백질 아미노산 잔기 170 번부터 카르복실기 말단까지 부위(Int c170)의 결정 구조와 비교 분석하였다. 비교한 결과에 의하면 사용된 대부분의 방법이 α-helix를 예측하는 경우 α-helix를 이룰 가능성이 80% 이상이며 다른 구조에 비하여 β 구조의 예측률이 낮음 (35%)을 알 수 있다. 그러나 예측된 β 구조의 64%가 실제 β 구조를 나타내었으며, 이는 helix(66%)나 coil 구조(63%)의 예측률과 비슷하다. 그러므로 예측된 Int 단백질 구조 중에서 강한 DNA binding specificity를 보이는 아미노말단 부분에서는 47-50 번 아미노산 잔기 부근만이 α-helix를 이루고, 이 부분을 제외한 영역은 유동적인 구조를 보일 가능성이 매우 높음을 알 수 있다. 또한 아미노산 잔기 65 번부터 169 번까지의 영역은 40% 이상이 α-helix 구조를 가지고 있을 것으로 예상된다. Secondary structure of Bacteriophage λ Intergrase (356 amino acid residues) was predicted from the amino acid sequence by using methods given by internet on-line service. The predicted secondary structure was compared to the known x-ray crystal structure of Int c170 (residues 170 to carboxyl terminal). Based on this result, we have analyzed the prediction accuracy of the secondary structure of λ Intergrase. When all the prediction methods used in this paper predicted a α-helix structure, the estimated prediction accuracy(the ratio of the number of correctly predicted to be α-helix to the number of all residues predicted to be α-helix) was > 80%. For β structure, the prediction accuracy was lower than that of other secondary structure with a value of 35%. However, the observed accuracy (the ratio of the number of correctly predicted to be β to the number of all residues observed to be β) was 64% while that of α-helix and coil were 66% and 63%, respectively. Therefore we concluded that the amino terminal domain of integrase forms a very flexible structure without any long patches of secondary structure. In the middle part of Integrase from the amino acid residues 65 to 169, over 60% of the domain was predicted to be in α-helical structure.