Identification Of Specific HIV-1Mutations On Drug Resistance And Replication Capacity

Our government put forward free antiretroviral therapy （ART） in2003. By theend of September,2009the coverage rate of ART had been up to73.5%. Along withthe time of ART, treatment failure has been emerging during HIV-1infected person.Virus hiding resistance is the major reason for failure. One feature of ART in ourcountry is that four subtypes are dominant in China including CRF07_BC,CRF01_AE, CRF08_BC, and subtype B, different from subtype B predominating inU.S.-European. Second is that the drug of ART is imitated in China. Third, we takethe medical model that township health and Centers for Disease Control andPrevention （CDC） unified medicine and management of the patient. All these featurescontribute to drug resistance of HIV-1strain having its own characteristics and rulesin our country. We have been carried out epidemiology surveillance of HIV-1drugresistance strain and obtained a series of important data, such as prevalence,influential factor and effect of ART. During ART, we have been identifying mutationsas novel resistance mutations. However, impact of novel mutations on HIV-1drugresistance, pattern of mutations, interaction of mutations and impact of novelmutations on HIV-1fitness did not be study in-depth. In this study，we analyzed thebaseline HIV-1drug resistance in treatment-na ve HIV-1-infected individuals anddrug resistance in antiviral-failure HIV-1-infected individuals in some areas of China.Subsequently, we constructed plasmids carrying mutation or combination ofmutations associated with antiviral therapy. After transfected into human embryonickidney （HEK293T）, we test resistant phenotype in the presence of antiviral drugscommonly used in our country using recombinant virus. Then, we analyzed thecontribution of single mutation to resistance and the interaction between any twomutations via appropriate analysis method. Finally, we studied the replication capacityof HIV-1strains carrying associated mutations. The aims of all the research to masterthe prevalence status of HIV-1drug-resistant strains in our country, to clarify theinteraction between mutations and the biocharacteristics of drug-resistant viruses, toprovide theoretical basis for recognizing mechanism of HIV-1drug-resistant in clinical, to give practical guidance to ensure the effect of antiviral treatment.Part I Cross-Sectional studies of HIV-1genotypic resistance insome Chinese areasPresently, HIV-1is appearing and circulating widely around the country. It is thefoundation of the effective treatment for patients in clinic to master the prevalence ofHIV-1resistance viruses. The purpose of this study is to understand the molecularepidemiology characteristics and to guide further research and antiviral therapy inclinical. We collected304plasma samples of treatment-na ve HIV-infectedindividuals from8provinces/cities in2009and476plasma samples oftreatment-failure HIV-1-infected individuals from Henan and Hebei provinces in2010.We performed HIV-1drug resistance by genotypic drug resistance assays. The polgene2.0kb fragment was amplified through a nested polymerase chain reaction（PCR）, including99amino acid of protease （PR） and560amino acid of transcriptase（RT）. We calculate the prevalence of drug-resistant strains and to analyze the factorsassociated with drug resistance.Result:（1） We obtained321sequences successfully in treatment-failureHIV-1-infected individuals of Henan and Hebei. Prevalence of HIV-1drug resistancewas up to74.6%in treatment-failure HIV-1-infected individuals. NRTIs and NNRTIsresistance mutations were54.21%（174of321） and65.42%（210of321）, respectively.TAMs and M184V dominated in NRTIs, while K03N and Y181C were the mostcommon NNRTIs resistance mutations followed by G190A/S and H221Y.Combination of Y181C and H221Y were23.33%（7of30） and16.58%（31of187）in Hebei and Henan, respectively. Tri-mutation K103N/Y181C/H221Y was6.67%（2of30）and11.76%（22of187） in Hebei and Henan, respectively.（2） We obtained269sequences successfully in treatment-naive HIV-1-infected individuals. Baselinedrug resistance was7.43%in treatment-na ve HIV-1-infected individuals andShenzhen held the highest baseline drug resistance with17.86%, significant higherthan other areas （χ2=4.9327, P=0.0264）. Baseline drug resistance was significanthigher in subtype B than in subtype non-B （P=0.0177）. K103N, Y181C, V179D/E,G190E and K238N were indentified in NNRTIs resistance mutations, while mutations（N88S, D30N and L90M） leading to high-level resistance NFV were identified in PIresistance mutations. In these samples, CRF55₀1B the novel recombinant form was prevalent with9.84%in MSM in Guangzhou and Shenzhen in2009.Conclusion: Affected by sample size and saved method, the result may beaffected by the low rate of amplification. This study gives an overview of resistance intreatment-failure and treatment-na ve HIV-1-infected individuals. Drug resistance hasbeen serious in treatment-failure HIV-1-infected individuals in Henan and Hebei andaffected subsequent ART. Baseline drug resistance is affected by areas and subtypes.Subtypes circulating in MSM are becoming more and more complex. Following theART result of baseline resistance individuals contribute to clarify the impact ofbaseline drug resistance on ART.Part II Identification of specific HIV-1mutations on drugresistanceMutations of HIV-1resistance are becoming complex. By2008, it has beenreported more than200mutations associated with HIV-1resistance and novelmutations are detected and identified constantly. In the presence of drugs, some nativepolymorphic sites of virus gene shifted to mutations associated with HIV-1resistanceeasily. Presently, the role of major primary mutations is clear in resistance. However,the role of many mutations which are novel identified or shifted from polymorphicand the interaction between them or the interaction between them and major primarymutations are unclear. Due to the major subtypes circulating in China different fromEurope-US and uniquely therapy model of group treatment with domestic genericdrugs, emerging and circulating of resistance mutations are endowed with distinctivefeatures. The purpose of this study is to clarify the role of H221Y in HIV-1resistantand interaction with other mutations. We obtained5HIV-1pol gene hiding mutationsassociated with resistance from HIV-1-infected individuals’plasma samples. Then, weconstructed recombinant virus in backbone of HIV-1pNL4-3. On this basis, weconstructed28HIV-1virus carring different combinations of mutations viasite-directed mutagenesis, transfected HEK293T, harvest virus.21Virus containingdifferent combinations of mutations were constructed and were cultured in MT2cells.After we measured50%tissue culture infectious dose （TCID50）, we performed thesusceptibility of virus to four anti-HIV agents in TZM-b1cell with wild-type pNL4-3as reference. Each drug sets11concentration gradients and each concentration sets2repeat wells and all experiments were performed in duplicate in three different dates. The50%inhibition concentration （IC50） was calculated and factorial design was usedto analyze the contribution of each mutaion to resistance and the interaction betweenthe various mutations.Result:（1） K103N/T215Y resulted in an88.98-fold significantly increases ofIC50of EFV, followed by V179E/T215Y, Y181C and K101Q with18.46-fold,6.12-fold and6.76-fold, respectively. H221Y single mutation hardly affected the IC50value of EFV （1.41-fold）. There were no interaction between H221Y andK103N/T215Y and V179E/T215Y whose IC50of EFV was increased88.98-fold and18.46-fold, respectively. While, there were interaction between H221Y and Y181Cand K101Q whose IC50were increased6.12-fold and6.76-fold, respectively（F=12.49, P=0.0028and F=28.10, P<0.0001）. H221Y may antagonize the resistanceof K101Q to EFV and leaded to the IC50drop to the same with wild-type from6.76-fold.（2） K103N/T215Y and V179E/T215Y diaplayed significantly increase ofIC50of AZT with30.66-fold and13.72-fold, respectively. Y181C significantlyreduced IC50of AZT with0.335-fold. The IC50affected by H221Y were changingwith basic mutations. There were no interaction between H221Y and K103N/T215Yand V179E/T215Y whose IC50of AZT was increased30.66-fold and13.72-fold,respectively. While, there were interaction between H221Y and Y181C and K101Qwhose IC50were changed0.335.12-fold and1.50-fold, respectively （F=18.71,P=0.0051and F=10.53, P=0.0011）. H221Y may antagonize the resistance of Y181Cto AZT and leaded to the IC50increase to the same with wild-type from0.335-fold.（3） K101Q single mutation decreases the IC50of3TC to0.48-fold, whileK103N/T215Y, V179E/T215Y and Y181C hardly affected the IC50value （1.35-fold,0.80-fold and1.29-fold）. The IC50affected by H221Y were changing with basicmutations. H221Y may antagonize the resistance of K101Q and V179E/T215Y to3TC. K101Q, K103N/T215Y, V179E/T215Y and Y181C did not affect the IC50ofd4T. However, H221Y significantly improved the IC50of d4T （4.71-fold）. There wasno interaction between181and221（P=0.9708）.Conclusion:（1） This is the first time that we introduce factorial experimentaldesign and analytical methods to find the contribution of H221Y mutation to drugresistance and interactions with other sites.（2） H221Y significantly increasesresistance to d4T and significantly smaller increase of EFV resistance. However,effects of H221Y on resistance to AZT and3TC are different with other mutations.（3）Y181C significantly improves the sensitivity of AZT.（4） Interaction between basic mutations which significantly change the IC50of EFV and AZT and H221Y are notsignificant.Part III Replication capacity study of HIV-1viruses carryingresistance associated mutationsHIV-1resistance mutations may cause damage to the virus activity and lead todecrease of replicate ability. However, Compensation mutations are to make up forthe loss of replicate ability. The purpose of this study was to elucidate the replicateability of HIV-1carring commonly mutations. We adopt a parallel culture to inoculateequal HIV-1viruses constructed in Part II in MT2cells. Then, we cultured ten days inabsent of drugs and harvested the supernatant to TZM-bl. At48hours post-infectionTZM-b1, relative luminescence units （RLU）/well were measured with wild-typepNL4-3as reference. We draw the replication kinetics and analyzed the result withrepeated measures of two-factor quantitative data hypothesis testing.Results:（1） Replicate ability of pNL4-3179/181/215was the strongest in MT2with71.69and132.94folds of wild-type pNL4-3in the third and fourth day, respectively.（2） Dating the fifth day, the replicate ability of almost all the HIV-1viruses carryingmutations were stronger than wild-type （2.21-145.69folds）.（3） Y181C and H221Yincreased the replicate ability of pNL4-3179/215, respectively. While, the two sitessiaplayed decreases results of pNL4-3103/215.Conclusions:（1） This is the first time to find the replicate ability ofV179E/Y181C/T215Y, V179E/H221Y/T215Y, V179E/T215Y, K103N/T215Y andK103N/Y181C/T215Y is stronger than wild-type pNL4-3. All this may be affected bythey belong to NNRTIs resistance mutations.（2） The increase of replicate ability maybe one of the factors of resistance.（3） We should be on high alert the HIV-1viruseswith high-level resistance and high replicate ability. It is important to monitor theseclosely.