The Impact Of S68G On Drug Resistance And Replication Fitness Of K65R Mutants Of HIV-1 CRF01_AE

Objective:Acquired Immune Deficiency Syndrome(AIDS)is one of the infectious diseases that serious threat to human health and still unable to be cured,it is caused by the Human Immunodeficiency Virus(HIV).The genetic variability of HIV-1 results from the high rates of mutation,genetic recombination and virus replication.HIV-1 is divided into different groups,subtypes,circulating recombinant forms(CRFs)and unique recombinant forms(URFs).CRF01_AE is HIV-1 recombinant strains earliest found in the world,originating from Central Africa and now widespread worldwide.CRF01_AE was first introduced to China in the mid-1990s,since 2007 it was rapidly transmit through different routes and widely distributed in various regions of China,become the main HIV-1 epidemic strain in our country.Highly Active Antiretroviral Therapy(HAART)can effectively inhibit the replication of the virus and significantly reduced the mortality related to AIDS.As a result of the accessibility to HAART and the increasing of class of antivirals,most patients can achieve complete inhibition of virology.However,there are still some patients who have failed in antiviral treatment(ART)because of the drug resistance.Infection caused by drug-resistant strains will also made treatment-naive resistance to the ART.Therefore,the International AIDS Treatment Guidelines recommend monitoring drug resistance before and during HAART.Genotypic resistance interpretation systems is the most common method for detecting HIV drug resistance.However,the genotype resistance systems can only be used to indirectly evaluate the resistance level of the virus.On the one hand,most available drug resistance data are obtained from studies on subtype B viruses,this is mainly because of the early availability of drugs and drug-resistance testing in developed countries where subtype B viruses predominate.Currently,genotype resistance interpretation of different subtypes and recombinant viruses is mainly based on the data obtained from HIV-1 B.However,previous studies has indicated that the significant differences of genetic variability between HIV-1 subtypes and that may affect the drug resistance pathway,non-B subtype strains may have unique resistance characteristics.On the other hand,there have study show that discordance between genotypic resistance and phenotypic resistance in vitro and in vivo,especially when the genotypic resistance interpretation are applied for non-B subtype viruses.The discordant phenotypic and genotypic results can also indicate the characteristics of drug resistance of subtype B virus may not be fully applicable to non-B subtype viruses.All of the above studies suggest that to estimate clinical drug resistance,optimize the treatment plan and improve prognosis of the patients,further study the rule of subtype specific resistance mutation and understand the drug resistance mechanisms among different subtype viruses will be key.Although CRF01_AE has become one of the major popular recombinant strains in the world and the characteristics of drug resistance also differ from the subtype B strains,there is no systematic study on viral resistance,drug-resistant mutations and viral adaptability of CRF01_AE strains.Preliminary studies of our group have found that the mutation rate of S68G in patient populations infected with CRF01_AE strains increased obviously after ART and it has no clear mutation meanings.The correlation of each mutation site was analyzed by bioinformatics software,and indicated that there is a significant correlation between K65R and S68G.Previous studies have also suggested that K65R and S68G have a certain correlation,but the significance of S68G has not been clarified.Based on the above background,in this study,we first used the Stanford Drug Resistant Database to compare the mutation rate of S68G before and after antiviral treatment in different subtypes and recombinant strains,and the frequency of K65R combined with S68G;next,to determine whether the increase of mutation rate of S68G after ART was related to K65R,the sequential changes of K65R and S68G and the relationship between them in the follow-up samples of the same study subjects were analyzed by Next Generation Sequencing(NGS);furthermore,the pseudovirus contain the pol of CRF01_AE was constructed that maximumly reserved the characteristics of the gene sequences of CRF01_AE strain,and the effects of S68G and K65R on virus phenotype were further explored through phenotype resistance and growth competition experiment,to provide laboratory basis for interpret the drug-resistant mutation scientifically and use antiviral drugs rationally of CRF01_AE-infected patients.Methods:1.Study subjectsAs of July 2016,a total of 2,300 HIV-1 infected patients received HAART in the red ribbon clinic of the First Affiliated Hospital of China Medical University,and among56 patients with K65R mutation,14 were also carrying S68G mutations.Because this study intends to observe dynamic evolution process of mutation,so,the patient of follow-up time is short were excluded.At the same time,considering that reply multiple mutation can affect the activity of the virus in the phenotypic resistance test,the patient contains a variety of NRTIs related mutation patients were excluded.Finally,four patients were included in the study.2.Analysis of the characteristics of viral sequence in Stanford University DatabaseUsed the Stanford Drug Resistant Database(https://hivdb.stanford.edu/)to compare the mutation rate of S68G before and after antiviral treatment,and the frequency of K65R combined with S68G in different subtypes and recombinant strains.3.Genotype resistance testingHIV-1 RNA was extracted from plasma according to QIAamp Viral RNA Mini Kit operation instructions.In order to observe the development of 65 and 68 amino acid mutations in the HIV pol genome,the coding region sequence of 48~96 amino acids in RT gene was amplified,and the PCR products were purified and quantified.Constructed and standardized libraries and sequenced on the MiSeq?.The mutation rate of 48~96 amino acids in RT gene was calculated by online analysis software HyDRA(https://hydra.canada.ca).4.Phenotype resistance testingPhenotypic drug resistance in vitro testing reference PhenoSense?method.The pol gene fragments of virus were amplified with the coding region of HIV protease 1-99amino acid and reverse transcriptase 1-240 amino acid.Reversed S68G mutation to wild type by Overlap-PCR.Pseudovirus plasmid were constructed by con-transfecting an envelope plasmid p VSV-G and a backbone plasmid pNL4-3-?E-Luc with CRF01_AE infected patient's pol gene fragment that contain K65R+S68G or K65R into 293T cells.Determined infectious titer of viral stocks according to Spearman-Karber.TZM-bl cells were used as target cells to Phenotype resistance testing.The resistance level based on the fold change(FC)of IC50(half maximal inhibitory concentration)of the mutants compared with the wild-type strain.Paired t test with SPSS18.0 statistical software,FC were compared of the K65R+S68G mutants with and the K65R+S68G mutants,P<0.05 was considered statistically significant.5.Contrast genotype with phenotype resistanceSusceptibility of patient virus is expressed as the fold change,and the fold change is compared to cutoff values determined through correlation of drug susceptibility and clinical outcomes data(From Monogram Biosciences).The reference range is:AZT(1.9),TDF(1.4~4),3TC(3.5).Less than threshold of critical value is read as sensitive(S),greater than the threshold of critical value is read as resistance(R),between of them read as partial resistance(P).Genotype resistance interpretation reference to HIVdb 8.4,ANRS 2016,Rega 9.0.1 and GRADE 2017.6.Growth Competition AssayIntroduce the insert fragment contain K65R+S68G or K65R into full-length infectious pNL4-3.Determined infectious titer of viral stocks according to Spearman-Karber.Initial infection ratio of mono-infections is 50%:50%,90%:10%and 40%:60%respectively,three parallel experiments per ratio.Mixed Viral growth kinetics in mono-infections begun at an MOI=0.005,3×10~5 PBMC from HIV-negative person were infected.The culture was removed,and the same volume of cell culture medium was added at the 5,7,10 and 13 days after infection.HIV-1 RNA was extracted and sequencedfromplasma.UsetheChromatQuanwebtool(https://indra.mullins.microbiol.washington.edu/)to calculate viral ratio at each time point.Results:1.There is a significant correlation between K65R and S68G in the CRF01_AE strainWe analyzed the characteristics of virus sequences with and without received ART in Stanford University Drug Resistant Database and compared changes of S68G mutation rate of different subtype strains.It shows that the frequency of S68G increased significantly after receiving ART in most of HIV-1 subtypes except subtype F,from 3.5%(2617/73806)of untreated increased to 6.5%(4690/72522)of after treatment.On the top list of rising proportion of S68G are CRF01_AE strain,it increased by about 10%after treatment.Moreover,we also found that all the subtypes and recombinant strains with the K65R mutation after antiviral treatment the 68th amino acid had extremely high mutation rates at the same time,the most common is from S mutate to G/N/D/K/R,the frequency of the mutation to G is the highest.The frequency of S68G with K65R mutation in the CRF01_AE strain as well,it is even up to 61.8%.2.K65R helps the virus evolve S68GIn this study,the virus gene sequences of 4 CRF01_AE infected patients were analyzed by deep sequencing by NGS to determine the sequential changes of K65R and S68G and the relationship between them.The results showed that in 3 patients,the K65R mutation was first selected under the pressure of drug,2 of which detected K65R mutation some strains were detected with S68G mutation at the same time,finally,almost all virus sequences carry these two mutations,and the other detected the K65R mutation first,and then some strains further produced S68G mutation.In another patient,the natural polymorphism of S68G was found in almost all virions without treatment,some of the virus strains produced K65R in 7 months after treatment,the time of detection K65R mutation is later than the other 3 cases.3.S68G is not affect the drug sensitivity of K65R mutantsIt is suggested that S68G is often selected in combination with K65R,in order to determine whether the S68G mutation change the resistance levels of K65R mutant to most NRTIs,we returned S68G to wild type as control,the model of pseudovirus was constructed for phenotype resistance detection.Due to the low frequency of K65R in the fourth follow-up point of 301635 patients,it was failed to obtain inferior species by clone and not included in the phenotype resistance assays.In vitro phenotype resistance experiment show that the S68G mutation did not significantly change the resistance levels of K65R mutant to AZT,TDF and 3TC.4.Genotype resistance interpretation overestimated the resistance level of K65R to TDFThe comparison between phenotype resistance and genotype resistance interpretation found that both methods suggested that the mutant strain was sensitive to AZT and was resistant to 3TC.Interesting,the genotypic interpretation algorithm considers that the virus with the K65R mutation is highly resistant to TDF,however,the phenotype results show K65R mutation is partial sensitivity to TDF,and the genotype resistance interpretation algorithm significantly overestimate the resistance level of the K65R mutant to TDF.5.The strain with double mutation has stronger replication adaptabilityBecause the S68G mutation did not significantly change the resistance levels of K65R mutant to most NRTIs,we hypothesized that S68G was coselected with K65R to restore the replication fitness of the K65R virus.To test our hypothesis,a growth competition assay was developed that allowed determination of the replication fitness for each mutant in combination with K65R viruses.As expected,the proportion of K65R single-mutation virus were gradually decreased with prolonged incubation time,indicating reduced replication capacity of the K65R virus in the absence of drug.However,the K65R/S68G double-mutation virus were gradually increased,it is suggested that the strain with double mutation has stronger replication adaptability which can gain the advantage of growth.Conclusions:In the CRF01_AE strain,K65R was first selected under the drug selection pressure that helps the virus evolve S68G.S68G is a natural polymorphism of CRF01_AE strain,it did not affect the drug sensitivity of K65R mutants but restore the replication defect associated with the K65R mutation.Furthermore,we found that genotype resistance interpretation overestimated the resistance level of K65R to TDF in CRF01_AE strains,further assessment of the extent of K65R impact on drug sensitivity is needed.