Comparision Of HIV-1 Quasispecies Variations Between Viral RNA And Proviral DNA And Antiviral Activities Of Pyridinone Compounds On HIV-1 Infection

Background and objectiveHIV-1 reservoir is the main obstacle to HIV-1 cure. Before achieving HIV-1 cure, highly active antiretroviral therapy (HAART) is the most effective treatment method. National Free Antiretroviral treatment Promgram was sacled up in China at 2003, and there were more than 470000 patients receiving antiretroviral therapy at the end of 2015. However, the drug-resistant muations are the major obstcal to the success of antiretroviral therapy. HIV-1 quasispecies dynamically changed under the drug pressure. In the progress of HIV-1 infection, the muatations of plasma viruses could intergrate into provirus persistently. Currently, studies on the quasispecies difference of proviral DNA and viral RNA are limited. Therefore, we analyze the HIV-1 provirus and plasma viruses quasispecies and their impact on long-term antiretroviral therapy using deep sequencing. We also tested antiviral activities of the pyridinone compounds HIV-1 infection. The results could provide more detailed and precise information for drug resistance monitoring and rational design of optimal antiretroviral therapy regimens.Methods1. The study population consisted of 43 HIV-infected patients who received antiretroviral therapy with a 3TC-based first-line regimen from a multicenter HIV-1 therapy cohort in China. According to the treatment outcome of first-line regimen, forty-three HIV-1 infected patients were divided into two groups, including virologic failure group (VF group) and virologic suppression group (VS group)2. The Taqman real-time PCR assay was established to quantify HIV-1 DNA from whole blood. Before treatment, the correlation of proviral DNA load with plasma viral load was analyzed. Deep sequencing was used to detect drug-resistant mutations (DRMs) in reverse transcriptase of pol gene of proviral DNA and viral RNA. The correlation of baseline drug-resistant quasispecies and treatment outcome was analyzed.3. The variants of VF-related positive selection mutations screened by CorMut package and treatment failure were included in Bayesian network to screen the mutations which directly related to treatment failure (direct VF-related mutations), as well as its dynamic changes during long-term antiretroviral therapy.4. The antiviral activities of all of pyridinone compounds on infection by HIV-1 were tested in TZM-bl cells.Results1. Comparision of HIV-1 drug-resistant quasispecies between viral RNA and proviral DNA revealed by deep sequencing in reverse transcriptaseDRMs in reverse transcriptase were detected from plasma and blood of 66 samples of 43 HIV-infected patients using deep sequencing. Among 37 samples with mutations detected,67.57%(25/37) samples harbored minority drug-resistant mutations. The majority DRMs of M41L, M184I, E138A, G190E and M230I were only detected in proviral DNA. The minority DRMs of M184V/I, T215I, K219R, E138K, H221Y and M230I were only detected in proviral DNA.By considering majority DRMs,32.43%(12/37) samples showed differences in drug-resistance interpretation between viral RNA and proviral DNA.33.33%(4/12) samples, with different DRMs in proviral DNA compared with the paired viral RNA, showed a higher level of drug-resistance to the first-line drugs. By considering minority DRMs,62.16%(23/37) samples showed differences in drug-resistance interpretation between viral RNA and proviral DNA.69.57%(16/23) samples were associated with a higher level of drug resistance to tested RTIs for proviral DNA when compared with paired RNA.2. The correlation of baseline drug-resistant quasispecies and treatment outcomeBefore treatment, HIV-1 DNA load was significantly correlated with plasma viral load (p= 0.003). The changes of HIV-1 DNA load were similar to plasma RNA during long-term therapy in 15 patients who experienced treatment failure. The baseline total mutations (RNA&DNA) were difined when the majority DRMs or minority DRMs in either proviral DNA or plasma RNA detected prior to treatment. To evaluate variables that contributed to treatment outcome at baseline, the multivariable logistic regression analysis was performed. In multivariate analysis, baseline total mutations (RNA&DNA) was associated with treatment failure [OR=5.9 (95% CI 1.4-24.7),p=0.01]. VL, CD4+ T cells count, proviral DNA load, DRMs only detected in viral RNA and DRMs only detected in proviral DNA were not significantly associated with the treatment failure. Further analisis indicated that the increasing of 1% in DRMs frequency was associated with 1.3% time the risk of treatment failure.3. Screening the mutations in reverse transcriptase of pol gene directly related to treatment failure (direct VF-related mutations).Before treatment, direct VF-related mutations of plasma RNA included NNRTIs mutations (K101E, K103N, E138K and V179D) and polymorphisms (K64R, K102Q, V111I, D123N, I135T, S162Y, E169D, Q174K, I202V, R211G, R211T, R211M and S251C). Direct VF-related mutations of proviral DNA included NNRTIs mutations (G190Aand V179D) and polymorphisms (E42K, K64R, K102Q, D123N, R125K, Q174K, V1891, R211T, R211M, R211Q, V245E and D250N).During long-term therapy, new direct VF-related mutations were emerged. The new emerged direct VF-related mutations of plasma RNA included NNRTIs mutations (K103S, H221Y and Y181C), NRTIs mutations (M184V) and polymorphisms (131L, K32R, V35I, T39A, K173R, I178M, Q197E and E203D). The new emerged direct VF-related mutations of proviral DNA included NNRTIs mutations (K103S, V179I and G190E), NRTIs mutations (D67N and M184V/I) and polymorphisms (K32R, V35I, T39A and G51R). During long-term therapy, the direct VF-related mutations including K103N, Y181C, K102Q, E169D, R211G,1202V, R211T and V245E were persisted with high frequency.4. Antiviral Activity of pyridinone compounds on HIV-1 infection3-Iodo-4-(2’-methylcyclohexyloxy)-6-phenethylpyridin-2(1H)-ones, as effective non-nucleoside reverse transcriptase inhibitors, were synthesized and resolved with different configurations, including 7-cis,7-mix,7-trans,7-trans-1 and 7-trans-2. For wild-type strain HIV-1 SF33 and double mutant strain A17,7-cis,7-mix,7-trans,7-trans-1, 7-trans-2 exhibited activities with SI ranging between 323 to 100000 and 8.2 to 2564 respectively, and without displaying obvious cytotoxicity on TZM-bl cells.7-trans-2 strongly inhibited the replication of the prevalent wild-type and mutant strain A17 with EC50 values at 3 nM and 117 nM respectively, and with high selectivity index (SI> 2500). All the tested isolates of the representative subtypes were sensitive to thetrans-isomers (except 7-cis) at a nanomolar range (EC50s:0.4 nM to 90 nM) with low cytotoxicity. It is noteworthy that the 7-trans-2 was susceptible to all tested strains with EC50 values ranging from 0.4 nM to 57 nM and showed lowcytotoxicitywith high SI values from 5,000 to 750,000.ConclusionsWe had study the the impact of HIV-1 drug-resistant quasispecies on long-term therapy based on plasma viral RNA and proviral DNA. HIV-1 proviral DNA was positive correlated with plasma RNA, and could assist to act as one of parameters for surveillance of the disease progression of HIV-1 infection. The provirus quasispecies are different from plasma viruses as revealed by deep sequencing. The drug-resistant quasispecies of plasma viruses and proviruses detected prior to ART cloud influence the treatment outcome. Moreover, the high frequency of DRMs in viral RNA could increase the risk for treatment failure. The DRMs and polymorphisms emerged before ART or during ART may influence treatment failure. Target to the mutations with high frequency, the pyridine ketone compounds, five conformation compounds have a relatively stronge inhibitory activities both for laboratory standard strains and drug-resistant strains, and also have a wide inhibition to the major epidemic strains of HIV-1 in China. Therefore, the combined analysis of viral RNA and proviral DNA using deep sequencing should be suggested to act as a meaningful method to increase the sensitivity of HIV-1 drug resistance testing for drug resistance monitoring. These results are important to drug resistance monitoring, rational design of optimal antiretroviral therapy regimens and drug development.