Study On The Impact Of Viral Mutations Generated During Early Infection Stage On HIV-1 Fitness

Human Immunodeficiency Virus(HIV)is the pathogen of Acquired Immune Deficiency Syndrome(AIDS).After HIV-1 infects the human body,escape mutations are generated in the viral genome under the selective pressures from host immune system,resulting in reduced viral replication capability.In other words,these mutations lead to fitness loss of HIV-1.However,this damage can be repaired by compensatory mutations.Recent studies have shown that in the early stage of HIV-1 infection,escape mutations generated under T cell immune responses can cause viral fitness loss,which leads to a reduction in plasma viral load from a peak to a set point level,and thereby decreases pathogenicity and the possibility of transmission from an AIDS patient to a healthy person.Therefore,studying the impact of viral mutations generated under the selective pressures of host immune system during early infection stage on HIV-1 fitness will help to understand the relationship between viral fitness and the disease progress,and provide a theoretical basis for the development of AIDS vaccine.Previous studies have used heterologous viral genomes,which are usually completely unrelated viral genomes,to detect the effect of immune escape mutations on viral fitness.However,such a detection system cannot accurately elucidate that effect of immune escape mutation due to the presence of different compensatory mutations in the heterologous viral genomes.Therefore,this method fundamentally can only detect the effects of immune escape mutations on the fitness of heterologous viruses.With the recognition of the mechanism of HIV infection and the maturity of technical means,it is currently being studied by obtaining the infectious molecular clone(IMC)of transmitted/founder virus(T/F)establishing the initial infection,and the real effects of immune escape mutations generated under immune selection pressures on the fitness of original T/F virus can be studied.In the detection system,the competitive infection method is widely accepted.But in this method,the viral genomes need to be inserted with different reporter genes away from the mutation site to distinguish each of them in a same culture.However,due to the high recombination rate of HIV-1 genome,it is difficult to rule out the impact of recombination between mutation sites and the reporter genes on the accuracy of fitness detection.Therefore,this paper uses a method of PCR and sequencing to determine the proportion of each single point mutation in a culture supernatant of competitive infection.It can accurately determine the impact of mutations on fitness.In our previous study,in order to study the evolution of the virus in patients more accurately,we focused on the homologous virus from the same host,inferred the sequence of T/F and 6-month(6-mo)viruses from the full-length genomes amplified from the longitudinal plasma samples of patient CH0185 who infected with HIV-1 by single genome sequencing,and synthesized the sequences of IMCs of the inferred T/F and 6-mo viruses,and found that the accumulation of mutations during this stage caused fitness loss.In this study,to investigate which of these accumulated mutations can affect the viral fitness,we constructed different IMCs with single-point mutations from the homologous T/F virus by molecular cloning technology.The infectivity of the prepared homologous viral mutants were determined,and then the replication capability of these viruses was evaluated by parallel infection experiments.The fitness of the mutant virus was evaluated by comparing the changes in the proportion of the T/F and the mutant virus by competitive infection experiments.First,the virus stocks of the T/F virus of patient CH0185(CH0185-T/F)and its seven homologous viral mutants were prepared by transfecting HEK293 T cells with their IMCs.Then,successful generation of these viruses were verified by examination of the expression of virion protein and the normal infectivity of these viruses were confirmed by p24 ELISA.To evaluate the fitness of the homologous viral mutants in host cells,we performed parallel and competitive infection experiments in healthy human PBMC cells.The results showed virus was able to replicate efficiently;In the competitive infection experiments,the replication capability of CH0185-T/F was significantly higher than Gag S66 P,GagY79H and GagQ182 G viral mutants,indicating that these three mutation sites generated during the early infection stage of HIV-1 would reduce the fitness of the virus;And there was no significant difference between the fitness of the remaining four viral mutants GagV118 E,GagG374R,RevP67 S or NefG63 E and CH0185-T/F.The Y79 H mutation occurred in the Gag epitope GTEELRSLY that was specifically recognized by the CTL response.And the Q179 G mutation occurred in the Gag epitope TPQDLNTML(TL9).And these two epitopes have been shown to be associated with immune escape mutations,presumably caused a fitness loss.Therefore,we found that Y79 H and Q179 G in the Gag can cause the viral fitness cost in the competitive infection experiments.In summary,based on the previous work,this paper established a more reasonable method and system for evaluating the fitness of HIV-1 virus mutations using IMC plasmids in PBMC cells,and preliminarily studied the impact of seven homologous viral mutants of patient CH0185 on fitness.In subsequent studies,we are going to study more T/F virus and its mutants of patients,and illustrate the impact of viral mutations generated during early infection stage on HIV-1 fitness.These results will help to understand the interaction between the virus and the host immune system.