Isolation And Characterization Of HIV-1 Circulating In China And The Effect Of The Rev101ptc On Replication And Evolution

As the pathogen of the Acquired Immunodeficiency Syndrome（AIDS）, Human Immunodeficiency Virus has a high degree of variability. Currently, the major source of the global pandemic is HIV-1 divided into four groups: M（Major）, O（Outlier）, N（Non-M and Non-O） and P. The M group includes 9 major subtypes（i.e., A-D, F-H, J, and K）, over 72 circulating recombinant forms（CRF）, and numerous unique recombinant forms（URF）. The phenotypic characteristics of HIV-1 are also complex, including the replication kinetics, Syncytia inducing effects, cell tropism（ability to grow on primary macrophages（M-tropism）, T cell lines（T-tropism） or both（Dual-tropism））, the chemokine receptors usage（CXCR4/CCR5 usage）, as well as the functional amino acid sequences encoded by envC2V3. Besides, The HIV-1 prevalence and diversity in China has its own characteristic: In China, not only strains of subtype B, but also circulating recombination forms like CRF01_AE, CRF07_BC and CRF08_BC spread widely. Among them, CRF07_BC and CRF08_BC are B/C recombination forms only circulating in China. The biological phenotypes are closely related to each other, as well as the pathogenicity, transmission, immune escape and disease progression of HIV-1.Because the HIV-1 primary isolates which can represent the real situation of the virus in vivo are hard to get, the analysis on phenotypes are more difficult than analysis on nuclear tide sequences. And the results of genic researches can’t be proved by phenotypic experiments. The epidemic of HIV-1 in China are diverse and complex. The major circulating subtypes of in China are CRF07_BC（35.5%）, CRF01_AE（27.5%）, CRF08_BC（20.2%） and B（9.6%）. A variety of new recombinant forms were also popular either recently. Nowadays, there are a large number of studies on gene mutation and evolution of Chinese prevalent HIV-1 strains, much more than the studies on phenotypes. Only a few phenotypic researches based on several isolates neither can made the biological characteristics of the HIV-1 strains circulating in China became understandable, nor explain the changing of pathogenesis. Researches on disease control and therapy like the vaccine development have been affected greatly, either. CRF07_BC is a circulating recombination form with frame of subtype C and several inserts of subtype B generated in Yunnan province. This recombination form circulated in Injecting Drug Users（IDUs） initially, and transmitted into sexually transmitted group recently, especially in Men who have Sex with Men（MSM） population where it might be spreading quickly. A large number of research have found that isolates of CRF07_BC have unique pathogenic characteristics, like the infected persons with HIV-1 of this subtype always have relative higher number of CD4+ cells but slower pathogenesis development. However, the related factors have not been found and analyzed yet.Therefore, we isolated and cultured primary strains circulating in China and determined their phenotypic characteristics fully, proved some correlation between the phenotypes exist. And we developed an HIV-1 subtype panel in China using the virus we isolated which can represent the current diversity of HIV-1 strains circulation in China and used it to evaluate the viral load assays and p24 assays which are commonly used in China. Based on the Rev101 premature termination codon we found in HIV-1 C-like virus like CRF07_BC, we analyzed its effects on replication and evolution. This study were performed in order to determine and understand the phenotypic characteristics of HIV-1 circulating in China fully to lay the foundation for researches on clinical control of the disease, and reveal the correlation between biologic characteristics and the pathogenesis. Part I: Isolation and Characterization on HIV-1 circulating in China and Development of an HIV-1 Subtype PanelThe prevalence of HIV-1 in China is complex and diverse, and the diversity of genotype and phenotype has a significant impact on the spread of the virus and the progression of the disease. At present, the research of HIV-1 diversity in our country mostly stays in the sequence and genotype level, and the lack of biological phenotype studies, the relationship between viral phenotypic diversity and virulence and pathogenicity needs to be clarified. The complex epidemic and significant diversity of HIV-1 strains pose serious challenges for surveillance and diagnostic assays development, vaccine development and clinical management. There is a lack of HIV-1 isolates in current canonical HIV-1 subtype panels that can represent the evolution of HIV-1 diversity in China; an HIV-1 subtype panel for China is urgently needed.Objectives: To isolate and culture the primary isolates of HIV-1 circulating in China, determine their replication capacity and phenotypic characteristics fully, and analyze the correlation between the virus phenotype and pathogenesis. To establish an HIV-1 subtype panel in China using the virus we isolated which can represent the current diversity of HIV-1 strains circulation in China and can be used for HIV-1 testing and treatment assays evaluation.Methods and Results:（1） Samples collection and virus isolation and culture: 48 anticoagulant peripheral venous blood samples from HIV infected persons from 10 provinces were collected by HIV-1 drug resistance and surveillance project. The peripheral blood mononuclear cells（PBMCs） from these samples were co-cultured with PBMCs from healthy blood donors. After 28 days’ culture and expanding culture by adding fresh cells on time in a biosafety level 3 laboratory（BSL-3）, 14 HIV-1 were isolated successfully. In addition, 16 primary isolates were subcultured steadily from 51 HIV-1 strains isolated and stored in liquid nitrogen year before. At last, 30 strains in total of high titer（106-109 copies/ml） /high-volume（>40 ml） viral culture were obtained and stored in liquid nitrogen. The subtypes of the isolates include three major subtypes circulating in China: B（13 cases）, CRF01 AE（12 cases） and CRF07 BC（4 cases）, and one rare subtype G（1 case）. The transmission routes include blood transmission（13 cases）, homosexual transmission（8 cases）, heterosexual transmission（6 cases）, intravenous drug users（1 case）, occupational exposure to infection（1 case） and unidentified（1 case）.（2） Sequencing and subtype identification: the viral genome were amplified and sequenced. Six full-length HIV-1 genome sequence, 25 gag gene, 27 pol gene and 25 envC2V3 region were sequenced successfully. Phylogenetic analysis confirmed 30 samples comprised by four subtypes mentioned before and the CRF01-AE strains belonged to the three transmission clusters.（3） Replication Kinetics: Eleven strains of volume of virus culture within 1ng p24 less than or equal to 500 μl were detected for replication kinetics together with NL4-3 of subtype B as the reference strain. Three strains（GX2005002, HN2002004, GD2005003） were determined to replicated fast and high（R/H） relatively and have the ability to induce syncytia（SI）. Replication capacity of GX2005002 determined to be extremely high and almost as 217 times as the capacity of the reference strain NL4-3.（4） Phenotypes prediction by envC2V3 sequences: 1) Co-receptor usage: 17 of 30 strains use of CCR5 as the co-receptor and regard as M-tropism. 12 strains use CXCR4/CCR5 as the coreceptor and regard as Double-tropism（M/T）. 1 case cannot be predicted. None of the strains use CXCR4 as the co-receptor and regard as T-tropism. 4 strains（3 isolates and the NL4-3） of fast / high replication and induced syncytia are all double-tropism using X4/R5 as co-receptor. SX2010001 of high replication but not induced syncytium formation（NSI） is M-tropism only use CCR5 as the co-receptor. 2) Four amino acids at the tip of the envV3-loop prediction: the four amino acids at the tip of the envV3-loop of the samples include GPGR, GPGQ, GQGR, GPGH and GWGR 5. The 5 strains with relatively high replication capacity all use GPGR. 3) Glycosylation sites on V3: The number of V3 glycosylation sites of all 30 strains ranged from 8 to 13. 17, 30, 36, 42, 98,131 and 137 are the glycosylation sites occurred in most of the strains. The phenomenon that 4 of the 5 strains with high replication capacity have an N-glycosylation at site 3 and this glycosylation never found in other strains may suggest that the N-glycosylation at site 3 may be associated with viral infection and replication capacity.（5） Drug-resistance mutations identified by pol sequences: Drug-resistance mutations were emerged in 16 of the 27 strains whose pol gene were sequenced successfully. Seven of them may cause high-level drug-resistance. Among the 16 strains with drug-resistance mutations, GD2005028 and SD2013001 had never been treated.（6） Evaluation of commonly used assays for HIV-1 testing based on the HIV-1 panel composed of the 30 isolates: Two kinds of viral load quantity assays: NucliSens EasyQ HIV-1 version 2.0 and Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 were evaluate by the virus culture of the 30 strains. Results show these two assays showed great concordance when testing samples of subtype B, while the concordance became less when test samples of CRF01_AE. And this may indicates that the assays still need improve to make the testing of samples of CRF01_AE much more corrective. 2 kinds of HIV-1p24 antigen assays had also been evaluated.Conclusion: Thirty primary strains of HIV-1 with high titer and high volume and stored in liquid nitrogen were obtained. They can represent the genotype and phenotype diversity of HIV-1 circulating in China to some extent. A CRF01_AE isolate with extremely high replication capacity were determined. The correlations between phenotypes like the syncytia inducing ability, replication capacity and co-receptor usage were proved to be exist. Two kinds of commonly used viral load assays were proved to have less concordance when testing samples of CRF01_AE, and that may indicate that their sensibility for testing samples of CRF01_AE need to be improved. This study laid the foundation for revealing the pathogenesis, as well as biological control and clinical treatment researches. Part II: Rev 101 PTC on HIV-1 C-like virus and its biological effect analysisRev is a regulatory protein of HIV-1 that promotes the nuclear export of the introncontaining viral mRNAs by specific binding the Rev Response Element（RRE） on the mRNAs to make the viral protein expression in the cytoplasm happen and it is one of the significant regulatory factors on HIV-1 replication. RRE is a part of RNA on the intron-containing mRNAs after the RNA-editing. It is the target of Rev-protein and by the interaction between Rev and RRE, the mRNA can be transferred from nuclear to the cytoplasm to translate and express. The alignment of the rev sequences of HIV-1 circulating in China showed that a premature termination codon（PTC）（at amino acid 101 in the HXB2 sequence） exist in the coding region of Rev in most subtype C and C-like recombination forms strains, while this PTC are rarely seen in strains of other subtypes.Objective: To determine the distribution of the Rev101 PTC among all the subtypes and circulating recombination forms. To analyze the effects that Rev101 PTC has on Rev-RRE function, HIV-1 replication, evolution and transmission.Methods and Results:（1） Comparison of the frequencies of Rev101 PTC that occurred in each subtype of HIV-1 and HIV-2 to determine whether it is subtype-specific characteristic of subtype C or C-like virus: 3041 available rev sequences of every subtype of HIV-1 and HIV-2 were downloaded from the Los Alamos HIV Database. Sequences of each subtype were corrected and translated into amino acid sequences. The frequency of Rev101 PTC occurred in each subtype were calculated. Results showed that the frequency of Rev101 PTC occurred in subtype C and C-like virus were 93.8%, while the frequency in other subtypes were only 0.9%, and the difference has statistical significance（P<0.001）. It has been proved that the Rev101 PTC is a subtype-specific characteristic of subtype C and C-like virus indeed.（2） Effect of Rev101 PTC on viral replication capacity: 1) the Rev101 codon on infectious clone of subtype B pNL4-3 were mutated from CAA（non-termination codon） to TAA（termination codon）. Plasmids before and after mutation were both transfected on HEK293 T cells to generate viral stocks. The replication capacity of infectious clones before and after mutation were determined. It is found that the replication capacity of NL4-3 decreased significantly（2 to 4 lg values） after Rev101 PTC were introduced. 2) CRF07_BC infectious clone pXJDC6291-13 were used as the representative of HIV-1 subtype C or C-like virus. The Rev101 codon were mutated from TAA（termination codon） to CAA（non-termination codon）. Plasmids before and after mutation were both transfected on HEK293 T cells. It is found that there were no difference between the expression efficiency of the p24 antigen（viral protein that encoded by gag-pol genetic region）. 3) The mRNA secondary structure encoded Rev protein of subtype B and CRF07_BC were predicted. Results showed that the introducing of Rev101 PTC in rev of subtype B caused the “stem-loop” structure alteration, while the secondary structure of Rev mRNA of CRF07_BC never changed whether the Rev101 PTC exist or not and only a base pair altered from GU to GC after the mutation. 4) To test whether the Rev-protein expression efficiency can be changed by Rev101 mutation or not. Rev-expression plasmids with or without the 101 PTC subtype B and CRF07_BC were transfected in HeLa cells with equal quantities, and the expression efficiency were determined by Western Blot. Results showed the expression efficiency were showed in the following order: Rev B > RevBmut（101PTC） while Rev07BC（101PTC） ≈ Rev07 BCmut. These results indicate that Rev101 PTC has effect on the Rev-expression of subtype B while has no effect on Revexpression of CRF07_BC.（3） Construction of GagPol-RRE reporter system to determine the effect that Rev101 PTC may has on Rev-RRE functional activity by Rev-RRE activity assay: 4 Rev-expression plasmids pcDNA3.1（+）-Rev-B, pcDNA3.1（+）-Rev-Bmut（101PTC）, pcDNA3.1（+）-Rev-07BC（101PTC） and pcDNA3.1（+）-07 BCmut were constructed, as well as 2 reporter plasmids pcDNA3.1（+）-GagPol-RRE_B and pcDNA3.1（+）-GagPol-RRE₀7BC。The 2 reporter plasmids were paried with the 4 Rev-expression plasmids respectively to constructed 8 Rev-RRE reaction systems. Different concentration of Rev-expression plasmids were co-transfected with GagPol-RRE reporter plasmids. The exogenous Rev protein help the Gag-Pol expression by binding to the linked RRE specifically. The p24（gag-pol express production） values were plotted to generate dose-response curves of Rev-RRE activity. The slope of the rising part of each Rev-RRE dose-response curve was regard as the relative activity of each Rev-RRE pair. The 8 groups of results of the 8 Rev-RRE reaction systems were compared and analysised in aspects of Rev and RRE respectively. 1) Effects of Rev101 PTC on Rev-RRE functional activity: Results showed that only if the Rev and RRE both came from genome of subtype B, Rev101 PTC can affect Rev-RRE functional activity significantly（P=0.0017）. For the other Rev-RRE pairs, Rev101 PTC didn’t affect their functional activities. The functional activities of Rev-RRE pairs with RRE of subtype B were higher than the activities of Rev-RRE with RRE of CRF07_BC whether they contain Rev101 PTC or not. It indicates that the subtype-specific characteristics of RRE may have greater effects on Rev-RRE functional activity. 2) Effects of subtype-specific characteristics on Rev-RRE functional activity: Only if binding with the wild-type Rev CRF07_BC contain the 101 PTC, the different activities caused by subtype difference of RRE have no statistical significance（P=0.074）. However, when binding with the mutant Rev CRF07_BC without the 101 PTC, the different activities caused by subtype difference of RRE became significant（P=0.017）. The different activities caused by subtype difference of RRE have even more statistical significance when binding to Rev B whether it has the 101 PTC or not（P=0.006 and 0.001 respectively）.（4） Effects of the subtype-specific sites of RRE on Rev-RRE functional activity: Four group of structural and functional key sites and their mutations of RREs of the 2 subtype（B and CRF07_BC） were introduced into the reporter plasmids respectively. Their effects on Rev-RRE functional activity were tested by binding the original and mutated RRE to the Rev-protein of the same subtype. Results showed that, for RRE B, T192 A and G262A-G264 A decrease the Rev-RRE of subtype B functional activity significantly（T192A: P=0.0005; G262A-G264A: P<0.0001）. For RRE CRF07_BC, A111G-G120 A and A262G-A264 G decrease the functional activity of Rev-RRE of subtype CRF07_BC significantly（P=0.0171 and 0.0019 respectively）, while A192 T increase the activity significantly（P=0.0024）. This phenomenon means the effect caused by the nucleotide usage of the 192 th base of RRE were according with the different activities of Rev-RRE of the 2 subtypes. And the each mutations on 262-264 can decrease the Rev-RRE functional activity of the 2 subtypes significantly.Conclusion: Rev101 PTC is specific for HIV-1 C or C-like strains. It may decrease the replication of HIV-1 B strains. And this could be related to its effect to disturb the mRNA structure and decreasing the expression efficiency of Rev itself. While it has no effect on HIV-1 C or C-like strains. The compatibility of C-like Rev with both B and C RREs may explain why the Rev of subtype C is kept in all the circulating recombinant B/C forms and Rev101 PTC may help the C-like Rev to maintain its compatibility. The nucleotide usage of the 192 th base of RRE have made a contribution to the different activities of Rev-RRE of the 2 subtypes. And the 262 th and 264 th base of RRE are significant to keep the functional activity of Rev-RRE.