Biological Characteristics And GP120, Tat Gene Analysis Of B'/C Recombinant And B' Subtype HIV-1 Isolates From China

Since the first HIV-1 infected patient was reported in 1985 in china,there have been 65 thousand patients with HIV-linfection and AIDS.In order to evade the surveillance of immunity system,viruses change themselves continously in the course of interaction with host.The factor of virus change,influence of environment and virus reverse transaction are responsible for HIV-1's many different subtypes and recombinant forms.There are seven kinds of HIV-1 subtype viruses of A,B,B',C,D,G,F and recombinant viruses prevalence in China.epidemiological studies showed CRF-B'C,B'and CRF01-AE were main epidemical viruses in 2000.The percentage of people infected with CRF07-B'C subtype increased from second to first and B' subtype decreased from first to second compared to the result of epidemic investigation in 1998.the percentage of people infected with CRF07-B'C subtype attain 50.20%in 2003.The result of epidemiological survey shows that B'C recombinant virus has stroger transmission effect.There are obvious difference in the ability of transmission and pathogenesis of HIV-1 different subtype.Some patients rapidly progress to AIDS from latent of HIV-1, even died in a few months,Contrariwise,some patients may live fifteen years with HIV-1.However,there are no clinical manifest and laboratory's changes.Pathogenicity of HIV-1 is result of interaction with host.Viruses changes their biological characteristics in the course of transmission.R5 viruses are associated with virus transmission which appear in the latent stage of infection.X4 viruses generally are associated with late stage. more severe stage of infection.Coreceptor switch of virus lead to change of infectivity and replication.HIV-1 can regulate itself to adapt to host's environment consecutively and assure the ability of replication and pathogenicity. Biological characteristics of Virus are associated with changes of gene.There are many complex reasons which can determine and affect the virulence of HIV-1.HIV-1 env glycoprotein is the place where virus invade and adhere to target cell.It is related to the body fluid of virus,the escapist of cell immunity,syncytium of cell,switch of coreceptor and pathogenicity.HIV-1 env gene has five changeable loops,three of which(V1/V2/V3) can determine whether virus combines with CCR5 or CXR4.it is well documented that positively charged,mutations of amino acids at positions 11 and 25,V3 tip or the loss of N-linked glycosylation of V3 Sequence can change coreceptor usage.The transformation of Co-receptor often follows with env mutations entail high risk,ranging from major loss of entry fitness to lethality.Nevertheless,mutations in or near V1/V2 were able to compensate for the deleteriousness and happened before the break of V3 so that virus can be alive and still keep the abilities of replication and pathogenicity.The N-linked glycosylation patterns of the gp120 protein also confer a significant effect on the HIV-1 infectivity and latent influence of transmission.With alterations in N-linked glycosylation patterns being able to successfully mask effective antibody neutralization responses. Several studies have shown that modification of proper glycosylation site of the envelope glycoprotein can affect viral infectivity.So number and site of glycosylation of the envelope glycoprotein has relationship with infectivity.Severity of disease is related to virus fusion and replication ability.Disease progress and severity are associated with replicative ability.High ability of replication lead to decrease of CD4 count and rapidly progress of disease.It is reported that rarefied tat protein can transactivate LTR gene express.Tat protein is an important regulative protein.It is well documented that replication would be influenced by change amino acid in key site of tat.So we can get conclusion that mutation of tat can influence virus biologic characteristics and ability of replication and transmission.So it has important purport to research GP120 and tat protein.B'and B'C subtype virus is main prevalence virus,especially ability B'C recombinant virus transmission is become a focus of disease prevention and control. Phenotype switch is caused by change of gene.Knowledge of Virus biological characteristics and molecule basis can be provided theory proof for prevention and controlHIV-1 prevalence.In order to show the characteristics of phenotype and genotype as well as difference between B'C recombinant virus and B' subtype isolates,we test co-receptor,infectivity viral replication.Biologic characteristics and the relationship with genotype of different subtype strains would be show through three aspect. 1 To investigate the co-receptor usage in different subtype virus and co-receptor switch and relationship with CD4 count and viral load in the progress of disease.2 To researching the major differences of infectivity and replication kinetics between B' and B'C subtype virus..3 G ene of GP120 is enlarged and assayed sequence.in order to analyses the relationship of co-receptor usage and infectivity with GP120.4 To analyze the relationship between viral replication kinetics and tat gene,tat were amplified by nest-polymerase chain reaction(nest-PCR)and sequenced.Methods:1 Sample population:Seventy-one samples were obtained from Xinjiang(n=25), Shanxi(n=8),Anhui(n=24)and Henan(n=14)provinces in the year from 2004 to the end of 2005.This study was approved by the Institutional Research Ethics Community and all subjects signed informed consent forms before blood collection.The average age of subjects was 37 years(range=27-50 years).Among them,40 were male and 21 female. Blood specimens were collected and fractionated by centrifngation.CD4 count and viral load were measured,and viruses were isolated.2 HIV isolates:Viruses were isolated from patients peripheral blood mononuclear cells (PBMCs)and co-cultured with PBMCs which stimulate by phytohemagglutinin(PHA)-stimulated 2-3 day from HIV-1-negative human donors.HIV-1 p24 was quantified using a commercial enzyme-linked immunosorbent assay(ELISA)kit once each week,and the cultures were maintained for 4 weeks.3 Co-receptor assay:GHOST cells were infected with same dose virus stocks.Cells were harvested48-72h post-infection.GFP expression was then analyzed with a flow cytometer.4 Assessment of infectivity:GHOST cells were infected in duplicate with 2 ng of p24 antigen equivalent of virus.The proportion of GFP-expressing cells determined by fluorescence activated cell sorting at 24 h post infection.5 Assessment of growth kinetics:Replication kinetics of viruses was generated in PBMCs from normal seronegative donors,those cells of which were stimulated with 1ug/ml PHA for 48 to 72h,and it were infected with 2 ng of p24 antigen equivalent of each viral isolate,Aliquots of virus supernatant were collected in 1,3,5,7,10.15 days and monitored for p24 antigen production over 15 days with a ELISA kit6 GP120 and Tat sequence analysis:DNA was harvested from PBMCs,,using a DNA Blood Mini Kit(QIAGEN;Hilden,Germany).Nested-polymerase chain reaction (Nest-PCR)was used to enlarge the gp120 region and tat gene.The PCR product was run on an agarosegel by electrophoresis.Purified products(Gel Extraction Kit,QIAGEN) were sequenced and analyzed using GCG Sequence Analysis software.Result:1 We isolated and co-cultured 71 viruses.The env and gag regions of viral sequence from 71 subjects were analyzed and showed that the subtype B' had 46 samples which came from Anhui,Shanxi,and Henan provinces.Twenty-five samples from the Xinjiang cohort belonged to the B'C recombinant viruses.Among 46 B' subtype isolates, thirty-one(67.4%)of B' subtype virus used CCR5 as co-receptor for entry,whereas 15 (32.6%)used both CCR5 and CXCR4.All 25(100%)B'C recombinant subtype samples used CCR5.There was a significant difference of co-receptor usage between B'C recombinant viruses and subtype B' viruses(x~2=10.336 p＜0.001).The relationship between virus co-receptor usage and CD4 and virol load results showed that:CD4 disperse in different range of B' subtype(CD4＞200/ul n=23,CD4 200-100/ul n=9, CD4＜100/ul n=14),proportion of dual tropic increases from 13.04%(CD4＞200/ul)to 50% (CD4＜100/ul)with decreases in CD4+ count in the in subtype B' viruses.This was not seen in the B'C recombinant viral pool.These viruses used CCR5 co-receptor regardless of CD4+ count.Viral loads were dispersed in different range and viruses co-receptor switched from R5 to R5/X4 increases from 0 to 45.83%with the decrease of VL from VL＜10~3 copies/ml to VL＞10~5 copies/ml in subtype B' viruses.Nevertheless,the B'C recombinant viruses used CCR5 co-receptor regardless of viral load(VL＜10~3 copies/ml, n=5;10~3-10~5 copies/ml,n=5;＞10~5 copies/ml,n=15).2 The result of V3 sequence showed that all of 11 B'C recombinant viruses had the GPGQ at the V3 tip,rarely had mutations at positions 11 and 25;positive charges disperse from 2 to 5,there was no loss of glycoprotein in V3 loop.In 20 subtype B' isolates,the GPGK,GPGQ and GPGR were seen at the V3 tip.The ratio of GPGR(70%) is the highest.Simultaneously,there are mutations at positions 11 and 25.Positive charges ranged from 3 to 6.10%glycoprotein was lost in B' subtype.There was a statistical difference of positive charges between two groups.3 The range of infectivity was expressed by x±SD.The infectivity of the B'C recombinant subtype((?)±SD,11.1154±8.1807)was higher than R5/X4 tropic virus ((?)±SD,4.4991±3.62312)and R5 tropic virus((?)±SD,5.0032±4.1502)of the B' subtype, there were significant differences(t=2.498,p=0.020)between B'C recombinant virus and B' subtype.4 The number of N-linked glycosylation is different in different region of GP120. Glycoprotein of V1,V2,V3 and adjoin region of V3 loop influence infectivity. Glycoprotein of V3 loop were lost in B' subtype.Nothing was lost in 11 B'C recombinant virus.There was a significant difference of the ratio of glycoprotein in N130,N133,N136,N144,N160 and N186 site of V1V2 region,N230,N234,N295 in C2 region and N362 site in C3 region between B' subtype and B'C recombinant virus.5 The replication kinetics of B' isolates were significantly increased on day-7.It reached 5000pg/ml and it increased to 10000-15000pg/ml on the day-15.Replication kinetics of B'C subtype began to increase on the day- 7,until day 10,it reached 5000pg/ml.It reached 5000-10000pg/ml in the day-15.B'subtype either R5/X4 strains or R5 isolates, replication kinetics is higher than B'C subtype on the day 7,10,15.There is a significant difference between B' subtype and B'C recombinants virus.6 Sequence of tat exon gene show C-tenn has a significant change,Core and Basic region of tat gene have the same changes.38-48 site is key dot for tat combining with cyclinl.B'C subtype change in 39 and 46 sites.A basic region RKKRRQRRR motif Confers tat binding TAR RNA,then connect with LTR.Our results showed 50,54 and 57 site were replaced by other amino in B'C subtype isolates;however B' subtype virus have little change.Possibly these sites influence B'C recombinant virus replication.Conclusion:1 Confirmed the biological characteristics of main prevalence strains and the regulation of their changes in HIV-1 infection and transmission.1.1 The present study showed B' subtype could use CCR5 and R5/X4 as co-receptor, the proportion of RS-to-RS/X4 augmented with the decrease of CD4+ counts and increase of VL in subtype B' isolates.No matter how CD4+ count and VL changed,all isolates used CCR5 co-receptor for viral entry in B'C subtype.1.2 It has advantage to B'C recombinant virus that persist use CCR5 as co-receptor Infectivity of B'C recombinant virus is higher than B' subtype and B'C recombinant virus persist use CCR5 as co-receptor,so it is beneficial to spread the B'C recombinant virus in Physiological,Virological and Immunological.1.3 Replication kinetics of B'C subtype decreased lower than those of B' subtype in vitro.It is the same as viral load in vivo.The result shows that the disease may progress slower than that of B' subtype infection.2 Founding the diversified genetical causes of HIV-1 are related with its pathogenesis.There are many complex reasons which can determine and affect the virulence of HIV-1.2.1 The third variable region(V3)of the gp120 amino acid is believed to play an important role in viral attachment to co-receptors such as CCR5 and CXCR4.Sequence of V3 shows B' subtype has obvious varieties in positively charged,mutations of amino acids at positions 11 and 25,V3 tip or the loss of N-linked glycosylation of V3 has been cited as one cause.B'C recombinant virus also is highly conservative compared to that in B' subtype of V3 sequence.This study suggests the high conservatism of the V3 sequence and the low charge established the basis to continue using CCR5 as co-receptor in B'C recombinant subtype.Change of V3 loop in B' subtype virus is associated with the phenotypic switch from R5 to X4 virus.2.2 HIV-1 envelope glycoprotein(gp120)of V1/V2 and V3 affect the binding of either CCR5 or CXCR4.Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4.V3 mutations entail high risk,ranging from major loss of entry fitness to lethality.Mutations in or near V1/V2 were able to compensate for the deleteriousness.The N-linked glycosylation of GP120 shows B'C recombinant virus is highly conservative in V3 sequence.Difference of glycoprotein in V1V2 and V3 adjoining region influence virus infectivity2.3 Tat exonl has changes in 39,46,50,54,57key dot of B'C subtype.These findings suggest that some mutations of tat exonl could decrease the ability of regulation transcription of Tat protein.Therefore,tat is an important factor influence the viral replication ability...