Studies Of Gene Polymorphism Of HIV-1 B'nef And Its Influence On Disease Progression In Northern China

PrefaceHIV-1 nef is one of the important factor to influence disease progression, and itspolymorphism may contribute to the infection of HIV-1 and AIDS disease progression.HIV regular factor Nef is a protein which having mutiple effective fuction. It has a sig-nificance effect for viral replication in vivo and pathogenesis of AIDS.It was said thatthe defection and deletion of nef was associated with long-term nonprogression afterinfecting with HIV by studying on subtype B, CRF01 AE and SIV. But it didn'tsupport this conclusion for subtype C in Indian. Because nef exists gene phylogenetic,and if there was difference of disease progression owing to mutational site of aminoacid sequence in different subtpye and different crowd? Subtype B' is the mostwidespread in China, and if gene phylogenetic of nef had effect on disease progressionin HIV-1 B' infected individuals in China. At present, we were studying on sequencevariability of nef in Chinese HIV-1 B' infected individuals preliminarily. It includesanalysis of provirus nef gene phylogenetic and amino acid sequence variability. Andstudy on that if there were defects or/and variation of nef in LTNPs and its influence onHIV-1 disease progression. Another purpose of this study was to assess the degree ofconservation of functionally important domains of nef. All these studies provided ascience evidence for comprehending HIV pathopoiesis mechanism deeply.Materials and methodsEpidemiological and clinical data and whole blood sample of Chinese HIVinfected individuals are collected and conversed at-80 (?). HIV-1 nef gene sequencing:QIAamp Blood Mini Kit (Germany) was used to extract genome DNA includingintegrated HIV-1 provirus DNA from whole blood samples. Nef gene specific primersO1,O2 were used as outer primers to process the first round nested-PCR and I1, I2 wasused as inner primers to process the second round nested-PCR on the extracted DNA. 1% agarose gel electrophoresis was used to detect target amplified DNA fragment, thenthe gel with the target amplified DNA were excised compared with the marker. The GelExtraction Kit was used to purify the PCR product. The BigDye Terminator Sequencingkit was used to detect the DNA sequences with ABI 3130 DNA Sequencing System.Sequences of HIV-1 nef gene nucleotides and amino acid in the HIV-1 infectedindividuals with different disease progression; HIV-1 infected individuals are groupedby different disease progression stage. CLUSTAL W program in the BIOEDIT softwareis used to do the alignment to produce consensus sequences and the gaps are inducedinto the sequences to keep complete translation. Based on it, DNADIST(Kimura's twoparameters method)program is used to compute nucleotides and amino acids variationdistances between different sequences and NEIGHBOR (neighbor-joining algorithm)program is used to produce phelogenetic tree with Mega 3．1．Position and type of theHIV-1 nef variations were analyzed and compared with that of foreign HIV-1 strainssearched from HIV Sequence Database. Statistical analysis included, when appropriate,P-values of the x test with Yates correction or the two-tail Fisher's exact test. Oddsratio(OR) was used to compare the fold association between variant frequency anddisease progression.Result1．Nef sequences phylogenetic analysis in Northern ChinaALL of 72 sequences were B' subtype. LTNPs and TPs distrbute together. And 72sequences were made tree without root with other subtypes of M group. It showed thatthey were at the same branch(yellow part), importing all of them were B' subtype.2．Comparing nef genetic variation distances between LTNPs and TPsin Northern ChinaComputing genetic variation distances of nef sequences with the west B, ThailandB and Chinese prevalence reference strains (B', A/E, B'/C) between LTNPs and TPs.And it showed that they all had a shortest genetic distances with Chinese B' referencestrain B.CN.02．02HNsq4, for 5．09(?)0．61 and 5．17(?)0．61 respectively, and briefly lowerthan the genetic distances within the two groups (5．12(?)0．37 and 5．28(?)0．38respectively).3．Analysis of important domain polymorphism and conservatism of nef acid amino sequences in Northern ChinaIt was showed that there was less variation, deletion and insert in importantdomain of nef acid amino sequences, and importing that it was fairly conservation ineach domain.4．Comparing nef sequences between LTNPs and TPs in NorthernChina(1) Comparing acid amino deletion and insert between LTNPs and TPs in NorthernChina: There was 1 sequence of deletion at site 8 and 9 in two groups respectively;There were 4 sequences of deletion at site 10 in TPs, moreover, only 1 in LTNPs; Therewere 3 sequences of deletion at site 11 in TPs, and only 1 in LTNPs; Position 38-41,there was a four amino acid deletion olny 1 sequence in LTNPs; There were 5sequences of insert after acid domain (EEEE) in TPs; Two sequences in TPs haddeletion at 156 site. There was no significant difference between two groups for acidamino deletion or insert deletion and insert.(2) Comparing acid amino variantion between LTNPs and TPs in Northern China:At position 15, the S(?)R/K/N substitution was happened, but the S(?) R substitution wasmost frequently. The frequency of TPs (64．29%) was higher than LTNPs' (33．33%),and there was significant difference between TPs and LTNPs (P<0．01, OR=3．60(CI95%, 1．21-10．97) ; In the variable length region, with R21(?)K/E/H/I/Q, but Kreplacement was the most frequent, and TPs and SPs mutation frequency was 59．52%and 93．33%, respectively P<0．005, OR=0．11(CI95% 0．02-0．55) ; Before the proteaseprocessing site (CAWLEAQ), K was substitute by E, R, N, and a replacement of R wasmost frequent at position 39．Only happened in TPs(23．81%), P<0．005．There were no significant difference of variantion at other positions betweenLTNPs and TPs.Conclusion1．There was no significant deletion or defect of HIV-1 B' nef gene in NorthernChina.2．The deletion or defects of HIV-1 B' nef gene were not association with disasenonprogression in Northern China.3．It suggests that they were association with delyaing disease progression at position 21 with a replacement of K, E, H, I, Q of HIV-1 B' nef gene in Northern China.4．It suggests that they were association with disease progression at position 15with a replacement of S by R, K, N, and at position 39 with a replacement of K by R, E,N of HIV-1 B' nef gene in Northern China.5．All domains of nef amino acids sequences were comparatively conservative.