HIV-1 gag与gp41基因片段的序列特征与亚型研究

本文对华北地区出入境39例HIV-1阳性样本(中国21例,非洲17例,东南亚1例)的gag和env两个基因片段进行了序列特征和亚型对比分析。发现了A、A1、A3、B、C、G亚型和重组亚型03_AB、01_AE、AG、02_AG、07_BC、08_BC、CD和06_CPX共14个亚型,其中重组亚型占57.2%(8/14)。表明HIV-1基因变异较快,亚型分布广泛,重组亚型有增多趋势。此外发现26.7%(8/30)的样本,其gag和env基因区亚型表现不一致。提示在研究HIV-1亚型中应综合gag和env两个基因区的序列特征进行亚型分析。     

Development of restriction fragment-length polymorphism method to differentiate five subtypes of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) isolates have been classified into five subtypes (A to E) based on the sequences of the env variable V3 to V5 region. In this study, we sequenced a partial gag region of 4 and 3 isolates belonging to subtypes C and E, respectively. Phylogenetic analysis revealed that the branching pattern based on the region was similar to that based on the env V3 to V5 region. Here, we propose a protocol to differentiate five subtypes by polymerase chain reaction amplifying 329 bp within the region followed by restriction fragment-length polymorphism analysis using four restriction enzymes.     

Comparative analysis: intracellular precursor polyproteins of baboon endogenous retroviruses and human viral isolate HL23V.

Intracellular precursor polyproteins of three baboon endogenous retrovirus (BaEV) isolates, m7, 455K, and BILN, were compared with the intracellular proteins of the type C human isolated HL23V by radioimmunoprecipitation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide analysis. Human and canine cells infected with m7-BaEV and canine thymus cells infected with BILN-BaEV were characterized by identical precursor polyproteins Pr85gag, Pr70-71gag, Pr65gag, and gPr85env. Canine cells infected with 455K-BaEV consistently showed a slightly different pattern of precursor polyproteins. These included Pr85gag, Pr70gag, Pr67gag, and gPR85env. By tryptic digest mapping of peptides containing [3H]leucine, m7-BaEV and 455K-BaEV were shown to be highly related. By comparison, mapping studies showed that BILN-BaEV was less highly related to m7-BaEV than ws 455K-BaEV. Differences in these related BaEV isolates presumably reflected virus-specific differential cleavage of core protein precursors or alterations in polyprotein primary structure or both. Chase-incubated cells infected with BaEV also contained a stable, p28-related polyprotein termed P72gag. This polyprotein migrated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than the major core protein precursor Pr70-71gag and appeared to arise by posttranslational modification of Pr70-71gag. Immunoprecipitation of extracts of HL23V-infected cells with antisera to simian sarcoma-simian-associated virus proteins and BaEV proteins confirmed that these cells contained two unrelated viral components, one that was similar to m7-BaEV or BILN-BaEV and a second that was related to simian sarcoma-simian-associated virus. Tryptic digest mapping of BaEV and HL23V prcursor polyproteins suggested that the BaEV-like component of HL23V weas more closely related to m7-BaEV than to 455K-BaEV or BILN-BaEV.     

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.     

Human immunodeficiency virus type 1 subtypes defined by env show high frequency of recombinant gag genes. The UNAIDS Network for HIV Isolation and Characterization.

Genetic subtypes of human immunodeficiency virus type 1 can be distinguished on the basis of phylogenetic analysis of their envelope (env) gene. A significant proportion of human immunodeficiency virus type 1 strains was retrospectively shown to result from recombination events between viruses belonging genetically to distinct subtypes (D. L. Robertson, P. M. Sharp, F. E. McCutchan, and B. H. Hahn, Nature [London] 374:124-126, 1995). To establish the frequency of natural infections with recombinant viruses and to exclude tissue culture artifacts, we analyzed plasma samples from the UNAIDS sample collection. The collection includes samples from 53 individuals infected with subtype A (n = 9), subtype B (n = 15), subtype C (n = 1), subtype D (n = 13), and subtype E (n = 15) on the basis of V3 region analysis. Phylogenetic analysis of the gag gene fragment showed intersubtype recombinant genomes in 23 cases: 3 of 9 (33%) of subtype A, 2 of 15 (13%) of subtype B, 3 of 13 (23%) of subtype D, and all of subtype E. Of the 23 recombinant viruses, 19 had a gag gene from one subtype and env from another (B(env)/C(gag), A(env)/C(gag), D(env)/A(gag), and E(env)/A(gag)). Phylogenetic analysis clustered the A(gag) of subtype E viruses as an outgroup of subtype A, suggesting that these viruses may belong to a distinct A' cluster. The remaining four recombinant viruses (B(env)/B(p17)F(p24), A(env)/A(p17)D(p24), A(env)/A(p17)C(p24), and D(env)/ D(p17)A(p24)) had breakpoint crossover sites in the proximity of the p17-p24 protein processing site. We conclude that recombination in the gag gene is highly frequent among the major env subtypes and that selection of recombinants is apparently based on particularly beneficial combinations of gag and env gene products.     

Three laboratory strains of spleen focus-forming virus: comparison of their genomes and translational products

The molecular properties of three laboratory strains of the spleen focus-forming virus were compared. All strains contain genetic sequences related to the env gene of mink cell focus-inducing murine type C leukemia viruses, and each strain codes for a glycoprotein of 50,000 to 52,000 daltons which shares specific immunological properties with the gp70's of mink cell focus-inducing viruses. In contrast to this constancy, gag gene products coded for by these strains vary significantly. The gag and env gene products are synthesized from separate mRNA's, and the mRNA for the env gene product is approximately 18S. Unlike other acute leukemia viruses, which can transform various undifferentiated cells, have large unique sequence cellular gene inserts fused to helper virus gag genes, and have one known genome-length intracellular mRNA, the spleen focus-forming virus transforms only specific hematopoietic stem cells, is an env gene rather than a gag gene recombinant virus, and has a second distinct and smaller class of intracellular mRNA. Our data therefore indicate that the Friend strain of the spleen focus-forming virus is a unique replication-defective acute leukemia virus.     

Evolution of alphaviruses in the eastern equine encephalomyelitis complex.

Evolution of viruses in the eastern equine encephalomyelitis (EEE) complex was studied by analyzing RNA sequences and oligonucleotide fingerprints from isolates representing the North and South American antigenic varieties. By using homologous sequences of Venezuelan equine encephalomyelitis virus as an outgroup, phylogenetic trees revealed three main EEE virus monophyletic groups. A North American variety group included all isolates from North America and the Caribbean. One South American variety group included isolates from the Amazon basin in Brazil and Peru, while the other included strains from Argentina, Guyana, Ecuador, Panama, Trinidad, and Venezuela. No evidence of heterologous recombination was obtained when three separate regions of the EEE virus genome were analyzed independently. Estimates of the overall rate of EEE virus evolution (nucleotide substitution) were 1.6 x 10(-4) substitution per nucleotide per year for the North American group and 4.3 x 10(-4) for the Argentina-Panama South American group. Evolutionary rate estimates for the North American group increased over 10-fold (from about 2 x 10(-5) to 4 x 10(-4)) concurrent with divergence of two monophyletic groups during the early 1970s. The North and South American antigenic varieties diverged roughly 1,000 years ago, while the two main South American groups diverged about 450 years ago. Analysis of multiple strains isolated from an upstate New York transmission focus during the same years suggested that, in certain locations, EEE virus may be relatively isolated for short time periods.     

The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region.

The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trs/art) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels of gag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.     

Evolution and biological characterization of human immunodeficiency virus type 1 subtype E gp120 V3 sequences following horizontal and vertical virus transmission in a single family

It has been suggested that immune-pressure-mediated positive selection operates to maintain the antigenic polymorphism on the third variable (V3) loop of the gp120 of human immunodeficiency virus type 1 (HIV-1). Here we present evidence, on the basis of sequencing 147 independently cloned env C2/V3 segments from a single family (father, mother, and their child), that the intensity of positive selection is related to the V3 lineage. Phylogenetic analysis and amino acid comparison of env C2/V3 and gag p17/24 regions indicated that a single HIV-1 subtype E source had infected the family. The analyses of unique env C2/V3 clones revealed that two V3 lineage groups had evolved in the parents. Group 1 was maintained with low variation in all three family members regardless of the clinical state or the length of infection, whereas group 2 was only present in symptomatic individuals and was more positively charged and diverse than group 1. Only virus isolates carrying the group 2 V3 sequences infected and induced syncytia in MT2 cells, a transformed CD4(+)-T-cell line. A statistically significant excess of nonsynonymous substitutions versus synonymous substitutions was demonstrated only for the group 2 V3 region. The data suggest that HIV-1 variants, possessing the more homogeneous group 1 V3 element and exhibiting the non-syncytium-inducing phenotype, persist in infected individuals independent of clinical status and appear to be more resistant to positive selection pressure.     

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.     

Molecular cloning and characterization of endogenous feline leukemia virus sequences from a cat genomic library.

Recombinant bacteriophage lambda clones from a cat genomic library derived from placental DNA of a specific pathogen-free cat were screened to identify endogenous feline leukemia virus (FeLV) sequences. Restriction endonuclease mapping of four different clones indicates that there are a number of similarities among them, notably the presence of a 6.0- to 6.4-kilobase pair (kbp) EcoRI hybridizing fragment containing portions of sequences homologous to the gag, pol, env, and long terminal repeat-like elements of the infectious FeLV. The endogenous FeLV sequences isolated are approximately 4 kbp in length and are significantly shorter than the cloned infectious FeLV isolates, which are 8.5 to 8.7 kbp in length. The endogenous elements have 3.3- to 3.6-kbp deletions in the gag-pol region and approximately 0.7- to 1.0-kbp deletions in the env region. These deletions would render them incapable of encoding an infectious virus and may therefore be related to the non-inducibility of FeLV from uninfected cat cells and the subgenomic expression of these endogenous sequences in placental tissue. It appears that there is conservation in the ordering of restriction sites previously reported in the proviruses of the infectious FeLVs in sequences corresponding to the pol and env boundary as well as the region spanning the env gene of the endogenous clones, whereas a greater divergence occurs among restriction sites mapped to the gag and part of the pol regions of the infectious FeLV. Such deleted, FeLV-related subsets of DNA sequences could have originated either by germ-line integration of a complete ecotropic virus followed by deletion, or by integration of a preexisting, defective, deleted variant of the infectious virus.     

Genetic characterization of new West African simian immunodeficiency virus SIVsm: geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a single feral sooty mangabey troop.

It has been proposed that human immunodeficiency virus type 2 (HIV-2) originated from simian immunodeficiency viruses (SIVs) that are natural infections of sooty mangabeys (Cercocebus torquatus atys). To test this hypothesis, SIVs from eight sooty mangabeys, including six new viruses from West Africa, were genetically characterized. gag and env sequences showed that while the viruses of all eight sooty mangabeys belonged to the SIVsm/HIV-2 family, each was widely divergent from SIVs found earlier in captive monkeys at American primate centers. In two SIVs from sooty mangabeys discovered about 100 miles (ca. 161 Km) from each other in rural West Africa, the amino acids of a conserved gag p17-p26 region differed by 19.3%, a divergence greater than that in four of five clades of HIV-2 and in SIVs found in other African monkey species. Analysis of gag region sequences showed that feral mangabeys in one small troop harbored four distinct SIVs. Three of the newly found viruses were genetically divergent, showing as much genetic distance from each other as from the entire SIVsm/HIV-2 family. Sequencing and heteroduplex analysis of one feral animal-derived SIV showed a mosaic genome containing an env gene that was homologous with other feral SIVsm env genes in the troop but having a gag gene from another, distinct SIV. Surprisingly a gag phylogenetic tree based on nucleotide sequences showed that the African relatives closest to all three household-derived SIVs were HIV-2 subtypes D and E from humans in the same West African areas. In one case, the SIV/HIV-2 cluster was from the same village. The findings support the hypothesis that each HIV-2 subtype in West Africans originated from widely divergent SIVsm strains, transmitted by independent cross-species events in the same geographic locations.     

Activation of cryptic splice sites in murine sarcoma virus-124 mutants.

We have examined splice site activation in relation to intron structure in murine sarcoma virus (MuSV)-124 RNA. MuSV-124 contains inactive murine leukemia virus env gene splice sites (termed 5' env and 3' env) as well as cryptic sites in the gag and v-mos genes (termed 5' gag and 3' mos) which are activated for thermosensitive splicing by a 1,487-base intronic deletion in the MuSV-124 derived MuSVts110 retrovirus. To determine conditions permissive for splice site activation, we examined MuSV-124 mutants deleted in the 1,919-base intron bounded by the 5' gag and 3' mos sites. Several of these deletions activated thermosensitive splicing either at the same sites used in MuSVts110 or in a previously unreported temperature-sensitive splice event between the 5' gag and 3' env sites. These data suggested that the thermosensitive splicing phenotype characteristic of MuSVts110 required neither a specialized intron nor selection of a particular 3' splice site. The 3' env and 3' mos sites were found to compete for splicing to the 5' gag site; the more upstream 3' env site was exclusively used in MuSV-124 mutants containing both sites, whereas selection of the 3' mos site required removal of the 3' env site. Branchpoint sequences were found to have a potential regulatory role in thermosensitive splicing. Insertion of a beta-globin branchpoint sequence in a splicing-inactive MuSV-124 mutant activated efficient nonthermosensitive splicing at the 3' mos site, whereas a mutated branchpoint activated less efficient but thermosensitive splicing.     

Genetic Structure of Rauscher Spleen Focus-Forming Virus

Rauscher spleen focus-forming virus contains functional gag and pol genes and a partially deleted env gene which is structurally related to the env genes of dual tropic murine leukemia viruses.     

Myristoylation of human immunodeficiency virus type 1 gag protein is required for efficient env protein transportation to the surface of cells

Highly conserved amino acids in the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) Pr55(gag) are recognized to be critical for the attachment of myristic acid. We previously reported that the env protein was not detected on the cell surface by blocking of N-myristoylation of Pr55(gag) with N-myristoyl glycinal diethylacetal. Here, we constructed a mutant by substituting the N-terminal glycine of Pr55(gag) with alanine to demonstrate that N-myristoylation of Pr55(gag) is required for efficient env protein transportation to the cell surface. The expression level of the env protein on the surface of Jurkat cells transfected with the myristoylation-defective phenotype was observed to be significantly reduced by electron microscopic analyses with a gold-labeled monoclonal antibody against the env protein. In addition, Jurkat cells transfected with the myristoylation-defective phenotype lost the ability of envelope-mediated cell-to-cell fusion. The results suggest that N-myristoylation of the HIV-1 gag protein is necessary for efficient env protein transportation to the cell surface.     

Contribution of the gag and pol sequences to the leukemogenicity of Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) is a highly leukemogenic replication-competent murine retrovirus. Both the F-MuLV envelope gene and the long terminal repeat (LTR) contribute to its pathogenic phenotype (A. Oliff, K. Signorelli, and L. Collins, J. Virol. 51:788-794, 1984). To determine whether the F-MuLV gag and pol genes also possess sequences that affect leukemogenicity, we generated recombinant viruses between the F-MuLV gag and pol genes and two other murine retroviruses, amphotrophic clone 4070 (Ampho) and Friend mink cell focus-inducing virus (Fr-MCF). The F-MuLV gag and pol genes were molecularly cloned on a 5.8-kilobase-pair DNA fragment. This 5.8-kilobase-pair F-MuLV DNA was joined to the Ampho envelope gene and LTR creating a hybrid viral DNA, F/A E+L. A second hybrid viral DNA, F/Fr ENV, was made by joining the 5.8-kilobase-pair F-MuLV DNA to the Fr-MCF envelope gene plus the F-MuLV LTR. F/A E+L and F/Fr ENV DNAs generated recombinant viruses upon transfection into NIH 3T3 cells. F/A E+L virus (F-MuLV gag and pol, Ampho env and LTR) induced leukemia in 20% of NIH Swiss mice after 6 months. Ampho-infected mice did not develop leukemia. F/Fr ENV virus (F-MuLV gag and pol, Fr-MCV env, F-MuLV LTR) induced leukemia in 46% of mice after 3 months. Recombinant viruses containing the Ampho gag and pol, Fr-MCF env, and F-MuLV LTR caused leukemia in 38% of mice after 6 months. We conclude that the F-MuLV gag and pol genes contain sequences that contribute to the pathogenicity of murine retroviruses. These sequences can convert a nonpathogenic virus into a leukemia-causing virus or increase the pathogenicity of viruses that are already leukemogenic.     

Nucleotide sequence of Mason-Pfizer monkey virus: an immunosuppressive D-type retrovirus

The genetic structure of Mason-Pfizer monkey virus (MPMV), a D-type retrovirus, has been determined. In addition to the viral gag, pol, and env genes is an ORF overlapping both gag and pol and that encodes the viral protease. Surprisingly, the MPMV env protein is highly homologous to that of the avian C-type virus, reticuloendotheliosis associated virus REV-A. The env sequence encodes an immunosuppressive peptide, which suggests that MPMV, like REV-A, may transiently induce a T-suppressor cell population. The different phylogenies of the MPMV pol and env genes indicate a recombinatorial origin for the D-type viruses. Sequence comparisons show that SRV-1, an MPMV-like virus etiologically linked to simian AIDS (SAIDS), is in fact a variant of MPMV. While MPMV-like viruses cannot be used as direct models for the AIDS/SAIDS associated with lentiviruses, they provide an important system for studying the molecular basis of immunosuppressive diseases in primates.     

Intracellular ribonucleoprotein complexes of visna virus are infectious.

Sheep choroid plexus cells infected with visna virus produce intracytoplasmic viral ribonucleoprotein complexes with sedimentation values of 120S to 200S and buoyant densities of 1.29 to 1.32 g/cm3. These ribonucleoprotein complexes display an endogenous RNA-directed DNA polymerase activity and contain all of the species of RNA associated with polysomes. An analysis of the polypeptides present in the ribonucleoproteins allowed us to identify the mature internal virion core proteins and their precursor, Pr55gag, as well as the glycosylated envelope precursor gPr150env and small amounts of mature glycoprotein gp135. Ultracentrifugation-purified ribonucleoproteins could infect sheep choroid plexus cells and led to a normal lytic cycle with virus production. Our results suggest that visna virus can propagate by means of intracellular infectious particles.