Medical Forum / Diseases and Disorders / AIDS / May 2004
Human immunodeficiency virus 1, complete genome.
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Baby Peanut - 15 May 2004 04:28 GMT LOCUS NC_001802 9181 bp ss-RNA linear VRL 17-APR-2004 DEFINITION Human immunodeficiency virus 1, complete genome. ACCESSION NC_001802 VERSION NC_001802.1 GI:9629357 KEYWORDS . SOURCE Human immunodeficiency virus 1 (HIV-1) ORGANISM Human immunodeficiency virus 1 Viruses; Retroid viruses; Retroviridae; Lentivirus; Primate lentivirus group. REFERENCE 1 (bases 1 to 9181) AUTHORS Martoglio,B., Graf,R. and Dobberstein,B. TITLE Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin JOURNAL EMBO J. 16 (22), 6636-6645 (1997) MEDLINE 98031891 PUBMED 9362478 REFERENCE 2 (bases 1 to 9181) AUTHORS Petropoulos,C.J. TITLE Appendix 2: Retroviral taxonomy, protein structure, sequences, and genetic maps JOURNAL (in) Coffin,J.M. (Ed.); RETROVIRUSES: 757; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, NY, USA (1997) REFERENCE 3 (bases 1 to 9181) AUTHORS Chappey,C. TITLE Direct Submission JOURNAL Submitted (15-MAR-1999) NIH, NLM, Rockville Pike, Bethesda, MD 20894, USA REMARK Sequence update by submitter REFERENCE 4 (bases 1 to 9181) AUTHORS Chappey,C. TITLE Direct Submission JOURNAL Submitted (12-NOV-1997) NIH, NLM, Rockville Pike, Bethesda, MD 20894, USA COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff. The reference sequence was derived from AF033819. The annotation of this sequence was corrected and updated with the kind help of Dr. Colombe Chappey (ViroLogic Inc., South San Francisco, CA USA) and Roger Ptak (Southern Research Institute, Frederick, MD USA). FEATURES Location/Qualifiers source 1..9181 /organism="Human immunodeficiency virus 1" /mol_type="genomic RNA" /db_xref="taxon:11676" /note="strain for reference annotation" misc_feature 1..96 /note="repeat; positions of RNA transcription initialization and polyadenylation; Region: R" polyA_signal 73..78 /note="both 5' and 3' poly A signals are transcribed into RNA, but the 5' one is suppressed" 5'UTR 97..181 primer_bind 182..199 gene 336..4642 /gene="gag-pol" /locus_tag="HIV1gp1" /db_xref="GeneID:155348" /db_xref="LocusID:155348" CDS join(336..1637,1637..4642) /gene="gag-pol" /locus_tag="HIV1gp1" /note="fusion protein consisting of the viral structural proteins and enzymes; cleaved by the viral protease into individual mature proteins; The processing products of the Gag and Gag-Pol polyproteins were annotated with the help of Pettit et al., 2003 and references therein; Pr160; ribosomal slippage at slippery sequence tttttta (1631..1637)" /codon_start=1 /product="Gag-Pol" /protein_id="NP_057849.4" /db_xref="GI:28872819" /db_xref="GeneID:155348" /db_xref="LocusID:155348" /translation="MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERF AVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALD KIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVE EKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRM YSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTIL KALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKC FNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLREDLAFLQGKAREFSS EQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNFPQVTLWQRPLVTIKIGGQLK EALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVG PTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEI CTEMEKEGKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIP HPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGW KGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLR WGLTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQ IYPGIKVRQLCKLLRGTKALTEVIPLTEEAELELAENREILKEPVHGVYYDPSKDLIA EIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKITTESIVIWGK TPKFKLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVD GAANRETKLGKAGYVTNRGRQKVVTLTDTTNQKTELQAIYLALQDSGLEVNIVTDSQY ALGIIQAQPDQSESELVNQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSAGIRKV LFLDGIDKAQDEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCS PGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHT DNGSNFTGATVRAACWWAGIKQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLK TAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSR NPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED" mat_peptide join(1632..1637,1637..1798) /gene="gag-pol" /product="Gag-Pol Transframe peptide" /note="the Glu-Asp-Leu tripeptide (positions 4-6) is a specific inhibitor of the HIV-1 protease. Involved in regulation of the protease-mediated polyprotein processing; p6*; alternative p6 protein" /protein_id="NP_787043.1" /db_xref="GI:28872821" mat_peptide 1655..4639 /gene="gag-pol" /locus_tag="HIV1gp1" /product="Pol" /note="unprocessed Pol polyprotein; includes part of the transframe peptide, protease, reverse transcriptase and integrase domains." /protein_id="NP_789740.1" /db_xref="GI:28872823" mat_peptide 1799..2095 /gene="gag-pol" /locus_tag="HIV1gp1" /product="protease" /note="The proteinase domain of Gag-Pol (in the form of homodimer) mediates all the cleavages in the polyprotein. Cleaves itself from the polyprotein late in particle assembly; aspartic peptidase" /protein_id="NP_705926.1" /db_xref="GI:25121906" mat_peptide 2096..3775 /gene="gag-pol" /locus_tag="HIV1gp1" /product="reverse transcriptase" /note="transcribes single stranded viral RNA genome into double stranded proviral DNA; HIV-1 reverse transcriptase is composed of the p66 subunit (this protein) and the p51 subunit that lacks the RNAse H domain of the larger subunit" /protein_id="NP_705927.1" /db_xref="GI:25121907" mat_peptide 2096..3415 /gene="gag-pol" /locus_tag="HIV1gp1" /product="reverse transcriptase p51 subunit" /note="HIV-1 reverse transcriptase is composed of the p66 subunit and the p51 subunit (this protein) that lacks the RNAse H domain of the larger subunit" /protein_id="NP_789739.1" /db_xref="GI:28872822" mat_peptide 3776..4639 /gene="gag-pol" /locus_tag="HIV1gp1" /product="integrase" /note="mediates integration of the viral DNA into the infected cell chromosome" /protein_id="NP_705928.1" /db_xref="GI:25121908" gene 336..1838 /gene="gag" /locus_tag="HIV1gp2" /db_xref="GeneID:155030" /db_xref="LocusID:155030" CDS 336..1838 /gene="gag" /locus_tag="HIV1gp2" /note="Pr55; The processing products of the Gag and Gag-Pol polyproteins were annotated with the help of Pettit et al., 2003 and references therein" /codon_start=1 /product="Gag" /protein_id="NP_057850.1" /db_xref="GI:9629360" /db_xref="GeneID:155030" /db_xref="LocusID:155030" /translation="MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERF AVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALD KIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVE EKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRM YSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTIL KALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKC FNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQ SRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ" mat_peptide 336..731 /gene="gag" /locus_tag="HIV1gp2" /product="matrix" /note="viral structural protein; forms the outer structural shell of HIV-1 virions; involved in the nuclear import of the HIV-1 preintegration complex; p17" /protein_id="NP_579876.2" /db_xref="GI:28876542" mat_peptide 732..1424 /gene="gag" /locus_tag="HIV1gp2" /product="capsid" /note="viral structural protein; forms the core of HIV-1 virions; p24" /protein_id="NP_579880.1" /db_xref="GI:19172948" mat_peptide 1425..1466 /gene="gag" /locus_tag="HIV1gp2" /product="p2" /note="Processing of Gag-Pol by the protease domain dimer starts with cleavage between the p2 and nucleocapsid proteins." /protein_id="NP_579882.1" /db_xref="GI:19172950" mat_peptide 1467..1631 /gene="gag" /locus_tag="HIV1gp2" /product="nucleocapsid" /note="viral structural protein; coats the genomic RNA inside the virion core; binds and delivers full-length viral RNAs into assembling HIV-1 virions; p7" /protein_id="NP_579881.1" /db_xref="GI:19172949" mat_peptide 1632..1679 /gene="gag" /locus_tag="HIV1gp2" /product="p1" /note="important for virus infectivity, protein processing, and genomic RNA dimer stability" /protein_id="NP_787042.1" /db_xref="GI:28872820" mat_peptide 1680..1835 /gene="gag" /locus_tag="HIV1gp2" /product="p6" /note="important for incorporation of Vpr into assembling HIV-1 virions; helps mediate efficient virus particle release from infected cells" /protein_id="NP_579883.1" /db_xref="GI:19172951" gene 4587..5165 /gene="vif" /locus_tag="HIV1gp3" /db_xref="GeneID:155459" /db_xref="LocusID:155459" CDS 4587..5165 /gene="vif" /locus_tag="HIV1gp3" /note="p23; viral infectivity factor; viral accessory protein important for virus replication in vivo" /codon_start=1 /product="Vif" /protein_id="NP_057851.1" /db_xref="GI:9629361" /db_xref="GeneID:155459" /db_xref="LocusID:155459" /translation="MENRWQVMIVWQVDRMRIRTWKSLVKHHMYVSGKARGWFYRHHY ESPHPRISSEVHIPLGDARLVITTYWGLHTGERDWHLGQGVSIEWRKKRYSTQVDPEL ADQLIHLYYFDCFSDSAIRKALLGHIVSPRCEYQAGHNKVGSLQYLALAALITPKKIK PPLPSVTKLTEDRWNKPQKTKGHRGSHTMNGH" gene 5105..5396 /gene="vpr" /locus_tag="HIV1gp4" /db_xref="GeneID:155807" /db_xref="LocusID:155807" CDS join(5105..5319,5321..5396) /gene="vpr" /locus_tag="HIV1gp4" /note="p15; viral protein R; viral accessory protein important for virus replication in vivo; involved in the nuclear import of the HIV-1 preintegration complex; induces G2 cell cycle arrest; influences mutation rates during viral DNA synthesis; An artificial frameshift eliminating the orf-disrupting nucleotide at position 5320 is introduced to obtain the typical HIV-1 Vpr protein sequence. For this particular HIV-1 strain, HXB2, only a short (78 amino acid long) variant of the Vpr sequence can be obtained by translation of nucleotides 5105 through 5341 without the frameshift" /codon_start=1 /exception="artificial frameshift" /product="Vpr" /protein_id="NP_057852.2" /db_xref="GI:28872817" /db_xref="GeneID:155807" /db_xref="LocusID:155807" /translation="MEQAPEDQGPQREPHNEWTLELLEELKNEAVRHFPRIWLHGLGQ HIYETYGDTWAGVEAIIRILQQLLFIHFRIGCRHSRIGVTRQRRARNGASRS" gene 5377..7970 /gene="tat" /locus_tag="HIV1gp5" /db_xref="GeneID:155871" /db_xref="LocusID:155871" CDS join(5377..5591,7925..7970) /gene="tat" /locus_tag="HIV1gp5" /note="p14; transcriptional activator; viral regulatory protein required for virus replication; transactivates the viral LTR promoter through interactions with cellular transcription factors; associated with pathogenic effects of the virus; the length of Tat varies depending on virus strain or clade" /codon_start=1 /product="Tat" /protein_id="NP_057853.1" /db_xref="GI:9629358" /db_xref="GeneID:155871" /db_xref="LocusID:155871" /translation="MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALG ISYGRKKRRQRRRAHQNSQTHQASLSKQPTSQPRGDPTGPKE" gene 5516..8199 /gene="rev" /locus_tag="HIV1gp6" /db_xref="GeneID:155908" /db_xref="LocusID:155908" CDS join(5516..5591,7925..8199) /gene="rev" /locus_tag="HIV1gp6" /note="p19; regulator of expression of virion proteins; prevents splicing of viral RNA; shuttles unspliced viral RNA to the cytoplasm for expression of viral proteins and incorporation of full length viral genomic RNA into virions" /codon_start=1 /product="Rev" /protein_id="NP_057854.1" /db_xref="GI:9629359" /db_xref="GeneID:155908" /db_xref="LocusID:155908" /translation="MAGRSGDSDEELIRTVRLIKLLYQSNPPPNPEGTRQARRNRRRR WRERQRQIHSISERILGTYLGRSAEPVPLQLPPLERLTLDCNEDCGTSGTQGVGSPQI LVESPTVLESGTKE" gene 5608..5856 /gene="vpu" /locus_tag="HIV1gp7" /db_xref="GeneID:155945" /db_xref="LocusID:155945" CDS 5608..5856 /gene="vpu" /locus_tag="HIV1gp7" /note="p16; viral protein U; viral accessory protein important for virus replication in vivo; promotes degradation of CD4 and down-regulates cell surface expression of MHC class I proteins; helps mediate efficient virus particle release from infected cells; reported to induce apoptosis by suppressing the nuclear factor kappaB-dependent expression of antiapoptotic factors; may attenuate the level of Env precursor(gp160) biosynthesis; Vpu and gp160 are translated from different reading frames of the same bicistronic mRNA" /codon_start=1 /product="Vpu" /protein_id="NP_057855.1" /db_xref="GI:9629366" /db_xref="GeneID:155945" /db_xref="LocusID:155945" /translation="MQPIPIVAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLID RLIERAEDSGNESEGEISALVEMGVEMGHHAPWDVDDL" gene 5771..8341 /gene="env" /locus_tag="HIV1gp8" /db_xref="GeneID:155971" /db_xref="LocusID:155971" CDS 5771..8341 /gene="env" /locus_tag="HIV1gp8" /note="gp160; envelope glycoprotein; envelope polyprotein; cleaved by cellular proteases into mature proteins gp120 and gp41" /codon_start=1 /product="Envelope surface glycoprotein gp160, precursor" /protein_id="NP_057856.1" /db_xref="GI:9629363" /db_xref="GeneID:155971" /db_xref="LocusID:155971" /translation="MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPV WKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVE QMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYC APAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVN FTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFN STWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNIT GLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQR EKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLL QLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWN HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGER DRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWN LLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL " sig_peptide 5771..5854 /gene="env" /protein_id="NP_579893.2" /db_xref="GI:28876543" mat_peptide 5855..7303 /gene="env" /locus_tag="HIV1gp8" /product="Envelope surface glycoprotein gp120" /note="mediates binding of HIV-1 to CD4 and cellular co-receptors; cooperates with gp41 to mediate fusion of viral membrane with cellular membrane during virus entry into cells; Envelope surface unit; SU" /protein_id="NP_579894.2" /db_xref="GI:28876544" mat_peptide 7304..8338 /gene="env" /locus_tag="HIV1gp8" /product="Envelope transmembrane glycoprotein gp41" /note="cooperates with gp120 to mediate fusion of viral membrane with cellular membrane during virus entry into cells; Envelope transmembrane domain; TM" /protein_id="NP_579895.1" /db_xref="GI:19172954" gene 8343..8963 /gene="nef" /locus_tag="HIV1gp9" /db_xref="GeneID:156110" /db_xref="LocusID:156110" CDS 8343..8963 /gene="nef" /locus_tag="HIV1gp9" /note="p27; negative factor; viral accessory protein; important for virus replication in vivo; determinant of HIV-1 pathogenesis; down-regulates cell surface CD4 and MHC class I molecules; enhances virus infectivity through interactions with multiple cellular signaling proteins; This particular nucleotide sequence has a premature stop codon in place of a well-conserved tryptophan codon at position 8712-8714 that truncates the HIV1 Nef protein sequence to a 123 amino acids-long N-terminal portion (not shown)" /codon_start=1 /transl_except=(pos:8712..8714,aa:Trp) /product="Nef" /protein_id="NP_057857.2" /db_xref="GI:28872818" /db_xref="GeneID:156110" /db_xref="LocusID:156110" /translation="MGGKWSKSSVIGWPTVRERMRRAEPAADRVGAASRDLEKHGAIT SSNTAATNAACAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIH SQRRQDILDLWIYHTQGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKIEEANKGE NTSLLHPVSLHGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC" 3'UTR 8631..9085 misc_feature 9086..9181 /note="repeat; positions of RNA transcription initialization and polyadenylation; Region: R" polyA_signal 9158..9163 /note="both 5' and 3' poly A signals are transcribed into RNA, but the 5' one is suppressed" ORIGIN
Yana - 18 May 2004 16:47 GMT Anyone can tell just by looking at that that it is not the genome of HIV they have presented.
It is obviously a green tree frog.
abinkum@yahoo.com - 19 May 2004 13:56 GMT CAn you please describe the source of the RNA for this analysis, and the methodology used.
Robert S. Holzman - 19 May 2004 23:57 GMT > CAn you please describe the source of the RNA for this > analysis, and the methodology used. Could not locate a descriptive paper for that particular clone in medline, but the two papers below are the original reports of HIV-1 cloning. Hope it is close enough for what you want.
Li, Y., J. C. Kappes, et al. (1991). "Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes." J Virol 65(8): 3973-85. All presently available replication-competent proviral clones of human immunodeficiency virus type 1 (HIV-1) are derived from cell culture-amplified virus. Since tissue culture is highly selective for viral strains with an in vitro growth advantage, such clones may not be representative of the biologically relevant virus present in vivo. In this study, we report the molecular cloning and genotypic characterization of 10 HIV-1 genomes directly from uncultured brain tissue of a patient with AIDS dementia complex. Targeting unintegrated circular HIV-1 molecules for recombinant lambda phage cloning, we obtained four full-length genomes with one or two long terminal repeats (LTRs), three defective genomes with internal deletions, two rearranged genomes with inverted LTR sequences, and one integrated proviral half with flanking cellular sequences. Nucleotide sequence analysis of these clones demonstrated chromosomal integration, circle formation, genomic inversion, and LTR-mediated autointegration of HIV-1 genomes in vivo. Comparison of a 510-bp hypervariable envelope region among 8 lambda phage-derived and 12 polymerase chain reaction-derived clones from the same brain specimen identified a predominant viral form as well as genetically divergent variants. Variability among 19 of 20 clones ranged between 0.2 and 1.2%. One clone exhibited 8.2% nucleotide sequence differences consisting almost exclusively of G-to-A changes. Transfection of the four full-length HIV-1 genomes identified one clone (YU-2) as replication competent and exhibiting growth characteristics similar to those of tissue culture-derived macrophage tropic strains of HIV-1. These results demonstrate, for the first time, that replication- competent HIV-1 genomes, complex mixtures of defective viral forms, and chromosomally integrated provirus persist in vivo. In addition, the brain-derived viral clones are expected to prove valuable for future studies of macrophage and neurotropism as well as for the analysis of other viral properties that are subject to in vitro selection pressures.
Li, Y., H. Hui, et al. (1992). "Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation." J Virol 66(11): 6587-600. Previous studies of the genetic and biologic characteristics of human immunodeficiency virus type 1 (HIV-1) have by necessity used tissue culture-derived virus. We recently reported the molecular cloning of four full-length HIV-1 genomes directly from uncultured human brain tissue (Y. Li, J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn, J. Virol. 65:3973-3985, 1991). In this report, we describe the biologic properties of these four clones and the complete nucleotide sequences and genome organization of two of them. Clones HIV-1YU-2 and HIV-1YU-10 were 9,174 and 9,176 nucleotides in length, differed by 0.26% in nucleotide sequence, and except for a frameshift mutation in the pol gene in HIV-1YU-10, contained open reading frames corresponding to 5'-gag-pol-vif-vpr-tat-rev-vpu-env-nef-3' flanked by long terminal repeats. HIV-1YU-2 was fully replication competent, while HIV-1YU-10 and two other clones, HIV-1YU-21 and HIV-1YU- 32, were defective. All three defective clones, however, when transfected into Cos-1 cells in any pairwise combination, yielded virions that were replication competent and transmissible by cell-free passage. The cellular host range of HIV-1YU-2 was strictly limited to primary T lymphocytes and monocyte-macrophages, a property conferred by its external envelope glycoprotein. Phylogenetic analyses of HIV-1YU-2 gene sequences revealed this virus to be a member of the North American/European HIV-1 subgroup, with specific similarity to other monocyte-tropic viruses in its V3 envelope amino acid sequence. These results indicate that HIV-1 infection of brain is characterized by the persistence of mixtures of fully competent, minimally defective, and more substantially altered viral forms and that complementation among them is readily attainable. In addition, the limited degree of genotypic heterogeneity observed among HIV-1YU and other brain-derived viruses and their preferential tropism for monocyte- macrophages suggest that viral replication within the central nervous system may differ from that within the peripheral lymphoid compartment in significant and clinically important ways. The availability of genetically and biologically well characterized HIV-1 clones from uncultured human tissue should facilitate future studies of virus-cell interactions relevant to viral pathogenesis and drug and vaccine development.
Yana - 21 May 2004 07:14 GMT “All presently available replication-competent proviral clones of human immunodeficiency virus type 1 (HIV-1) are derived from cell culture-amplified virus. Since tissue culture is highly selective for viral strains with an in vitro growth advantage, such clones may not be representative of the biologically relevant virus present in vivo.”
May I repeat that last statement…..
“.... Such clones may not be representative of the biologically relevant virus present in vivo………..”
========================================== Additionally, why do they need to use cell cultured-amplified virus to clone? Why not use the isolated virus itself?
Oh I remember…because there IS no isolated virus!
So again I ask, what are they cloning? (It still could be a green tree grog for all we know)
Nick Bennett - 27 May 2004 18:00 GMT > All presently available replication-competent proviral clones of human > immunodeficiency virus type 1 (HIV-1) are derived from cell [quoted text clipped - 6 lines] > .... Such clones may not be representative of the biologically relevant > virus present in vivo
.. This is a simple statement of the limitations of the process. It's well known that HIV exists as a mixture of quasispecies, in the same way as most humans differ in their genetic make-up over the planet. Some clones isolated from infected people are in fact defective: not always immediately obvious from culture (the genes aren't required) but sometimes. The odds are though that anything you find from a patient with HIV will be a dominant strain (the numbers game) and presumably such a strain will be replication competant. It's not always been true, at least one strain that I'm aware of in common lab usage has a deletion from true "wild type" HIV-1.
> ========================================== > Additionally, why do they need to use cell cultured-amplified virus to > clone? Why not use the isolated virus itself? Not enough, basically. However you do it, you'll have to amplify the genetic material. Either through PCR or through culture. Culture has the advantage that the genetic code is less likely to be corrupted through normal error-generation mechanisms, and that it'll happily deal with a full-length construct. It does however have the disadvantage that you might grow out a clone that is defective for a gene that is required in vivo but not in vitro (see above).
I have read one report which I'm fairly sure used sequences derived from peripheral blood samples of an infected patient, but due to the limitations of PCR (in order to get enough DNA to work with) they had to clone seperate chunks and then line them up in order to get the full sequence. This is the same process used in detecting a gene from a linkage study (chromosome walking) but with a different aim. Not ideal really, but it was done from a patient's sample as I recall.
> Oh I remember
because there IS no isolated virus! *sigh*. When are you going to listen to the science, and try to learn how it works, instead of repeating rubbish ad nauseum? You don't need an isolate to find new genetic material (if you did, then the entire DNA-based forensics business would collapse overnight!), just the material. You can then use the genetic "isolate" to produce virus isolates, true purified virus, to confirm your suspicion that the material produces virus.
> So again I ask, what are they cloning? (It still could be a green tree > grog for all we know) The methods used for HIV don't differ from any used for other viruses like herpes, adenovirus, measles etc etc. "Isolating" it usually involves some method of amplification, either PCR or culture.
If you were to fill a grain of salt (1mm cube) with HIV particles you would have over 1 trillion of them. That would be enough to fill the blood stream of 200 AIDS patients with viral loads of 1 million per ml. So...in order to get enough virus to even begin to attempt to isolate RNA directly from it, are you suggesting we bleed several hundred people dry, in the name of science?
I don't think so. Seriously, those are the numbers we're talking about. Viruses are small. My back-of-the-envelope math says that the total RNA you'd get from that would be 64ng. Just enough to even see on an ethidium gel (typical amounts you might run from, say, a plasmid prep are 1 microgram, or 15 times as much). From 200 fully bled AIDS patients. Sheesh.
THAT puts it into perspective. If you want to isolate your genome using that method, go right ahead. I won't hold my breath.
Bennett
Dr. Phillip S. Duke - 20 May 2004 01:42 GMT > CAn you please describe the source of the RNA for this analysis, and > the methodology used. That is the NCBI "Reference Sequence" for HIV-1. It is from the Lambda-HXB-2 proviral genomic clone produced by George Shaw and coworkers in the Gallo lab in 1984 (1), sequenced in 1985 (3) and 1987 (4) after proving to be an infectious molecular clone in 1985 (2). The complete methodology is clearly spelled out in those papers. The source was proviral DNA, not viral RNA.
1: Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC, Wong-Staal F. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome. Science. 1984 Dec 7;226(4679):1165-71.
2: Fisher AG, Collalti E, Ratner L, Gallo RC, Wong-Staal F A molecular clone of HTLV-III with biological activity. Nature. 1985 Jul 18-24;316(6025):262-5.
3: Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B, Josephs SF, Doran ER, Rafalski JA, Whitehorn EA, Baumeister K, et al. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24-30;313(6000):277-84.
4: Ratner L, Fisher A, Jagodzinski LL, Mitsuya H, Liou RS, Gallo RC, Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57-69.
Baby Peanut - 20 May 2004 19:10 GMT > CAn you please describe the source of the RNA for this analysis, and > the methodology used. Oh that's easy. I used blatent copyright violation to aquire the data.
BASE COUNT 3272 a 1642 c 2225 g 2042 t ORIGIN 1 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 61 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 121 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 181 gtggcgcccg aacagggacc tgaaagcgaa agggaaacca gaggagctct ctcgacgcag 241 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc 301 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa 361 gcgggggaga attagatcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 421 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 481 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 541 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 601 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 661 acaaaagtaa gaaaaaagca cagcaagcag cagctgacac aggacacagc aatcaggtca 721 gccaaaatta ccctatagtg cagaacatcc aggggcaaat ggtacatcag gccatatcac 781 ctagaacttt aaatgcatgg gtaaaagtag tagaagagaa ggctttcagc ccagaagtga 841 tacccatgtt ttcagcatta tcagaaggag ccaccccaca agatttaaac accatgctaa 901 acacagtggg gggacatcaa gcagccatgc aaatgttaaa agagaccatc aatgaggaag 961 ctgcagaatg ggatagagtg catccagtgc atgcagggcc tattgcacca ggccagatga 1021 gagaaccaag gggaagtgac atagcaggaa ctactagtac ccttcaggaa caaataggat 1081 ggatgacaaa taatccacct atcccagtag gagaaattta taaaagatgg ataatcctgg 1141 gattaaataa aatagtaaga atgtatagcc ctaccagcat tctggacata agacaaggac 1201 caaaggaacc ctttagagac tatgtagacc ggttctataa aactctaaga gccgagcaag 1261 cttcacagga ggtaaaaaat tggatgacag aaaccttgtt ggtccaaaat gcgaacccag 1321 attgtaagac tattttaaaa gcattgggac cagcggctac actagaagaa atgatgacag 1381 catgtcaggg agtaggagga cccggccata aggcaagagt tttggctgaa gcaatgagcc 1441 aagtaacaaa ttcagctacc ataatgatgc agagaggcaa ttttaggaac caaagaaaga 1501 ttgttaagtg tttcaattgt ggcaaagaag ggcacacagc cagaaattgc agggccccta 1561 ggaaaaaggg ctgttggaaa tgtggaaagg aaggacacca aatgaaagat tgtactgaga 1621 gacaggctaa ttttttaggg aagatctggc cttcctacaa gggaaggcca gggaattttc 1681 ttcagagcag accagagcca acagccccac cagaagagag cttcaggtct ggggtagaga 1741 caacaactcc ccctcagaag caggagccga tagacaagga actgtatcct ttaacttccc 1801 tcaggtcact ctttggcaac gacccctcgt cacaataaag ataggggggc aactaaagga 1861 agctctatta gatacaggag cagatgatac agtattagaa gaaatgagtt tgccaggaag 1921 atggaaacca aaaatgatag ggggaattgg aggttttatc aaagtaagac agtatgatca 1981 gatactcata gaaatctgtg gacataaagc tataggtaca gtattagtag gacctacacc 2041 tgtcaacata attggaagaa atctgttgac tcagattggt tgcactttaa attttcccat 2101 tagccctatt gagactgtac cagtaaaatt aaagccagga atggatggcc caaaagttaa 2161 acaatggcca ttgacagaag aaaaaataaa agcattagta gaaatttgta cagagatgga 2221 aaaggaaggg aaaatttcaa aaattgggcc tgaaaatcca tacaatactc cagtatttgc 2281 cataaagaaa aaagacagta ctaaatggag aaaattagta gatttcagag aacttaataa 2341 gagaactcaa gacttctggg aagttcaatt aggaatacca catcccgcag ggttaaaaaa 2401 gaaaaaatca gtaacagtac tggatgtggg tgatgcatat ttttcagttc ccttagatga 2461 agacttcagg aagtatactg catttaccat acctagtata aacaatgaga caccagggat 2521 tagatatcag tacaatgtgc ttccacaggg atggaaagga tcaccagcaa tattccaaag 2581 tagcatgaca aaaatcttag agccttttag aaaacaaaat ccagacatag ttatctatca 2641 atacatggat gatttgtatg taggatctga cttagaaata gggcagcata gaacaaaaat 2701 agaggagctg agacaacatc tgttgaggtg gggacttacc acaccagaca aaaaacatca 2761 gaaagaacct ccattccttt ggatgggtta tgaactccat cctgataaat ggacagtaca 2821 gcctatagtg ctgccagaaa aagacagctg gactgtcaat gacatacaga agttagtggg 2881 gaaattgaat tgggcaagtc agatttaccc agggattaaa gtaaggcaat tatgtaaact 2941 ccttagagga accaaagcac taacagaagt aataccacta acagaagaag cagagctaga 3001 actggcagaa aacagagaga ttctaaaaga accagtacat ggagtgtatt atgacccatc 3061 aaaagactta atagcagaaa tacagaagca ggggcaaggc caatggacat atcaaattta 3121 tcaagagcca tttaaaaatc tgaaaacagg aaaatatgca agaatgaggg gtgcccacac 3181 taatgatgta aaacaattaa cagaggcagt gcaaaaaata accacagaaa gcatagtaat 3241 atggggaaag actcctaaat ttaaactgcc catacaaaag gaaacatggg aaacatggtg 3301 gacagagtat tggcaagcca cctggattcc tgagtgggag tttgttaata cccctccctt 3361 agtgaaatta tggtaccagt tagagaaaga acccatagta ggagcagaaa ccttctatgt 3421 agatggggca gctaacaggg agactaaatt aggaaaagca ggatatgtta ctaatagagg 3481 aagacaaaaa gttgtcaccc taactgacac aacaaatcag aagactgagt tacaagcaat 3541 ttatctagct ttgcaggatt cgggattaga agtaaacata gtaacagact cacaatatgc 3601 attaggaatc attcaagcac aaccagatca aagtgaatca gagttagtca atcaaataat 3661 agagcagtta ataaaaaagg aaaaggtcta tctggcatgg gtaccagcac acaaaggaat 3721 tggaggaaat gaacaagtag ataaattagt cagtgctgga atcaggaaag tactattttt 3781 agatggaata gataaggccc aagatgaaca tgagaaatat cacagtaatt ggagagcaat 3841 ggctagtgat tttaacctgc cacctgtagt agcaaaagaa atagtagcca gctgtgataa 3901 atgtcagcta aaaggagaag ccatgcatgg acaagtagac tgtagtccag gaatatggca 3961 actagattgt acacatttag aaggaaaagt tatcctggta gcagttcatg tagccagtgg 4021 atatatagaa gcagaagtta ttccagcaga aacagggcag gaaacagcat attttctttt 4081 aaaattagca ggaagatggc cagtaaaaac aatacatact gacaatggca gcaatttcac 4141 cggtgctacg gttagggccg cctgttggtg ggcgggaatc aagcaggaat ttggaattcc 4201 ctacaatccc caaagtcaag gagtagtaga atctatgaat aaagaattaa agaaaattat 4261 aggacaggta agagatcagg ctgaacatct taagacagca gtacaaatgg cagtattcat 4321 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 4381 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa 4441 ttttcgggtt tattacaggg acagcagaaa tccactttgg aaaggaccag caaagctcct 4501 ctggaaaggt gaaggggcag tagtaataca agataatagt gacataaaag tagtgccaag 4561 aagaaaagca aagatcatta gggattatgg aaaacagatg gcaggtgatg attgtgtggc 4621 aagtagacag gatgaggatt agaacatgga aaagtttagt aaaacaccat atgtatgttt 4681 cagggaaagc taggggatgg ttttatagac atcactatga aagccctcat ccaagaataa 4741 gttcagaagt acacatccca ctaggggatg ctagattggt aataacaaca tattggggtc 4801 tgcatacagg agaaagagac tggcatttgg gtcagggagt ctccatagaa tggaggaaaa 4861 agagatatag cacacaagta gaccctgaac tagcagacca actaattcat ctgtattact 4921 ttgactgttt ttcagactct gctataagaa aggccttatt aggacacata gttagcccta 4981 ggtgtgaata tcaagcagga cataacaagg taggatctct acaatacttg gcactagcag 5041 cattaataac accaaaaaag ataaagccac ctttgcctag tgttacgaaa ctgacagagg 5101 atagatggaa caagccccag aagaccaagg gccacagagg gagccacaca atgaatggac 5161 actagagctt ttagaggagc ttaagaatga agctgttaga cattttccta ggatttggct 5221 ccatggctta gggcaacata tctatgaaac ttatggggat acttgggcag gagtggaagc 5281 cataataaga attctgcaac aactgctgtt tatccatttt cagaattggg tgtcgacata 5341 gcagaatagg cgttactcga cagaggagag caagaaatgg agccagtaga tcctagacta 5401 gagccctgga agcatccagg aagtcagcct aaaactgctt gtaccaattg ctattgtaaa 5461 aagtgttgct ttcattgcca agtttgtttc ataacaaaag ccttaggcat ctcctatggc 5521 aggaagaagc ggagacagcg acgaagagct catcagaaca gtcagactca tcaagcttct 5581 ctatcaaagc agtaagtagt acatgtaatg caacctatac caatagtagc aatagtagca 5641 ttagtagtag caataataat agcaatagtt gtgtggtcca tagtaatcat agaatatagg 5701 aaaatattaa gacaaagaaa aatagacagg ttaattgata gactaataga aagagcagaa 5761 gacagtggca atgagagtga aggagaaata tcagcacttg tggagatggg ggtggagatg 5821 gggcaccatg ctccttggga tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac 5881 agtctattat ggggtacctg tgtggaagga agcaaccacc actctatttt gtgcatcaga 5941 tgctaaagca tatgatacag aggtacataa tgtttgggcc acacatgcct gtgtacccac 6001 agaccccaac ccacaagaag tagtattggt aaatgtgaca gaaaatttta acatgtggaa 6061 aaatgacatg gtagaacaga tgcatgagga tataatcagt ttatgggatc aaagcctaaa 6121 gccatgtgta aaattaaccc cactctgtgt tagtttaaag tgcactgatt tgaagaatga 6181 tactaatacc aatagtagta gcgggagaat gataatggag aaaggagaga taaaaaactg 6241 ctctttcaat atcagcacaa gcataagagg taaggtgcag aaagaatatg cattttttta 6301 taaacttgat ataataccaa tagataatga tactaccagc tataagttga caagttgtaa 6361 cacctcagtc attacacagg cctgtccaaa ggtatccttt gagccaattc ccatacatta 6421 ttgtgccccg gctggttttg cgattctaaa atgtaataat aagacgttca atggaacagg 6481 accatgtaca aatgtcagca cagtacaatg tacacatgga attaggccag tagtatcaac 6541 tcaactgctg ttaaatggca gtctagcaga agaagaggta gtaattagat ctgtcaattt 6601 cacggacaat gctaaaacca taatagtaca gctgaacaca tctgtagaaa ttaattgtac 6661 aagacccaac aacaatacaa gaaaaagaat ccgtatccag agaggaccag ggagagcatt 6721 tgttacaata ggaaaaatag gaaatatgag acaagcacat tgtaacatta gtagagcaaa 6781 atggaataac actttaaaac agatagctag caaattaaga gaacaatttg gaaataataa 6841 aacaataatc tttaagcaat cctcaggagg ggacccagaa attgtaacgc acagttttaa 6901 ttgtggaggg gaatttttct actgtaattc aacacaactg tttaatagta cttggtttaa 6961 tagtacttgg agtactgaag ggtcaaataa cactgaagga agtgacacaa tcaccctccc 7021 atgcagaata aaacaaatta taaacatgtg gcagaaagta ggaaaagcaa tgtatgcccc 7081 tcccatcagt ggacaaatta gatgttcatc aaatattaca gggctgctat taacaagaga 7141 tggtggtaat agcaacaatg agtccgagat cttcagacct ggaggaggag atatgaggga 7201 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 7261 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 7321 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcct caatgacgct 7381 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 7441 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 7501 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 7561 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 7621 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 7681 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 7741 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 7801 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 7861 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 7921 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 7981 tggagagaga gacagagaca gatccattcg attagtgaac ggatccttgg cacttatctg 8041 ggacgatctg cggagcctgt gcctcttcag ctaccaccgc ttgagagact tactcttgat 8101 tgtaacgagg attgtggaac ttctgggacg cagggggtgg gaagccctca aatattggtg 8161 gaatctccta cagtattgga gtcaggaact aaagaatagt gctgttagct tgctcaatgc 8221 cacagccata gcagtagctg aggggacaga tagggttata gaagtagtac aaggagcttg 8281 tagagctatt cgccacatac ctagaagaat aagacagggc ttggaaagga ttttgctata 8341 agatgggtgg caagtggtca aaaagtagtg tgattggatg gcctactgta agggaaagaa 8401 tgagacgagc tgagccagca gcagataggg tgggagcagc atctcgagac ctggaaaaac 8461 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgcttgtgcc tggctagaag 8521 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga 8581 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 8641 taattcactc ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct 8701 acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg 8761 gatggtgcta caagctagta ccagttgagc cagataagat agaagaggcc aataaaggag 8821 agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag 8881 tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc 8941 cggagtactt caagaactgc tgacatcgag cttgctacaa gggactttcc gctggggact 9001 ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat 9061 aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag 9121 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt 9181 c //
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