ABSTRACT We studied the evolution of human immunodeficiency virus type 1 (HIV-1) in a cohort of long-term survivors infected with an attenuated strain of HIV-1 acquired from a single source. Although the cohort members experienced differing clinical courses, we demonstrate similar evolution of HIV-1 nef /long-terminal repeat (LTR) sequences, characterized by progressive sequence deletions tending toward a minimal nef /LTR structure that retains only sequence elements required for viral replication. The in vivo pathogenicity of attenuated HIV-1 is therefore dictated by viral and/or host factors other than those that impose a unidirectional selection pressure on the nef /LTR region of the HIV-1 genome.

The transcription and splicing of human immunodeficiency virus type 1 (HIV-1) mRNA in primary blood monocyte-derived macrophages (MDM) and CD4(+) peripheral blood lymphocytes (PBL) were compared to determine whether any differences might account for the slower noncytopathic infection of cells of the macrophage lineage. The expression of regulatory mRNAs during acute infection of MDM was delayed by about 12 h compared to that of PBL. In each cell type, an increase in spliced viral mRNAs slightly preceded virus production from the culture. Following the peak of productive infection, there was a proportional decrease in the expression of all regulatory mRNAs detected in PBL. In MDM, a dramatic additional decrease specifically in the tat mRNA species heralded a reduction in virus production. This decline in tat mRNA was reflected by a concomitant decrease in Tat activity in the cells and occurred with the same kinetics irrespective of the age of the cells when infected. Addition of exogenous Tat protein elicited a burst of virus production from persistently infected MDM, suggesting that the decrease in virus production from the cultures is a consequence of the reduction in tat mRNA levels. Our results show that modulation of HIV-1 mRNAs in macrophages during long-term infection, which is dependent on the period of infection rather than cell differentiation or maturation, results in a selective reduction of Tat protein levels at the commencement of a persistent, less productive phase of infection. Determination of the mechanism of this mRNA modulation may lead to novel targets for control of replication in these important viral reservoirs.

Primary HIV-1 infection (PHI) is associated with a period of viremia, the resolution of which generally coincides with the development of both humoral and cellular immune responses. In this study replication-competent quasispecies were derived from virus isolated from an individual before and after seroconversion. Virus was also isolated from the presumed donor. Phenotypic and genotypic analysis of biological clones identified transmission of an R5/M-tropic phenotype. However, the ability of clones derived from the recipient to replicate in primary macrophages and PBMCs was restricted after transmission. This apparent selection process was supported by analysis of molecular clones derived from the isolated virus. Analysis of the ratio of synonymous and nonsynonymous substitutions predicted the existence of selective pressure soon after transmission, coincident with the development of HIV-1-specific antibodies. An Env trans-complementation assay demonstrated that the infectivity of a clone derived from the recipient after seroconversion was enhanced in the presence of a selected neutralizing antibody, indicating that the developing humoral immune response may have at least in part contributed to the selective pressure identified.

Background: The Sydney Blood Bank Cohort (SBBC) was infected between 1981 and 1984 with a nef/LTR defective strain of HIV-1. Different responses to HIV-1 infection have emerged between cohort members in the last 5 years. Three recipients (C135, C64 and C49) remain asymptomatic, have normal CD4 T cell counts, below detection (BD) viral loads (VL), remain therapy naive and are termed long-term non-progressors (LTNP). The donor (D36) and the two recipients (C98 and C54) have significantly declining CD4 T cell counts, detectable VL and are now long-term survivors (LTS). In contrast, in the SA cohort, comparison study group for the SBBC, five of 24 remain therapy naive after 15 years infection with HIV-1 and all have detectable VL. Objectives: This paper examines different outcomes to long-term infection with HIV-1 in the SBBC and provides a brief overview of the therapy naive in a comparison study group, the SA cohort. Study design: Retrospective epidemiological follow-up of the SBBC and the SA cohort has been conducted for > 15 years. Analysis of CD4 T cell counts, VL and intermittent monitoring of HIV-specific proliferative responses are reviewed. Viral sequence changes in the SBBC will be considered. Results: Prior to therapy D36 had a CD4 T cell count of 160/mm(3) and plasma VL of 9900 copies/ml while C98 had a CD4 T cell count of 387/mm(3) and plasma VL of 11 491 copies/ml. After 1 month of therapy, plasma VL was BD ( < 400 copies/ml) and both showed significant increase in CD4 T cell counts. Molecular changes have occurred in D36 and C98 viral strains, the most recently evolved quasispecies have larger deletions in the nef/LTR region. Conclusions: Infection with nef/LTR deleted HIV-1 has resulted in slower disease progression for the SBBC. The three LTNP have maintained normal low levels of activated CD8 T cells and strong HIV-specific proliferative responses to HIV-1 p24, which are associated with control of viral replication. (C) 2001 Elsevier Science BN. All rights reserved.

c-Myb is expressed in proliferating T cells. Fifteen c-Myb-binding sites can be identified in the HIV-1 long terminal repeat (LTR), suggesting that c-Myb may regulate HIV-1 gene expression and virus replication. Increasing the cellular levels of c-Myb by transient transfection of CEM cells resulted in a 10- to 20-fold activation of HIV-1 LTR-driven gene expression and mutation of one high-affinity Myb-binding site within the LTR reduced this activation by 60 to 70%. Conversely, inhibition of c-Myb expression in MT-2 cells by treatment with c-myb antisense oligonucleotides decreased HIV-1 replication by 85%, as measured by reverse transcriptase activity and cytopathic effects. The effect of c-myb antisense oligonucleotides on HIV-1 gene expression and virus particle production appeared to be independent of cell proliferation, but dependent on the presence of c-Myb activity mediated through the HIV-1 LTR. These data show that c-myb expression affects HIV-1 replication in CD4(+) T cells.

An explosive epidemic of human immunodeficiency virus type 1 (HIV-1) has been documented among the injecting drug user population of Kathmandu, Nepal, whose seropositivity rate has risen from 0 to 40% between 1995 and 1997. By using Catrimox to preserve whole-blood RNA at ambient temperature for transportation, HIV-1 envelope V3-V4 sequences were obtained from 36 patients in this group. Analysis of the sequences indicated a homogenous epidemic of subtype C virus, with at least two independent introductions of the virus into the population, Viral diversity was restricted within two transmission subclusters, with the majority of variation occurring in V4. Calculation of the synonymous-to-nonsynonymous mutation ratio (Ks:Ka) across this region showed that significant evolutionary pressure had been experienced during the rapid horizontal spread of the virus in this population, most strongly directed to the region between V3 and V4.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) represents a model promoter system and the identification and characterisation of cellular proteins that interact with this region has provided a basic understanding about both general eukaryotic and HIV-1 proviral transcriptional regulation. To date a large number of sequence-specific DNA-protein interactions have been described for the HIV-1 LTR. The aim of this report is to provide a comprehensive, updated listing of these HIV-1 LTR interactions. It is intended as a reference point to facilitate on-going studies characterising the identity of cellular proteins interacting with the HIV-1 LTR and the functional role(s) of specific regions of the LTR for HIV-1 replication.

Background and Methods The Sydney Blood Bank Cohort consists of a blood donor and eight transfusion recipients who were infected before 1985 with a strain of human immunodeficiency virus type 1 (HIV-1) with a deletion in the region in which the nef gene and the long terminal repeat overlap. Two recipients have died since 1994, at 77 and 83 years of age, of causes unrelated to HIV infection; one other recipient, who had systemic lupus erythematosus, died in 1987 at 22 years of age of causes possibly related to HIV. We present longitudinal immunologic and virologic data on the six surviving members and one deceased member of this cohort through September 30, 1998.Results The five surviving recipients remain asymptomatic 14 to 18 years after HIV-1 infection without any antiretroviral therapy; however, the donor commenced therapy in February 1999. In three recipients plasma concentrations of HIV-1 RNA are undetectable (<200 copies per milliliter), and in two of these three the CD4 lymphocyte counts have declined by 9 and 30 cells per cubic millimeter per year (P=0.3 and P=0.5, respectively). The donor and two other recipients have median plasma concentrations of HIV-1 RNA of 645 to 2850 copies per milliliter; the concentration has increased in the donor (P<0.001). The CD4 lymphocyte counts in these three cohort members have declined by 16 to 73 cells per cubic millimeter per year (P<0.001). In the recipient who died after 12 years of infection, the median plasma concentration of HIV-1 RNA was 1400 copies per milliliter, with a decline in CD4 lymphocyte counts of 17 cells per cubic millimeter per year (P=0.2).Conclusions After prolonged infection with this attenuated strain of HIV-1, there is evidence of immunologic damage in three of the four subjects with detectable plasma HIV-1 RNA. The CD4 lymphocyte counts appear to be stable in the three subjects in whom plasma HIV-1 RNA remains undetectable. (N Engl J Med 1999;340:1715-22.) (C) 1999, Massachusetts Medical Society.

585 WITHIN THE MAJOR a Mo group of HIV-1, phylogenetic analysis permits subclassification into the subtypes A to J. In 1996, a reference set of HIV-1 sequences representing the different subtypes was proposed and this list has been updated and modified as more full-length HIV-1 genomic sequences, including an increasing number of complex recombinant isolates, have become available.2 An analysis of four full-length HIV-1 genomes from Africa (DJ263, DJ264, HH8793, and SE6165) has proposed a new classification of strains previously defined as subtype G or as containing regions of subtype G.3 Isolates with a characteristic genomic pattern of recombination between subtypes A and G cluster together into a distinct clade exemplified by the strain IbNg. This recombinant virus shows an extensive geographic spread, having been isolated in Nigeria, Djibouti, and, by extrapolation of more limited sequence data from isolates that cluster closely with these examples, in Ivory Coast, the Democratic Republic of Congo (DRC), and Gabon.3,4 It is proposed that such circulating recombinant HIV-1 strains should be designated as a circulating recombinant formso (CRFs) of the virus, rather than as separate subtypes. Thus, sequences of the IbNg CRF type are termed a AG-IBNG.o By contrast, the two novel isolates HH8793 and SE6165, from Kenya and Congo, respectively, carry genomes that appear to be nonrecombinant and cluster together in subtype G. This sequence note presents the full-length genomic sequence of a subtype G HIV-1 originating in Kinshasa, DRC (termed DRCBL). The clinical history of the infected patient and initial subtyping of the virus in gag have been previously reported as isolate BL.5 It has become apparent that classification of HIV-1 on the basis of the analysis of small genomic regions is sometimes inadequateÐparticularly when dealing with isolates of African origin. Complex recombinant forms of HIV1 are increasingly being identified and accurate classification requires the examination of large, continuous regions of the genome. For this reason the entire genomic sequence of the virus was determined from patient peripheral blood mononuclear cells (PBMCs) by a previously described method.6 All reading frames were open and intact, apart from a 7-nucleotide (nt) deletion in pol at position 2948, resulting in a premature termination of the predicted protein product. This deletion was most probably the result of deriving the genomic sequence from a single integrated provirusÐmany of which contain defects. Its presence had no effect on the subsequent phylogenetic analysis. Initial phylogenetic analysis of p17gag, pol, the C4±V4 region of env and nef of DRCBL, by comparison with reference strain sequences, showed clustering in subtype G with a high degree of confidence in all regions (data not shown). Neighborjoining phylogenetic analysis was then performed using fulllength genomic sequences from reference strains of subtypes A to J. DRCBL clustered with subtype G isolates with 100% bootstrap value (Fig. 1). The LTR of DRCBL shows the described structural features of subtype G. Interestingly, the 3-nucleotide TAR bulge has the sequence UUUÐ in common with the isolates SE6165, HH8793, DJ263, DJ264, 92NG083.2,7 92NG003.1,7 and 92RU1318; but not IbNg, which has UCU. To examine DRCBL for evidence of intersubtype recombination, maximum parsimony bootscanning was performed as described with a sliding window of 300 nt advancing in 20-nt increments (Fig. 2). DRCBL was compared with the two reference subtype G strains SE6165 and HH8793 (mentioned above). One reference strain of each subtype was included, excepting E, because the A-type portions of this recombinant sub-

OBJECTIVE:To investigate whether members of a transfusion-linked cohort (the Sydney Bloodbank Cohort) infected with a nef-deleted strain of HIV-1 could be differentiated from individuals infected with wild-type strains of HIV-1 by characterizing the Nef antibody response of cohort members. DESIGN:Retrospective and prospective analysis of the nef gene sequence and the antibody response to Nef peptides in HIV-infected subjects. METHODS:Plasma was obtained from all individuals of the Sydney cohort, and from a variety of HIV-1-infected and uninfected controls. Antibodies recognizing full-length recombinant HIV-1NL43 Nef protein and synthetic peptide analogues were assessed by enzyme-linked immunosorbent assay. RESULTS:All 34 individuals infected with wild-type HIV-1 had antibodies reacting with full-length Nef protein as well as with a series of synthetic peptides (6-23-mers) spanning most of the Nef protein of HIV-1NL43. Although the HIV-1 quasispecies infecting the Sydney cohort had a consensus deletion of the nef gene corresponding to amino-acids 165-206, HIV-1 strains from individual members of the cohort had additional deletions comprising up to 80% of the nef gene. Members of the cohort had antibodies to peptides homologous to all regions of the Nef protein tested, except for a single peptide (amino-acids 162-177) that lies within the consensus nef deletion for the cohort quasispecies. CONCLUSION:These data show that nef-deleted strains of HIV-1 can be detected serologically. In the Sydney cohort, detection of antibodies to all regions of Nef tested, except that corresponding to amino-acids 162-177, suggests that observed deletions outside this domain occurred after this virus had infected these subjects and stimulated an immune response. A Nef peptide serological assay may be useful for identifying further examples of individuals infected with nef-deleted, attenuated HIV-1 quasispecies and for assessing the evolution of those variants in vivo.

AIDS Research and Human RetrovirusesVol. 14, No. 16 Sequence Note: A Novel Subtype A/G/J Recombinant Full-Length HIV Type 1 Genome from Burkina FasoROBERT B. OELRICHS, CASSY WORKMAN, TIINA LAUKKANEN, FRANCINE E. McCUTCHAN, and NICHOLAS J. DEACONROBERT B. OELRICHS, CASSY WORKMAN, TIINA LAUKKANEN, FRANCINE E. McCUTCHAN, and NICHOLAS J. DEACONPublished Online:15 Mar 2009https://doi.org/10.1089/aid.1998.14.1495AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byMolecular Epidemiology of HIV-1 in African Countries: A Comprehensive Overview21 December 2020 | Pathogens, Vol. 9, No. 12Reconstructing the HIV-1 CRF02_AG and CRF06_cpx epidemics in Burkina Faso and West Africa using early samplesInfection, Genetics and Evolution, Vol. 46Recombinant Viruses Initiated the Early HIV-1 Epidemic in Burkina Faso19 March 2014 | PLoS ONE, Vol. 9, No. 3First Report on a T69-ins Insertion in CRF06_cpx HIV Type 1 Young-Keol Cho, Ba-Reum Kim, Jung-Eun Kim, June-Hee Woo, and Brian T. Foley13 June 2013 | AIDS Research and Human Retroviruses, Vol. 29, No. 7Phylogenetic Analysis of Near Full-Length HIV Type 1 Genomic Sequences from 21 Korean Individuals Young-Keol Cho, Jung-Eun Kim, and Brian T. Foley5 April 2013 | AIDS Research and Human Retroviruses, Vol. 29, No. 4Drug Resistance Pattern of HIV Type 1 Isolates Sampled in 2007 from Therapy-Naive Pregnant Women in North-Central Nigeria Hannah O. Ajoge, Michelle L. Gordon, Sani Ibrahim, Oladapo S. Shittu, Thumbi Ndung'u, and Stephen O. Olonitola5 January 2012 | AIDS Research and Human Retroviruses, Vol. 28, No. 1Changing patterns in HIV-1 non-B clade prevalence and diversity in Italy over three decades*3 September 2010 | HIV Medicine, Vol. 11, No. 9Complex Mosaic Composition of Near Full-Length Genomes of Two NED (NIH-ENVA-DOD) Subtype Panel HIV Type 1 Strains, BCF-Dioum and BCF-Kita, Originating from the Democratic Republic of Congo (DRC) Diana D. Huang, Brian T. Foley, Catlin A. Tolzmann, Annastasia Ouma, and James W. Bremer20 October 2009 | AIDS Research and Human Retroviruses, Vol. 25, No. 10Characterization of drug-resistance mutations in HIV-1 isolates from non-HAART and HAART treated patients in Burkina Faso1 January 2006 | Journal of Medical Virology, Vol. 78, No. 11Two Viral Strains and a Possible Novel Recombinant Are Responsible for the Explosive Injecting Drug Use-Associated HIV Type 1 Epidemic in Estonia Veera Zetterberg, Valentina Ustina, Kai Zilmer, Kirsi Liitsola, Nelli Kalikova, Ksenia Sevastianova, Henrikki Brummer-Korvenkontio, Pauli Leinikki, and Mika O. Salminen19 November 2004 | AIDS Research and Human Retroviruses, Vol. 20, No. 11Molecular epidemiology of HIV in Ghana: Dominance of CRF02_AG1 January 2004 | Journal of Medical Virology, Vol. 73, No. 2Rising Prevalence of HIV-1 Non-B Subtypes in Belgium: 1983???2001JAIDS Journal of Acquired Immune Deficiency Syndromes, Vol. 35, No. 3The Genetic Diversity and Global Molecular Epidemiology of HIVSegregation of Human Immunodeficiency Virus Type 1 Subtypes by Risk Factor in AustraliaJournal of Clinical Microbiology, Vol. 41, No. 10Field Evaluation of the gag -Based Heteroduplex Mobility Assay for Genetic Subtyping of Circulating Recombinant Forms of Human Immunodeficiency Virus Type 1 in Abidjan, Côte d'IvoireJournal of Clinical Microbiology, Vol. 41, No. 7CRF06-cpx is the predominant HIV-1 variant in AIDS patients from Ouagadougou, the capital city of Burkina FasoAIDS, Vol. 17, No. 3HIV Type 1 Group M Clades Infecting Subjects From Rural Villages in Equatorial Rain Forests of CameroonJAIDS Journal of Acquired Immune Deficiency Syndromes, Vol. 31, No. 5Prevalence and origin of HIV-1 group M subtypes among patients attending a Belgian hospital in 1999Virus Research, Vol. 85, No. 1CRF06-cpx: A New Circulating Recombinant Form of HIV-1 in West Africa Involving Subtypes A, G, K, and JJAIDS Journal of Acquired Immune Deficiency Syndromes, Vol. 29, No. 5Genotypic and Phenotypic Analysis of HIV Type 1 Primary Isolates from Western Cameroon Denis M. Tebit, Léopold Zekeng, Lazare Kaptué, Mika Salminen, Hans-Georg Kräusslich, and Ottmar Herchenröder5 July 2004 | AIDS Research and Human Retroviruses, Vol. 18, No. 1Construction and Characterization of a Full-Length HIV-192UG001 Subtype D Infectious Molecular Clone P. Novelli, C. Vella, J. Oxford, and R.S. Daniels5 July 2004 | AIDS Research and Human Retroviruses, Vol. 18, No. 1Impact of Human Immunodeficiency Virus Type 1 Subtype on HIV Antibody Detection in Burkina FasoJAIDS Journal of Acquired Immune Deficiency Syndromes, Vol. 28, No. 2A complex human immunodeficiency virus type 1 A/G/J recombinant virus isolated from a seronegative patient with AIDS from Benin, West AfricaJournal of General Virology, Vol. 82, No. 5A New Human Immunodeficiency Virus Type 1 Circulating Recombinant Form from Tanzania Irene N. Koulinska, Thumbi Ndung'u, Davis Mwakagile, Gernard Msamanga, Charles Kagoma, Wafaie Fawzi, Max Essex, and Boris Renjifo5 July 2004 | AIDS Research and Human Retroviruses, Vol. 17, No. 5The public health significance of HIV-1 subtypesAIDS, Vol. 15Sequence Note: HIV Type 1 A/J Recombinant with a Pronounced pol Gene Mosaicism Vladimir A. Novitsky, Sarah Gaolekwe, Mary F. McLane, Thumbi P. Ndung'u, Brian T. Foley, Fredrik Vannberg, Richard Marlink, and Max Essex5 July 2004 | AIDS Research and Human Retroviruses, Vol. 16, No. 10Molecular Characterization of a Recombinant HIV Type 1 Isolate (A/G/E/?): Unidentified Regions May Be Derived from Parental Subtype E Sequences D. Paraskevis, M. Magiorkinis, V. Paparizos, G. N. Pavlakis, and A. Hatzakis5 July 2004 | AIDS Research and Human Retroviruses, Vol. 16, No. 9Rapid full-length genomic sequencing of two cytopathically heterogeneous Australian primary HIV-1 isolatesJournal of Biomedical Science, Vol. 7, No. 2Near-Full-Length Genome Sequencing of Divergent African HIV Type 1 Subtype F Viruses Leads to the Identification of a New HIV Type 1 Subtype Designated K Karine Triques, Anke Bourgeois, Nicole Vidal, Eitel Mpoudi-Ngole, Claire Mulanga-Kabeya, Nzila Nzilambi, Ndongo Torimiro, Eric Saman, Eric Delaporte, and Martine Peeters5 July 2004 | AIDS Research and Human Retroviruses, Vol. 16, No. 2Intersubtype Recombinant HIV Type 1 Involving HIV-MAL-Like and Subtype H-Like Sequence in Four Norwegian Cases Tom O. Jonassen, Bjorn Grinde, Birgitta Asjo, Gunnar Hasle, and Olav Hungnes5 July 2004 | AIDS Research and Human Retroviruses, Vol. 16, No. 1Sequence Note The Identification of a Complex A/G/I/J Recombinant HIV Type 1 Virus in Various West African Countries Celine Montavon, Frederic Bibollet-Ruche, David Robertson, Brahim Koumare, Claire Mulanga, Eka Esu-Williams, Coumba Toure, Souleymane Mboup, Eric Saman, Eric Delaporte, and Martine Peeters15 July 2004 | AIDS Research and Human Retroviruses, Vol. 15, No. 18Molecular Characterization of Human Immunodeficiency Virus (HIV)-1 and -2 in Individuals from Guinea-Bissau with Single or Dual Infections: Predominance of a Distinct HIV-1 Subtype A/G Recombinant in West AfricaVirology, Vol. 262, No. 2Full-Length Genomic Sequence of an HIV Type 1 Subtype G from Kinshasa Robert B. Oelrichs, Anne-Mieke Vandamme, Kristel Van Laethem, Zeger Debyser, Francine E. Mccutchan, and Nicholas J. Deacon5 July 2004 | AIDS Research and Human Retroviruses, Vol. 15, No. 6Inaccurate HIV-1 viral load quantification by three major commercially available methodsAIDS, Vol. 13, No. 6Virtually Full-Length Sequences of HIV Type 1 Subtype J Reference Strains Tiina Laukkanen, Jan Albert, Kirsi Liitsola, Stacie D. Green, Jean K. Carr, Thomas Leitner, Francine E. Mccutchan, and Mika O. Salminen5 July 2004 | AIDS Research and Human Retroviruses, Vol. 15, No. 3HIV-1 Subtypes and Recombinants Volume 14Issue 16Nov 1998 To cite this article:ROBERT B. OELRICHS, CASSY WORKMAN, TIINA LAUKKANEN, FRANCINE E. McCUTCHAN, and NICHOLAS J. DEACON.Sequence Note: A Novel Subtype A/G/J Recombinant Full-Length HIV Type 1 Genome from Burkina Faso.AIDS Research and Human Retroviruses.Nov 1998.1495-1500.http://doi.org/10.1089/aid.1998.14.1495Published in Volume: 14 Issue 16: March 15, 2009PDF download

Human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in vitro in differentiated macrophages than in freshly isolated monocytes. We investigated whether this may be partly explained by changes in expression of NF-kappaB with monocyte differentiation. We demonstrated that constitutive expression of NF-kappaB in primary human monocytes changed significantly with differentiation in vitro to monocyte-derived macrophages (MDMs) and differentiation in vivo to alveolar macrophages (AMs). Freshly isolated monocytes constitutively expressed high levels of transcriptionally inactive p50 homodimer which decreased with time in culture in favor of the transcriptionally active p50/p65 and p50/RelB heterodimers. As in MDMs, AMs constitutively expressed p50/p65 and p50/RelB although at lower levels. HIV infection of fresh monocytes failed to induce p50/p65 as seen in MDMs. The replacement of p50 homodimers with transcriptionally active heterodimers following time in culture may partially explain the progressive increase in susceptibility of monocytes to HIV infection during in vitro culture. The change in NF-kappaB components with monocyte differentiation in vivo may also explain the different transcriptional activities of these cell populations in HIV-infected individuals.

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which we refer to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. We show that EBS4 is, required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4, and a mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner.

We report the discovery of a fourth Sp1 binding site at the 5' end of the U3 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (HIV-1 Sp1 IV), localized to HXB2 nucleotides -433 to -441. This site is shown to bind Sp1 protein specifically in electrophoretic mobility shift assays. Sp1 protein appears to bind to HIV-1 Spl IV with 5 to 10 times lower affinity than to a consensus Sp1 site. Mutation of HIV-1 Sp1 IV in an HXB2-derived long terminal repeat-chloramphenicol acetyltransferase reporter construct gave no significant change in positive-sense transcription but abolished both basal and phorbol myristate acetate-activated negative-sense transcription. Taken together, the results further define the HIV-1 negative-sense promoter as an Sp1-dependent, phorbol myristate acetate-responsive, and Tat-inhibited promoter initiating at HXB2 nucleotide -450.

BioTechniquesVol. 21, No. 3 BenchmarksOpen AccessElimination of Sequence Ambiguities by a Single-Step Modification of a Solid-Phase, Single-Stranded Sequencing ProtocolVictoria Alice Lawson, Dale Alan McPhee & Nicholas John DeaconVictoria Alice Lawson*Address correspondence to Victoria A. Lawson, AIDS Cellular Biology Unit, Macfarlane Burnet Centre for Medical Research, P.O. Box 254, Fairfield, Victoria, 3078, Australia.Macfarlane Burnet Centre for Medical Research Fairfield, Victoria, Australia, Dale Alan McPheeMacfarlane Burnet Centre for Medical Research Fairfield, Victoria, Australia & Nicholas John DeaconMacfarlane Burnet Centre for Medical Research Fairfield, Victoria, AustraliaPublished Online:2 Aug 2018https://doi.org/10.2144/96213bm01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByAdaptive Changes after Human Immunodeficiency Virus Type 1 TransmissionAIDS Research and Human Retroviruses, Vol. 18, No. 8Amplification and Direct Sequence Analysis of the 23S rRNA Gene from Thermophilic BacteriaAshraf Ibrahim, Jacob Hofman-Bang & Birgitte K. Ahring5 September 2018 | BioTechniques, Vol. 30, No. 2Systematics and Evolution of the Algae. I. Genomics Meets PhylogenyApproaches to DNA Mutagenesis: An OverviewAnalytical Biochemistry, Vol. 254, No. 2Direct Sequencing of PCR-Amplified 23S rDNAAshraf Ibrahim & Anders Sjöstedt7 August 2018 | BioTechniques, Vol. 23, No. 2Method to Reverse the Order of Multiple Cloning SitesChing-Shwun Lin7 August 2018 | BioTechniques, Vol. 23, No. 2 Vol. 21, No. 3 Follow us on social media for the latest updates Metrics Downloaded 92 times History Published online 2 August 2018 Published in print September 1996 Information© 2018 Author(s)PDF download

The relationship between virus and host cells is multifactorial and nonlinear. This indicates that the effect of an immune response on infection can lead to several different outcomes. These include severe immunopathology. We seek to define properties of CTL-induced pathology in viral infections and examine the implications for HIV disease progression. We find that CTL-induced pathology is observed if the rate of viral replication is fast relative to the CTL responsiveness of the host. Theoretical predictions are consistent with empirical data on LCMV infection. These conditions are also sufficient to induce pathology in HIV infection. However, the absence of HIV-specific CTL can result in an equivalent depletion of the CD4 T cell pool as a consequence of the short life span of activated T cells. A mathematical model describing the evolution of HIV coreceptor usage in the context of lytic and nonlytic CD8 cell responses might account for the relatively long time span required to result in disease. Viral evolution toward parameter ranges allowing CTL-induced pathology is difficult to achieve. It requires the emergence of fast viral replication together with escape from nonlytic CTL responses. However, according to the model, fast viral replication can result in the evolution of virus strains that are susceptible to chemokine-mediated inhibition of viral replication.

We would like to state two caveats about this proposal. The death of the HIV-infected patient C83, who died with Pneumocystis carinii pneumonia (1), is attributed to her underlying autoimmune disease, SLE, which required immunosuppressive therapy. However, neither CD4+ T cell counts nor data on viral load are provided in the report. Deacon et al. mention that DNA extracted from the only blood sample of patient C83 available did not yield HIV nef gene sequences, even though single copy cellular genes could be amplified. The sensitivity of the polymerase chain reaction (PCR) assays is not given in the report. As patient C83 died with an opportunistic infection, readers need to see all CD4+ T cell counts available, more information regarding the intactness of this DNA sample, and the time of its collection during the patient's illness. Thus, the data presented by Deacon et al. for patient C83 are inconclusive and do not exclude the possibility that nef-deleted HIV contributed significantly to her death. The pathogenicity observed in infant macaques infected with SIVA3, a mutant of the simian immunodeficiency virus (SIV) containing large deletions in the nef and vpr genes and in the negative regulatory element of the long terminal repeat (2), is not discussed by Deacon et al. Two of the original four SIVA3-infected macaque infants have died of AIDS, and the survivors now have high viral loads. Although we have concerns regarding the safety and efficacy of the proposed use of nef-deleted viruses as anti-AIDS vaccines, nef-deleted, live attenuated viruses still could play a major role in determining the correlates of immune protection in animal models, which could contribute to the design of safer, more effective vaccines. Ruth M. Ruprecht Timothy W. Baba Vladimir Liska Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA References

Sequencing of the reverse transcriptase (RT) region of 26 human immunodeficiency virus type 1 (HIV-1) isolates from eight patients treated with 3'-azido-3'-deoxythymidine (AZT) revealed a mutation at codon 210 from TTG (leucine) to TGG (tryptophan) exclusively in association with resistance to AZT. The mutation Trp-210 was observed in 15 of the 20 isolates phenotypically resistant to AZT, being more commonly observed than resistance-associated mutations at codons 67, 70, and 219. Trp-210 was never observed before the emergence of resistance-associated mutations Leu-41 and Tyr-215, and in a sequential series of five isolates from one patient the order of emergence of mutations was found to be Tyr-215, Leu-41, and then Trp-210. Trp-210 was also found in association with the Leu-41, Asn-67, Arg-70, and Tyr-215 resistance genotype. To define the role of Trp-210 in AZT resistance, molecular HIV-1 clones were constructed with various combinations of RT mutations at codons 41, 67, 70, 210, and 215 and tested for susceptibility to AZT. In clones with polymerase genes derived either from HXB2-D or clinical isolates, Trp-210 alone did not increase AZT resistance, whereas in conjunction with Leu-41 and Tyr-215, Trp-210 contributed to high-level resistance (50% inhibitory concentration of >1 microM). In HXB2-D, Trp-210 with Tyr-215 generated a virus with resistance comparable to one with Leu-41, Tyr-215, and Trp-210. Inserting Trp-210 into the genetic context of mutations at codons 41, 67, 70, and 215 further enhanced resistance from a 50% inhibitory concentration of 1.44 microM to 8.41 microM. Molecular modeling of the tertiary structure of HIV-1 RT revealed that the distance between the side chains of Trp-210 (in helix alphaF) and Tyr-215 (in strand beta11a) approximated 4 A (1 A = 0.1 nm), sufficiently close to result in significant energetic interaction between these two aromatic side chains. In conclusion, Trp-210 contributes significantly to phenotypic AZT resistance of HIV-1 by augmenting resistance at least three- to sixfold in the context of two resistant genotypes, and its effect may require an interaction with an aromatic amino acid at position 215.