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Division of Microbiology, Kanagawa Prefectural Institute of Public Health
Atsugi City Hospital, Kanagawa
AIDS Research Center, National Institute of Infectious Diseases, Musashimurayama
AIDS Research Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
We identified an unusual case of human immunodeficiency virus type 1 (HIV-1) infection in a patient (GM43) who exhibited a persistently low antibody response and undetectable viral load during a 5-year follow-up period. GM43 harbored HIV-1 circulating recombinant form 01_AE with gross deletions in the nef/long terminal repeat (LTR) region. The sizes of the deletions increased progressively from 84 to >400 bp during the 5-year period. GM43 appeared to have acquired defective variants from her husband. The genetic alterations in the nef/LTR region were remarkably similar to those that have been reported in slow progressors (such as the slow progressors in the Sydney Blood Bank Cohort). The present study is the first report of slow disease progression due to gross genetic alterations in the nef/LTR region in a person infected with an HIV-1 nonsubtype B strain.
Rates of disease progression vary among individuals infected with HIV-1, because of the complex interplay between host genetic and immunologic factors and the pathogenic potential of the infecting virus. The viral nef gene is one of the crucial determinants of disease progression, as has been demonstrated in animal models [13]. That the nef gene is a key factor for disease progression in humans is strongly supported by the finding that some long-term nonprogressors (LTNPs) with low viral loads (despite 1014 years of HIV-1 infection) carry viruses with gross deletions [46] or small structural defects and mutations [7] in the nef gene.
The nef gene is known to have pleiotropic functions, including down-regulation of the cell-surface expression of CD4 and class I major histocompatibility complex (MHC) molecules, enhancement of viral replication and infectivity, induction of cytokine and chemokine expression by T cells and macrophages, and blockage of proapoptotic signaling by HIV-1infected cells (reviewed in Geyer et al. [8]). A large number of cellular interaction partners critical to nef gene functions have been identified, and the binding sites have been mapped to distinct locations within the Nef protein (reviewed in Geyer et al. [8]).
Although the genetic alterations in the nef gene that are associated with slow disease progression have been identified in HIV-1 subtype B in US and European populations [47], it remains unclear whether these alterations are found only in the subtype B lineage. In the present study, we identified attenuated variants of HIV-1 circulating recombinant form 01_AE (CRF01_AE) that harbored gross deletions in the nef/long terminal repeat (LTR) region in an asymptomatic patient (GM43) who had an unusually weak antibody response and an undetectable viral load during a 5-year follow-up period, demonstrating the association between nef/LTR deletions and slow disease progression with respect to infection with a nonsubtype B strain.
Patients, materials, and methods.
Informed consent was obtained from the patients, and the study was conducted in accordance with the clinical research guidelines of Japan. Antibodies to HIV-1 were detected by use of the Serodia HIV-1 gelatin particle agglutination (PA) test (Fujirebio), and Western-blot (WB) analysis (LAV Blot I; Bio-Rad) was used for confirmation. Plasma HIV-1 RNA loads were measured by the ultrasensitive method with the Amplicor HIV-1 Monitor Kit (version 1.5; Roche Diagnostics), which allows the sensitive detection of HIV-l RNA from both HIV-1 subtype B and nonsubtype B strains, including CRF01_AE [9]. Replication-competent HIV-1 strains were isolated by cocultivation with CD8-depleted peripheral-blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated by use of anti-CD3 antibody (CLB-CD3; PeliCluster), instead of the standard activation stimuli of phytohemagglutinin and interleukin-2, to improve the efficiency of isolation [10, 11].
The nef/LTR regions of HIV-1 provirus genomes were amplified by nested polymerase chain reaction (PCR), with the outer primers Env43F14 (sense; 5-GAGTTAGGCAGGGATACTCAC-3; positions 78927912 of the genome of CM240, the HIV-1 CRF01_AE reference strain [12]) and 3LTR43R16 (antisense; 5-TAAGCACTCAAGGCAAGC-3; positions 92029185 of the genome of CM240) and the inner primers Env43F15 (sense; 5-AGCCTGTGCCTCTTCAGCTACCA-3; positions 80528083 of the genome of CM240) and MSR5 (antisense; 5-GCACTCAAGGCAAGCTTTATTGAGGCT-3; positions 91999173 of the genome of CM240). The nucleotide sequences of both strands were determined by the BigDye Terminator cycle sequencing method, using a Prism 310 DNA Sequencer (Applied Biosystems). Nucleotide sequences (GenBank accession numbers AB193797AB193800) were aligned by use of CLUSTAL W (version 1.4). Phylogenetic trees were constructed by the neighbor-joining method, based on Kimura's 2-parameter distance matrix with 100 bootstrap replicates. Analyses were implemented by use of PHYLIP (version 3.573) [13].
Results.
GM43 is a 28-year-old Thai woman who has lived in Japan for the past 6 years. GM43 was infected with HIV-1 via her husband, GM46. GM46 had contracted HIV-1 via heterosexual contact before 1995, presumably in Thailand. Both GM43 and GM46 remain healthy and do not have any clinical symptoms. There is no indication of HIV-2 infection. In January 1996, serologic tests conducted for GM43 at her 18th week of pregnancy (her first visit) showed low marginal HIV-1 seropositivity, with a PA antibody titer of 1 : 16 (figure 1A). Indeterminate WB results (1+ reactivity for gp160 only) persisted for GM43 throughout an 18-month observation period, whereas GM46 was unequivocally positive by WB (figure 1B). Empirically, in most HIV-1infected patients, PA antibody titers exceed 1 : 103 within 2 weeks of seroconversion and reach 1 : 104 one month after seroconversion. However, in GM43, low PA antibody titers (<1 : 103) persisted for 1.5 years, and it took >4.5 years until her PA antibody titer reached 1 : 104 (figure 1A).
In parallel with this slow process of seroconversion, GM43's plasma HIV-1 RNA load was persistently below the limit of detection by PCR (<50 copies/mL) during a 4.5-year period (figure 1A). HIV-1 proviral DNAs were amplified by nested PCR for the env (C2/V3) region only in July 1997 and for both the gag (p17) and env (C2/V3) regions in December 1998 (figure 1A), providing conclusive evidence that GM43 was infected with HIV-1. During the first 3 years after seroconversion, GM43's CD4+ cell count gradually decreased, from 1074 to 600 cells/L, as her plasma HIV-1 RNA load gradually increased, but her CD4+ cell count remained stable thereafter, at 400600 cells/L (figure 1A).
To further characterize the genetic alterations in the nef/LTR region for GM43, we molecularly cloned (by the TA cloning method) the PCR products and determined the nucleotide sequences of 1639 independent PCR clones at each time point. Nucleotide-sequence alignment revealed progressive deletions in the nef/LTR region over time (figure 2). Plasma HIV-1 RNA load was detectable after the increased deletions in the nef/LTR region (time points IIIV). A replication-competent HIV-1 strain (GM43-23) was isolated for the first time at time point V. The HIV-1 quasispecies with the 391-bp nef/LTR deletion (391) appeared to constitute a major functional (replication-competent) segment of the proviral population in GM43.
(1 of 2 images)
The overall structural configuration of the nef/LTR deletions found in the CRF01_AE variant infecting GM43 was remarkably similar to that of the attenuated HIV-1 variant C18 (which belongs to HIV-1 subtype B) detected in the Sydney Blood Bank Cohort [5] (figure 2A and 2B). Of note, the sequence features unique to CRF01_AEincluding the GABP motif (5-ACTTCCG-3), a single NF-B [14], an unusual TATA box (5-TAAAA-3), and a 2-nt bulge in TAR stems (figure 2)were detected in GM43. No appreciable direct repeats that may have caused the deletion in the nef/LTR region [6] were detected in GM43.
Discussion.
We have identified a unique case of CRF01_AE infection, in which a patient, GM43, experienced an unusually slow increase in HIV-1 antibody titers and had an undetectable viral load over a prolonged period of time. GM43 carried attenuated viral variants with a range of nef/LTR deletions that were similar to those found in LTNPs infected with HIV-1 subtype B [46]. The present study is the first report demonstrating the association between gross nef/LTR deletions and slow disease progression in a patient infected with a nonsubtype B strain.
Phylogenetic-tree analysis demonstrated that GM43 acquired CRF01_AE from her husband, GM46. Interestingly, GM46 was also found to harbor unique sets of nef/LTR deletions, although the profiles of the deletions detected in GM46 were not identical to those detected in GM43 (figure 2). It is tempting to speculate that the defective genomes detected in GM43 may have evolved from a minor viral quasispecies carried by GM46 that was not detected in the present study or that was present only transiently at the time of transmission to GM43. If this is the case, the lack of selection for functional nef alleles in GM43 during transmission and/or establishment of infection from GM46 is rather surprising, because functional forms of nef alleles are quickly and efficiently selected for in rhesus monkeys infected experimentally with nef-defective simian immunodeficiency virus [1]. This suggests that, in certain patients, attenuated viral variants might have a selective advantage over HIV-1 strains with an intact nef allele. For instance, an efficient immune response may contribute to the selection of nef-defective viruses that could escape the cytotoxic T lymphocyte recognition that is critical to the effective control of viral replication [15]. In light of the identification of this unique case of CRF01_AE infection, a systematic search for the viral and host factors that influence disease progression may be warrantedespecially in less-studied regions of the HIV-1 epidemic, such as in developing countries in Asiawith a slow increase in antibody titer used as a convenient marker.
Acknowledgments
We thank the staffs of the participating clinics for their help and Dr. Shingo Kato for his valuable advice on virus isolation.
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