Human immunodeficiency virus type 1 DNA sequences genetically damaged by hypermutation are often abundant in patient peripheral blood mononuclear cells and may be generated during near-simultaneous infection and activation of CD4(+) T cells

Abstract

G-to-A hypermutation has been sporadically observed in human immunodeficiency virus type 1 (HIV-1) proviral sequences from patient peripheral blood mononuclear cells (PBMC) and virus cultures but has not been systematically evaluated. PCR primers matched to normal and hypermutated sequences were used in conjunction with an agarose gel electrophoresis system incorporating an AT-binding dye to visualize, separate, clone, and sequence hypermutated and normal sequences in the 297-bp HIV-1 protease gene amplified from patient PBMC. Among 53 patients, including individuals infected with subtypes A through D and at different clinical stages, at least 43% of patients harbored abundant hypermutated, along with normal, protease genes. In 70 hypermutated sequences, saturation of G residues in the GA or GG dinucleotide context ranged from 20 to 94%. Levels of other mutants were not elevated, and G-to-A replacement was entirely restricted to GA or GG, and not GC or GT, dinucleotides. Sixty-nine of 70 hypermutated and 3 of 149 normal sequences had in-frame stop codons. To investigate the conditions under which hypermutation occurs in cell cultures, purified CD4(+) T cells from normal donors were infected with cloned NL4-3 virus stocks at various times before and after phytohemagglutinin (PHA) activation. Hypermutation was pronounced when HIV-1 infection occurred simultaneously with, or a few hours after, PHA activation, but after 12 h or more after PHA activation, most HIV-1 sequences were normal. Hypermutated sequences generated in culture corresponded exactly in all parameters to those obtained from patient PBMC. Near-simultaneous activation and infection of CD4(+) T cells may represent a window of susceptibility where the informational content of HIV-1 sequences is lost due to hypermutation.

FIG. 1

Calibration of the HA yellow gel system. HIV-1 protease sequences amplified from patient PBMC and representing a range of G-to-A hypermutations were used to explore the performance of 1% agarose gels containing HA yellow, a dye that preferentially binds to AT-rich regions in DNA. The alignment of the 297-bp sequences, amplified with primers DP16 and DP17 and ranging from normal (62% A+T) to maximally hypermutated (78% A+T) is shown at the top. The GA and GG dinucleotides that are susceptible to hypermutation are shaded. The gel on the left shows the migration of these PCR products as a single band at 297 bp without HA yellow. On the right, a gel incorporating HA yellow is shown, illustrating the direct relationship between AT content and mobility. Sequences with as little as 10% G-to-A substitution (2nd lane) migrated differently than normal sequences. PCR products representing the normal and maximally hypermutated sequences were used as migration standards in subsequent HA yellow gels.

FIG. 2

Relative recovery of hypermutated sequences with normal and hypermutated PCR primers. Primers were designed to incorporate G-to-A substitutions in GA or GG dinucleotides (Materials and Methods) and compared to primers matched to normal sequences for their ability to amplify hypermutated sequences from the PBMC of 10 patients. A side-by side comparison of the PCR products using HA yellow gels is shown. N, normal primers; H, primers hyp and hypa. Hypermutated bands detected with primers hyp and hypa but not with normal primers are indicated for patients 3, 4, 5, 6, and 8 (asterisks). The positions of normal and fully hypermutated standards are indicated by arrows.

FIG. 3

Abundance and variety of hypermutated PCR products amplified from patient PBMC. DNA extracted from patient PBMC was amplified in a touchdown, nested PCR with primers hyp and hypa as described in Materials and Methods. PCR products were compared to normal and hypermutated standards on HA yellow gels. While all products migrated as a single band at 297 bp without HA yellow (left), they were often split into several bands in the presence of HA yellow (right). Products were visualized by staining with ethidium bromide.

FIG. 4

Classification of sequences from patient PBMC. HIV-1 protease genes were amplified using DNA extracted from the PBMC of 53 patients ranging from the early, asymptomatic stage of HIV-1 infection to late-stage AIDS, and including infections with HIV-1 subtypes A through D (Table 2). Products PCR amplified with primers hyp and hypa were molecularly cloned and sequenced. In total, 287 sequences were obtained, of which 219 were unique. Among the 219 sequences, the percent A+T distribution is shown in the top graph. Using the sequence with the lowest percent A+T from each patient as a normal reference, the percent G in the GA or GG context that was mutated to A was calculated for each sequence, and the distribution of percent G-to-A mutation was examined (bottom graph). Based on the bimodal distribution of these parameters, 149 sequences were classified as normal (less than or equal to 66% A+T and less than 20% G-to-A mutation) and 70 were classified as hypermutated (67% A+T and more than 21% G-to-A mutation).

FIG. 5

Parameters of hypermutation in sequences from patient PBMC. One hundred forty-nine normal and 70 hypermutated sequences from patient PBMC were examined for base composition (top). The normal sequences had a narrow distribution of percent G and percent A, centered on 22 and 37%, respectively. Hypermutated sequences showed a broad range of G content from 5 to 20% and a range of A content from 40 to 54%. The distribution of percent C and percent T was the same in normal and hypermutated sequences. Hypermutated sequences were arranged in order of increasing hypermutation (bottom two graphs). While G-to-A mutations in the GA or GG context increased 10-fold, other mutations (G to A in the GC and GT context and all other mutations) showed no discernible increase. The percentages of available G replaced by A in the GA and GG contexts were calculated separately and compared over the range of G-to-A substitution (bottom). More of the G residues in the GA context than in the GG context were replaced by A at all levels of hypermutation.

FIG. 6

Relationship of hypermutation to T-cell activation in virus cultures. CD4⁺ T cells isolated from the PBMC of normal donors were infected with an NL4-3 virus stock for 2 h at a multiplicity of infection of 0.02 (stippled gray bar) under varying conditions of PHA activation (gray bars). Condition I, infection without addition of PHA; condition II, simultaneous infection and PHA addition; conditions III to VIII, infection with prior PHA activation for 3 to 72 h respectively; condition IX, infection and addition of PHA 24 h later. Cultures were sampled at intervals and assayed for the presence of p24 antigen in the culture supernatant (solid lines) and for the presence of hypermutated HIV-1 DNA sequences. Filled stars, samples where hypermutated sequences were abundant; open stars, samples with rare hypermutated sequences. The appearance of PCR products on HA yellow gels is shown on the right. Time points are labeled “a” through “n” at the tops of the gels and in the diagram. Samples a, b, c, d, e, g, and h are bulk PCR products. Samples from time points at which hypermutation was rare (f, i, j, k, and l) are shown both as bulk PCR products (B) and after pre-enrichment by ScrF1 and AvaII digestion (A). Time points not marked by stars or asterisks yielded only normal sequences both before and after enrichment (not shown).

FIG. 7

Parameters of hypermutation in virus cultures. A total of 227 HIV-1 protease sequences, 131 of which were unique, were obtained from virus cultures. The unique sequences were compared for their distribution of percent A+T (A). Seventy-three were normal and 58 were hypermutated. (B) Distribution of G-to-A mutation in sequences from different culture conditions. Roman numerals refer to the culture conditions described in the legend to Fig. 6. Filled symbols, normal sequences; open symbols, hypermutated sequences. The cultures where abundant hypermutants covering the full range of G-to-A replacement were found are shaded. Below, the average ratio of percent G-to-A substitutions in the GA versus GG context is plotted for the sequences (all except six) in which both contexts were used. (C) Base composition of normal and hypermutated sequences from virus cultures. (D) Specificity for G-to-A substitutions in the GA or GG context in contrast to all other mutations, and the preferential use of GA over GG over the range of hypermutation.