Predominance of distinct viral genotypes in brain and lymph node compartments of HIV-1-infected individuals

Abstract

A modified polymerase chain reaction protocol was used to amplify the entire envelope-coding region of HIV-1 directly from brain and lymph node tissue obtained at autopsy from three HIV-1-infected individuals. Molecular analysis of amplified DNA by digestion with 18 restriction endonucleases, singly and in combination, revealed different HIV-1 genotypes in the brain and lymph node compartments in each of the three individuals. This anatomic compartmentalization of HIV-1 populations may reflect different viral genomic sequences that determine tropism or differences in host immune selection pressures in the brain and lymphoid compartments that drive the emergence of distinct viral populations.

FIG. 1.

PCR-mediated amplification of complete HIV-1 envelope-coding region. A. Region of the genome targeted by nested PCR primers (numbering according to the HXB2 sequence [30|). DNA sequence of the primer-binding regions is shown below the representation of the ORFs. B. Effect of primer concentration on 3.1-kb env amplification. One picogram of cloned HXB2 DNA (30) was subjected to a preliminary 2-min denaturation step at 95°C and amplified by 30 PCR cycles, each consisting of 95°C for 30 sec (denaturation) followed by 58°C for 30 sec (annealing) and then by 72°C for 2 min (extension). PCR reaction buffer was as described by Perkin-Elmer Cetus except that the MgCl₂ concentration was increased to 2.0 mM. Amount of each primer (pM) in the PCR reaction is indicated. C. Comparative efficiency of two rounds of nested PCR amplification (“Nested”; primer 8/3, 1st round followed by 7/9, 2nd round) versus two rounds of amplification with the same primer set (“Reused”; primers 8/3); 0.5 μg cellular DNA from brain (Br) and lymph node (LN) of subject Wi were subjected to PCR as described for Panel B. One fiftieth of the 1st-round PCR products (1 μl) was diluted to 100 μl for 2nd-round amplification. Reaction conditions were identical in both rounds. D. Efficiency of single-v double-round PCR: 0.5 μg of lymph node DNA from subject St or 0.5 μg of cellular DNA from an HI V-l -negative CD4⁺ T-cell line CEM (“cont.”) were amplified by one round of PCR (primers 8/3) or an additional second round (primers 7/9) as described in Panel C. M, 1-kb DNA ladder (BRL); Mλ, HindIII-digested λ-DNA mol. wt. markers.

FIG. 2.

Distinct restriction fragment polymorphisms between PCR-amplified env DNA from brain and lymph node tissue (subject Wi). Total cellular DNA was amplified by two rounds of nested PCR as described in Figure 1C. Then 100 to 200 ng of PCR-amplified, gel-purified, 3.1-kb env DNA was digested overnight with the indicated restriction enzyme, electrophoresed, transferred to nylon membranes, and hybridized to a subgenomic HIV-1 env probe (35). Additional DNA fragments resulting from extra-endonuclease cleavage sites (arrows) and loss of fragments secondary to absence of a cleavage site (asterisk) are shown. Higher mol. wt. bands are overexposed to allow visualization of lower mol. wt. fragments.

FIG. 3.

Restriction endonuclease cleavage maps of brain- and lymph node-specific PCR-amplified envelope-coding regions. DNA was prepared and analyzed as described for Figure 2, and the locations of restriction enzyme cleavage sites were deduced from single and double digests and compiled. Only polymorphisms detected exclusively in that particular tissue compartment are indicated on each map. The restriction maps of seven North American isolates (HXB2 [30], BRU [36], SF2 [32], CDC 451 [10], MN [16], BRVA [1], JFL [22]) and one European isolate (HAN [33]) are shown for comparison. Restriction endonuclease cleavage sites are indicated as A, AvaI; V, AvaII; B, BamHI; G, BglII; E EcoRI; H. HphI; I, HindIII; K, KpnI; D, NdeI; N, NsiI; P, PstI; U, PvuII; L, SalI; S, SacI; C, ScaI; Z, SspI; T. StuI; X. XhoI. Derivation of cleavage patterns for all characterized isolates was facilitated by information contained within the Human Retroviruses and AIDS Sequence Database (24).