Memory in retroviral quasispecies: experimental evidence and theoretical model for human immunodeficiency virus

Abstract

Viral quasispecies may possess a molecular memory of their past evolutionary history, imprinted on minority components of the mutant spectrum. Here we report experimental evidence and a theoretical model for memory in retroviral quasispecies in vivo. Apart from replicative memory associated with quasispecies dynamics, retroviruses may harbour a "cellular" or "anatomical" memory derived from their integrative cycle and the presence of viral reservoirs in body compartments. Three independent sets of data exemplify the two kinds of memory in human immunodeficiency virus type 1 (HIV-1). The data provide evidence of re-emergence of sequences that were hidden in cellular or anatomical compartments for extended periods of infection, and recovery of a quasispecies from pre-existing genomes. We develop a three-component model that incorporates the essential features of the quasispecies dynamics of retroviruses exposed to selective pressures. Significantly, a numerical study based on this model is in agreement with the experimental data, further supporting the existence of both replicative and reservoir memory in retroviral quasispecies.

Figure 1

Flux diagram of quasispecies components in a typical HIV-1 infection and the possible sources of genetic memory. The contribution of different infected cells to the circulating HIV-1 quasispecies is as follows: 98% of the circulating virions are produced by actively replicating CD4+ lymphocytes (which constitute 1% of the total number of CD4+ lymphocytes), about 1% of the virions come from macrophages, another 1% from follicular dendritic cells and, finally, less than 1% are produced upon activation of latently infected CD4+ lymphocytes (which represent 9% of the total number of CD4+ lymphocytes). Most (about 90%) of the CD4+ lymphocytes are infected with defective viruses, and they do not contribute to the circulating quasispecies. These three types of CD4+ lymphocytes are similarly distributed between lymph nodes (98%) and the bloodstream (2%) (based on data from , , , , , , and references therein).

Figure 2

Neighbor-Joining trees of pol ((a) case 1; (c) case 2) and env ((b) case 1; (d) case 2) sequences. Amplification of the env region was not possible for samples 7.95 and 9.96 in case 1 (b) and for sample 8.96 in case 2 (d). Bootstrap values (1000 replicas) higher than 0.65 per unit are indicated in parenthesis. Clearly defined groups of sequences have been encircled. The bar represents 0.01 ((a) and (c)) or 0.1 ((b) and (d)) substitutions per nucleotide. Genetic distances can be calculated from the branch lengths; exact values of them will be provided upon request. The same topology was found using other phylogenetic approaches such as UPGMA, maximum parsimony and maximum likelihood (data not shown).

Figure 3

Maximum parsimony clustering of the 120 sequences of molecular clones from six sequential samples from case 3, using HIV-1 CAM-1 as reference sequence. Unique sequences are identified as sample-clone number (see Table 1). Groups of clones with identical sequences are labelled as sample-letter-number: the letter is consecutive for each group in one sample and the number indicates how many identical clones belong to that group. Sample 1 (February 1994) showed four groups of identical sequences: 1-A5 (clones 1-1, 1-4, 1-7, 1-14 and 1-16), 1-B3 (1-3, 1-9 and 1-12), 1-C3 (1-8, 1-18 and 1-19) and 1-D2 (1-13 and 1-15). Samples 2 (January 1995) and 3 (June 1995) had all their clonal sequences different from each other. Sample 4 (August 1995) showed five groups of identical sequences: 4-A3 (4-2, 4-6 and 4-20), 4-B2 (4-4 and 4-12), 4-C2 (4-7 and 4-16), 4-D2 (4-8 and 4-9) and 4-E3 (4-10, 4-14 and 4-15). Sample 5 (September 1995) had three groups of identical sequences: 5-A2 (5-4 and 5-12), 5-B2 (5-7 and 5-20) and 5-C2 (5-14 and 5-18). Sample 6 (May 1996) showed one group of identical sequences: 6-A2 (6-4 and 6-5). Therefore, the 120 clones were represented by 100 different sequences in the clustering. Five defective clones detected in sample 5 were 5-2, 5-9, 5-11 and those in group 5-C2. Colour code: blue, sequences with the RT insertion T69SSS; green, sequences with the RT insertion T69SSG; red, sequences without RT insertions. Asterisks indicate clusters for which a bootstrap analysis (1000 replicas) gave a significance higher than 0.98 per unit. Minority sequences in samples 2 (2-17) and 3 (3-3, 3-5 and 3-8) have been underlined.

Figure 4

Dynamics of three-component viral quasispecies. The plot gives relative fractions of viral components (n_j(t)/κ) as a function of time for μ=10⁻³. The left y axis corresponds to n₁(t)/κ (green lines), and the right y axis corresponds to both n₂(t)/κ (blue lines) and n₃(t)/κ (pink lines). Notice the different scales on the left and right y axes. Values at the right y axis decrease by a power of 10 each time μ decreases by a power of 10 (simulations with μ=10⁻⁴ and μ=10⁻⁵, not shown). The parameters chosen for this simulation are as follows: at time t₀=0, n₁(t₀)=500, n₂(t₀)=500, k₁=1.0 and k₂=0.02; at time t₁=250 the system has reached its steady-state (corresponding to the values of k_j at t₀); at time t₂=350, the two-component system is exposed to a selective pressure (antiviral treatment) and k₁=0.01 and k₂=1.0; at time t₂+δ=370 the selective pressure is switched off and k₁=1.0 and k₂=0.2; at time t₃=620 the system has reached its steady-state (corresponding to the values of k_j at t₂+δ), and the value of n₂(t₃)/κ corresponds to the level of replicative memory of component 2 (which has a level of 1.02×10⁻³ in this example, with μ=10⁻³); at time t₄=720 the system is exposed to a second selective pressure (a different antiviral treatment) that forces a new component 3 to move from a reservoir to the bloodstream (the initial condition for component 3 was chosen as n₃(t₄)=10⁻⁴[n₁(t₄)+n₂(t₄)]), and k₁=0.03, k₂=0.3, k₃=1.0; at time t₄+δ=740 the selective pressure is switched off and k₁=1.0, k₂=0.15, and k₃=0.25; at time t₅=990 the system has reached its steady-state (corresponding to the values of k_j at t₄+δ), and the value of n₃(t₅)/κ reflects the contribution of non-replicative (or reservoir) components to the quasispecies memory (which has a level of 1.17×10⁻³ in this simulation, with μ=10⁻³).