Accelerated evolution of SIV env within the cerebral compartment in the setting of morphine-dependent rapid disease progression

Abstract

Human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) have been shown to compartmentalize within various tissues, including the brain. However, the evolution of viral quasispecies in the setting of drug abuse has not been characterized. The goal of this study was to examine viral evolution in the cerebral compartment of morphine-dependent and control macaques to determine its role in rapid disease progression. To address this issue, we analyzed the envelope (env) gene from proviral DNA in our SIV/SHIV macaque model of morphine dependence and AIDS. Analyses of proviral DNA revealed a direct correlation between total genetic changes and survival time. However, the rate of evolution during disease progression was higher in morphine-dependent and rapid-progressor macaques than was the rate of evolution in the control animals. This study provides additional insight into SIV envelope variation in the CNS of morphine-dependent macaques and genotypes that may have evolved in the brain and contributed to disease progression.

Figure 1

Pathological changes in morphine-dependent rapid progressor with neurological symptoms. Immunohistochemical analyses were performed on paraffin-embedded tissues from macaque 1/28Q as described in the Materials and Methods. Panels: A, accumulation of macrophages in the perivascular region; B, focal histiocytic nodules; C, multi-nucleated giant cells.

Figure 2

Boostrap Neighbor-Joining trees of env V3-V5 sequences. Envelope sequences were obtained from brain tissue of morphine-dependent rapid progressors (red circles), morphine-dependent normal progressors (green circles) and control macaques (blue diamonds) infected with SIV/17E-Fr, SHIV_KU-1B and SHIV_89.6P. The phylogenetic trees were generated using the MEGA 3.1 program and boostrap values were performed on 500 replicates and those >50 are indicated at the nodes. Nodes where no value is indicated were not supported at this level. The trees were rooted using the corresponding sequence from SIV/17E-Fr (black square).

Figure 3

Viral molecular diversity and divergence of brain-derived env V3-V5 sequences obtained from morphine-dependent and control macaques infected with SIV/17E-Fr, SHIV_KU-1B and SHIV_89.6P. The proviral env sequences were aligned and used to calculate diversity and divergence using MEGA 3.1. The mean percent diversity and divergence are plotted for each group. Error bars indicate mean of standard error.

Figure 4

Viral molecular diversity and divergence of brain-derived env V4 sequences obtained from morphine-dependent and control macaques infected with SIV/17E-Fr, SHIV_KU-1B and SHIV_89.6P. The proviral env sequences were aligned and used to calculate diversity and divergence using MEGA 3.1. The mean percent diversity and divergence are plotted for each group. Error bars indicate standard error of the mean.

Figure 5

Rate of substitution at synonymous and non-synonymous sites. SIV env V3-V5 nucleotide sequences isolated from brain tissue of morphine-dependent and control macaques were compared to the sequence of the starting molecular clone of SIV/17E-Fr. The numbers of nucleotide changes leading to synonymous (dS) and non-synonymous (dN) mutations were tallied using SNAP, www.hiv.lanl.gov. Error bars indicate standard deviation.

Figure 6

Mean frequency of amino acid mutations and net charge changes within the V4 loop from brain sequences of morphine-dependent and control macaques infected with SIV/17E-Fr, SHIV_KU-1B and SHIV_89.6P. Panel A, The mean frequency of amino acid mutations was calculated as the total number of mutations in monkey divided by the number of clones. Represented are the averages of mutations per group. Panel B, The net charge of the V4 loop was calculated by subtracting the number of negatively charged amino acids from the number of positively charged amino acids. Represented are the averages of the net charge per group. Error bars indicate standard deviation.

Figure 7

Mean frequency of amino acid mutations and net charge changes within the GGDPE motif from brain sequences of morphine-dependent and control macaques infected with SIV/17E-Fr, SHIV_KU-1B and SHIV_89.6P. Panel A, The mean frequency of amino acid mutations was calculated as the total number of mutations in monkey divided by the number of clones. Represented are the averages of mutations per group. Panel B, The net charge of the GGDPE motif was calculated by subtracting the number of negatively charged amino acids from the number of positively charged amino acids. Represented are the averages of the net charge per group. Error bars indicate standard deviation.