Molecular dynamics in HIV-1 infection of the brain

Abstract

The focus of the present thesis was to investigate the molecular dynamics in HIV-l infection of the brain. HIV-l infects the brain early during the infection and causes neurological syndromes and neuropathological modifications. To understand how the virus replicates and evolves within the brain may be important to unravel pathogenesis of HIV-l associated neurological diseases. We analyzed HIV-lV3 sequences from the biological material of infected patients classified in different clinical stages of infection, we characterized the biological properties of the virus recovered from the brain and blood compartments and measured the viral load in these two compartments-moreover, we studied the tropism of HIV-l isolates from the blood and brain for microglia cells and astrocytes.

We analysed published and unpublished HIV-l sequences derived from the brains and CSF of neurologically healthy subjects to determine the amino acid present at position 305 of the V3 loop, which has been linked to development of AIDS dementia. Contrary to what previously reported by other authors, a proline was not present at position 305 in any of the nondemented subjects, while histidine, which was reported to be a hallmark for sequences derived from demented patients, was present in 16 of the 25 sequences. Furthermore, we studied whether the presence of neurological manifestations was accompanied by appearance of viral genetic polymorphism. For this purpose, we directly sequenced the V3 loop of the env gene obtained from brain tissues of asymptomatics and patients with various neurological manifestations including HIV-l encephalitis. The analysis of the V3 loop revealed the presence of very homogeneous viral populations in the brain tissues with different histopathological findings.

To evaluate whether active viral replication in the brain compartment correlates with the development of neurological symptoms, we selected eight brain tissues obtained from infected patients deceased at different stages of the disease and quantified the HIV-l DNA copies by using a semiquantitative PCR analysis. We were able to detect HIV-l proviral DNA in 7 of the 8 samples independently of the stage of the disease: the highest levels of viral DNA however were found in the brain of subjects with histopathological signs of HIV-l encephalitis. We quantified HIV-l RNA levels in paired CSF and plasma samples of 30 infected patients with various neurological disorders or without. The method used in this study was a RT-PCR technique and the aim to determine whether active replication of the virus in the brain is correlated to the neurological symptoms. Plasma viral load did not differ significantly among patients with or without HIV-l encephalitis; conversely, higher levels of HIV-l RNA were detected in CSF samples collected from patients with HIV-l encephalitis compared to subjects without CNS involvement or that presented with opportunistic infections of the CNS. These results showed a striking correlation between productive viral infection of CNS and the development of HIV-l encephalitis.

In order to understand whether a different evolution of HIV-l in the brain occurs with respect to the blood, we compared the biological characteristics of paired HIV-l isolates from CSF and PBMCs obtained from infected individuals. Using the MT-2 cell assay we detected that more than 50% of PBMC isolates were SI (syncytia inducing). The presence of SI variants in blood correlated to the advanced stage of the disease and to the low CD4 cell count of the patients studied. Conversely, the majority (77%)of primary HIV-I isolates from CSF were typed as NSI variants. In our study we observed discordant phenotype in 46% of paired CSF and PBMCs isolates - in particular NSI (nonsyncytia inducing) variants were cultured from CSF whereas the corresponding virus in the blood was characterized as SI. Our data suggest that the virus populations in the two compartments are different and may evolve apart from each other. That this may be what happens during disease progression is confirmed by sequence analysis of HIV-l isolates from CSF and blood of patients treated with Zidovudine.

To further investigate the properties of HIV-I isolates obtained from blood and brain compartment we infected cells derived from the brain. We found that a majority of blood and CSF isolates (9/10) obtained from infected patients at different stage of the disease, infected and replicated in primary cultures of microglial cells derived from the temporal lobe tissue resected surgically from patients with intractable epilepsy. Primary human astrocytes could also be infected in vitro by approximately 1/3 of the isolates used in the study; in these cells, however, the infection was latent and could be reactivated with cytokines.