Molecular dynamics in HIV-1 infection of the brain

Abstract: The focus of the present thesis was to investigate the molecular dynamics in HIV-linfection of the brain. HIV-l infects the brain early during the infection and causesneurological syndromes and neuropathological modifications. To understand how thevirus replicates and evolves within the brain may be important to unravel pathogenesisof HIV-l associated neurological diseases. We analyzed HIV-lV3 sequences from thebiological material of infected patients classified in different clinical stagesof infection, we characterized the biological properties of the virus recovered fromthe 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 microgliacells and astrocytes. We analysed published and unpublished HIV-l sequences derived from the brainsand CSF of neurologically healthy subjects to deterrnine the amino acid present atposition 305 of the V3 loop, which has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 presentat position 305 in any of the non demented subjects, while histidine, which was reportedto be a hallmark for sequences derived from demented patients, was present in 16of the 25 sequences. Furthermore, we studied whether the presence of neurologicalmanifestations was accompanied by appearance of viral genetic polymorphism. For thispurpose, we directy sequenced the V3 loop of the env gene obtained from brain tissuesof asymptomatics and patients with various neurological manifestations includingHIV-l encephalitis. The analysis of the V3 loop revealed the presence of very homogeneousviral populations in the brain tissues with different histopathological findings. To evaluate whether active viral replication in the brain compartment correlateswith the development of neurological symptoms, we selected eight brain tissues obtainedfrom infected patients deceaded at different stages of the disease and quantifiedthe HIV-l DNA copies by using a semiquantitative PCR analysis. We were able to detectHIV-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 withhistopathological signs of HIV-l encephalitis. We quantified HIV-l RNA levels inpaired CSF and plasma samples of 30 infected patients with various neurological disordersor without . The method used in this study was a RT-PCR tecnique and the aim to deterrninewhether active replication of the virus in the brain is correlated to the neurologicalsymptoms. Plasma viral load did not differ significantly among patients with or withoutHIV-l encephalitis; conversely, higher levels of HIV-l RNA were detected in CSF samplescollected from patients with HIV-l encephalitis compared to subjects without CNSinvolvement or that presented with opportunistic infections of the CNS. These resultsshowed a striking correlation between productive viral infection of CNS and the developmentof HIV-l encephalitis. In order to understand whether a different evolution of HIV-l in the brain occurswith respect to the blood, we compared the biological characteristics of paired HIV-lisolates from CSF and PBMCs obtained from infected individuals.Using the MT-2 cellassay 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 diseaseand 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 observeddiscordant phenotype in 46% of paired CSF and PBMCs isolates- in particular NSI (nonsyncytia inducing) variants were cultured from CSF whereas the corrisponding virusin the blood was characterized as SI. Our data suggest that the virus populationsin the two compartments are different and may evolve apart from each other. Thatthis may be what happens during disease progression is confirmed by sequence analysisof HIV-l isolates from CSF and blood of patients treated with Zidovudine. To further invetigate the properties of HIV-I isolates obtained from blood andbrain compartment we infected cells derived from the brain. We found that a majorityof blood and CSF isolates (9/10) obtained from infected patients at different stageof the disease, infected and replicated in primary cultures of microglial cells derivedfrom the temporal lobe tissue resected surgically from patients with intractableepilepsy. Primary human astrocytes could also be infected in vitro by approximately1/3 of the isolates used in the study; in these cells, however, the infection waslatent and could be reactivated with cytokines. ISBN 91-85910-65-1