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Animal models of human immunodeficiency virus 1, such as feline immunodeficiency virus (FIV), provide the opportunities to dissect the mechanisms of early interactions of the virus with the central nervous system (CNS). The aims of the present study were to evaluate viral loads within CNS, cerebrospinal fluid (CSF), ocular fluid, and the plasma of cats in the first 23 weeks after intravenous inoculation with FIV GL8 . Proviral loads were also determined within peripheral blood mononuclear cells (PBMCs) and brain tissue. In this acute phase of infection, virus entered the brain in the majority of animals. Virus distribution was initially in a random fashion, with more diffuse brain involvement as infection progressed. Virus in the CSF was predictive of brain parenchymal infection. While the peak of virus production in blood coincided with proliferation within brain, more sustained production appeared to continue in brain tissue. In contrast, proviral loads in the brain decreased to undetectable levels in the presence of a strengthening PBMC load. A final observation in this study was that there was no direct correlation between viral loads in regions of brain or ocular tissue and the presence of histopathology.

Ineffective transgene expression in a sufficient amount of target cells is still a limitation in retroviral vector mediated gene therapy. Thus, we systematically evaluated four genetic modulators, (i) the woodchuck posttranscriptional regulatory element (WPRE), (ii) the mouse RNA transport element (RTE), (iii) the constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), and (iv) the 5' untranslated region of the human heat shock protein 70 (Hsp70 5'UTR), all of them involved in the posttranscriptional control of mRNA nucleo/cytoplasmatic transport, RNA stability, and translation efficiency, in an MLV-based retrovirus vector context. Insertion of the WPRE into the retrovirus vector resulted in enhancement of transgene expression (EGFP) both in transfected virus producing cells as well as in infected recipient cells irrespective of the location in the vector. The best effect was observed with two copies of the WPRE, 3' of the transgene and in the 3' untranslated region of the vector backbone. However, oligomerization of this element does not further increase transgene expression. Presence of the WPRE resulted also in an increase in virus production. Introduction of the CTE and/or RTE in the retroviral vector did not alter transgene expression and infectious particle production. Positive effects were observed only in vectors harboring the CTE and/or RTE in combination with the WPRE. The activity of the Hsp70 5'UTR as a translational enhancer was found to be negligible in the context of the retroviral vector. However, interference of the Hsp70 5'UTR strong secondary structure with the packaging sequence of the viral RNA was experimentally excluded as being the cause of this. These data suggest that only the WPRE is a suitable element for the improvement of transgene expression and oncoretroviral vector production.

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