RESTRICTED HIV-1 INFECTION OF HUMAN ASTROCYTES - POTENTIAL ROLE OF NEF IN THE REGULATION OF VIRUS-REPLICATION

A small percentage of astrocytes are consistently infected in vivo by HIV-1 and may contribute to neuropathogenesis despite a non-productive infection. Overexpression of the nef gene pro duct has been associate d with their infection both in vivo and in vitro. We examined the role of the nef gene during HIV replication in astrocytes (U251MG cells) f ollowing transfection with pNL4-3 proviral plasmid or isogenic strains containing a deletion or point mutation in the nef gene (pNL4-3 Delta Nef; pNL4-3-nef-stop). We were able to initiate virus replication whi ch peaked at 5 days post-transfection and became nonproductive after 2 1 days. Nef protein expression by wild type pNL4-3 was observed at low levels compared to control HeLa cells at peak virus replication. At l ater time points after development of a non-productive infection, vira l antigen and Nef protein was not detectable however virus was readily recovered by co-culture with CD4+T-cells. Interestingly, virus produc tion was significantly enhanced by a 222 base pair deletion in the nef reading frame. This was not observed with a frame shifting point muta tion in nef, indicating a suppressive effect of nef on virus productio n in astrocytes. The enhanced virus production from nef-deleted pNL4-3 in U251MG cells was not reversed by coexpression of Nef from a second Nef-expressing plasmid, and in fact Nef expression in trans had a fur ther positive effect on virus production. This suggested opposing effe cts of the Nef protein and elements contained within the nef sequence on virus production in astrocytes. Despite the low expression of Nef b y U251MG astrocytes, relatively high amounts of multiply spliced 2 kb mRNA were present compared to HeLa cells. These data demonstrate that an acute low-level infection of astrocytes rapidly becomes a non-produ ctive infection and this process is assisted by sequences in nef. The low level Nef protein expression, despite high levels of mRNA, suggest s a block in translation of multiply spliced HIV mRNA in astrocytes, o r a translational control mechanism not yet characterised.