The objectives of this Concerted Action were:
- to obtain an insight into the basic molecular mechanisms which are involved in the attachment to receptor and fusion of HIV with the cell membrane leading to release of the viral genome into the cell;
- to obtain an insight into the basic molecular mechanisms involved in HIV assembly and budding leading to the production and release of new virus particles;
- to define strategies, based on the knowledge gained from investigation of the above processes, that are aimed at interfering with the infection of cells by HIV or with the budding of new virus particles from infected cells, and for specific targeting of cytotoxic drugs to HIV infected cells;
- to supply the participants of the program with methodologies and materials that are specifically required for the project, including synthetic peptides, poly- and monoclonal antibodies, fluorescently or radioactively labeled HIV, HIV proteins and recombinant CD4. In this respect, a centralized facility for the production, purification and characterization of peptides and antibodies has been established.
Themechanisms underlying human immunodeficiency virus (HIV) neuropathogenicity in the brain have been studied using chronically infected glial cell lines as an in vitro model system for HIV infection of the central nervous system. These cell lines exhibit restricted virus production although detailed analysis of a representative glial cell line confirmed expression of all major structural proteins, albeit at low levels, and of Vif, Tat, Rev and Nef. Expression of Nef exceeded intracellular p24 antigen levels about 10-fold. In order to evaluate a possible role of Nef in the development of HIV-1 associated neurological disorders, Nef was compared with known neuroactive proteins. It was found that HIV Nef shares sequence and structural features with scorpion peptides known to interact with potassium channels. Sequence similarity encompasses 2 distinct regions of scorpion peptides. Based on crystallography data, both regions in scorpion peptides cooperate in forming a common domain stabilized by ion pairs between charged amino acid residues. Recombinant Nef protein as well as a synthetic part of a scorpion channel active peptide reversibly increased the total potassium current of chick dorsal root ganglions in patch clamp experiments without killing the cells. Moreover, as found by other groups, sequence similarities to these scorpion toxins are present in other proteins of neuropathogenic viruses (HIV-1 gp 120, HIV-1 gp41 and VP1 protein of poliovirus) though at present experimental evidence for functional similarity is only available for HIV-1 Nef. These results make it tempting to speculate that regions similar to scorpion peptides, especially in HIV Nef, may affect neuronal cell function in a similar way.
The interaction of human immunodeficiency virus (HIV) HIV-1 gp 120 with the major receptor CD4 has been studied in detail leading to a detailed description of the conformational changes in gp 120 induced after attachment to CD4 and leading to the liberation of the fusion end of gp41. It could be shown that the interaction of HIV-2 and simian immunodeficiency virus with CD4 is following somewhat different pathways as with HIV-1. The necessity for secondary receptors and the possibility of HIV-1 to use other receptors for entry other than the CD4 receptor has been elaborated. A fine analysis of the processes and conditions involved in the insertion of the fusion peptides of lentiviruses (HIV-1, HIV-2, SIV) and some oncogenic retroviruses like bovine leukaemia virus (BLV) has been performed. The fusion peptides are interchangeable and are inserted into the membrane in alpha-helical conformation and in an oblique orientation. Furthermore, it could be shown that the process in virus-cell membrane fusion is different from that involved in cell-cell fusion of HIV infected cells. The HIV assembly process is primarily initiated by an insertion of envelope glycoproteins into the cell membrane and by induction of a budding process solely by the HIV core protein. This is basically different from the budding process of other enveloped viruses like Semliki Forest Virus (SFV) where a close interaction of the core proteins and the envelope proteins is needed to perform budding. The membrane associated accessory protein nef of HIV-1, HIV-2 and SIV has been found to be structurally similar to peptides (neurotoxins) which modulate ion channel functions. The HIV-1 nef protein could be shown to change the potassium ion flux in neuronal cells. The myristoylation dependent budding process has been inhibited by myristyl transferase inhibitors. Amphipathic alpha-helical peptides with a similar structure to the transmembrane region and the fusion peptide of gp41 have been found to exert an inhibitory effect on HIV-1 replication.
The interaction of human immunodeficiency viruses with cellular membranes during their entry into the target cell, in the process of virus assembly and budding as well as in the induction of cell fusion is the general interest of this collaborative research effort.