Determinants of HIV-1 involved in galactosylceramide/3\u27-sulfo-galactosylceramide mediated entry into the SK-N-MC neuroblastoma cell line

Although the CD4 molecule was identified as the primary receptor for the human immunodeficiency virus type 1, HIV-1, several other molecules have been demonstrated to serve as cellular receptors in vitro. Two molecules that may mediate viral entry are the glycolipids galactosylceramide (GalCer) and 3$\prime$-sulfo galactosylceramide (GalS). To further characterize this CD4-independent entry mechanism, I examined the early events of viral replication in the SK-N-MC cell line, a neuroblastoma cell line that expresses GalCer/GalS on the plasma membrane. To study events in the viral life cycle that are specific for GalCer/GalS-dependent entry, I prepared a SK-N-MC cell line expressing the CD4 molecule on the cell surface. Using both primary HIV-1 isolates and a panel of envelope chimeric viruses, I studied events in the viral replication cycle that occur in GalCer/GalS mediated infection. Viral nucleic acids, used as an indication of viral infection, were amplified from SK-N-MC cells exposed to some, but not all, primary HIV-1 isolates, laboratory adapted viruses, and molecularly cloned viruses. Restriction to viral infection in the SK-N-MC cells was generally overcome in the SK-N-MC/CD4 cell line suggesting the primary, though not sole, determinants to infection were early events in the HIV-1 life cycle including virus binding and entry. Using chimeras between a virus isolate with a positive infection phenotype, HIV-1$\sb{\rm HxB2}$ in the SK-N-MC cells, and a virus isolate with a negative infection phenotype, HIV-1$\sb{89.6}$ in the SK-N-MC cells, a 193 amino acid fragment from the envelope region of HIV-1$\sb{\rm HxB2}$ containing the V3, V4, and V5 regions was found to be sufficient to confer a positive infection phenotype in the HIV-1$\sb{89.6}$ background. These studies suggested that a stable GalCer/GalS-envelope interaction was dependent upon the conformation of the viral envelope protein in the context of the viral membrane. Binding of recombinant monomeric envelope proteins to GalCer/GalS did not directly correlate with viral entry in SK-N-MC cells reflecting the significance of envelope conformation in the GalCer/GalS entry pathway