Analysis of HIV Co-receptor CCR5 gene mutations obtained from an African-American Family

In order to be infected with HIV, the virus must bind to two cell receptors, the CD4 and one of two secondary receptors, either CXCR4 or CCR5, which allow viral RNA to enter the host cell.  Mutations have been documented that prevent viral entry into the host cell, most notably the CCR5 Δ32, which is found in people of European descent.  CCR5 Δ32 prevents viral entry by truncating the CCR5 gene.  Once truncated, the protein is no longer expressed on the cell surface.  A CCR5 gene of an African-American family is currently being studied.  It is known that this family does not have the CCR5 Δ32 mutation, however, the family has been found to express multiple, novel, unique mutations.  A mother and four out of five of her children have tested positive for HIV.  The virus was found to be genetically similar, which is indicative of vertical transmission.  The mother had previously been married to a now deceased IV drug user, who is believed to be the source of infection.  The mother, as well as the first, third, fourth, and fifth child were infected with HIV.  The mother and first child are long term AIDS non-progressors, showing no symptoms for over 20 years.  The second child was exposed to but not infected with HIV.  This child has a point mutation in the cytoplasmic domain of CCR5. This Lys to Arg missense mutation will be characterized by expressing it in a monocyte cell line.  The gene encoding the mutation was cloned into the expression vector pcDNA. This study will employ U-937 cells (which possess CCR5 receptors but do not express them) and DNA expression vectors of various CCR5 mutations will be transfected into the cells.  Some cells will then be exposed to retinoic acid, which has been used to promote expression of endogenous CCR5.  The transfected U-937 cells will then be tested for CCR5 expression and HIV infectivity.