It is well-established that human immunodeficiency virus (HIV) can be transmitted vertically from HIV-positive mothers to their infants with a frequency varying between 17 and 50%. We hypothesize that the placenta, as a target tissue for HIV infection, presents a formidable block to fetal infection. We propose that the HIV genome is interpreted differently in infected placental trophoblasts than in T lymphocytes or monocytes, resulting in novel patterns of RNA splicing of the small regulatory RNAs (tat/rev, nef) which control the timing of the viral life cycle. The possibility of a novel tat/rev mRNA is supported by preliminary data using reverse transcriptase-polymerase chain reaction amplification (RT/PCR) of first trimester placental RNA. A fundamental goal of this proposal is to define the molecular events involved in HIV infection of first trimester placenta, with an accent on the trophoblast in particular. These are the specific aims: 1) In situ RNA hybridization and p24 immunohistochemistry to identify which cell type(s) in placental tissue express the HIV genome.
Aim 2) Polymerase chain reaction (PCR)-mediated cloning and DNA sequencing of HIV transcripts isolated from first trimester and term placenta derived from infected mothers. Identification of spliced subgenomic RNAs expressed in placenta in order to determine which viral gene products may be expressed (gag, pol, env, tat, rev, vif, nef). Are the doubly-spliced mRNAs encoding the regulatory genes tat, rev, and nef being expressed? Aim 3) Laboratory infection of cytotrophoblast cultures derived from uninfected first trimester placentas; comparison of laboratory HIV strains and clinical isolates. Do certain strains show placental tropisms? Is the infection of trophoblast CD4-dependent? Is virus propagated on trophoblasts infectious? Does the temporal appearance of HIV RNA species differ from that described in one-step infection of T lymphocytes? Aim 4) Recovery of HIV proviral DNA from first trimester placenta via polymerase chain reaction (PCR). Can reconstructed PCR products be used to form an infectious DNA clone? It is possible that the temporal pattern of transcript appearance of the small RNAs encoding the regulatory proteins may result in an inefficient program of gene expression which either (1) delays the replication of virus in placenta, (2) results in the production of largely-defective particles, or (3) results in the production of virus with an altered host cell tropism unlikely to infect the typical HIV targets in the fetal blood.
