The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55 Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.

The Drosophila salivary gland is proving to be an excellent experimental system for understanding how cells commit to specific developmental programs and, once committed, how cells implement such decisions. Through genetic studies, the factors that determine where salivary glands will form, the number of cells committed to a salivary gland fate, and the distinction between the two major cell types (secretory cells and duct cells) have been discovered. Within the next few years, we will learn the molecular details of the interactions among the salivary gland regulators and salivary gland target genes. We will also learn how the early-expressed salivary gland genes coordinate their activities to mediate the morphogenetic movements required to form the salivary gland and the changes in cell physiology required for high secretory activity.

From a screen for genes expressed and required in the Drosophila salivary gland, we identified pasilla (ps), which encodes a set of proteins most similar to human Nova-1 and Nova-2. Nova-1 and Nova-2 are nuclear RNA-binding proteins normally expressed in the CNS where they directly regulate splicing. In patients suffering from paraneoplastic opsoclonus myoclonus ataxia (POMA), Nova-1 and Nova-2 proteins are present as auto-antigens. Consistent with a role in splicing, PS is localized to nuclear puncta. The salivary glands of ps mutants internalize normally and maintain epithelial polarity. However, the mutant salivary glands develop irregularities in overall morphology and have defects in apical secretion. The secretory defects in ps mutants provide a potential mechanism for the loss of motor function observed in POMA patients.