Viral protein U (Vpu) is a protein encoded by human immunodeficiency virus type 1 (HIV-1) that promotes the degradation of the virus receptor, CD4, and enhances the release of virus particles from cells. We isolated a cDNA that encodes a novel cellular protein that interacts with Vpu in vitro, in vivo, and in yeast cells. This Vpu-binding protein (UBP) has a molecular mass of 41 kDa and is expressed ubiquitously in human tissues at the RNA level. UBP is a novel member of the tetratricopeptide repeat (TPR) protein family containing four copies of the 34-amino-acid TPR motif. Other proteins that contain TPR motifs include members of the immunophilin superfamily, organelle-targeting proteins, and a protein phosphatase. UBP also interacts directly with HIV-1 Gag protein, the principal structural component of the viral capsid. However, when Vpu and Gag are coexpressed, stable interaction between UBP and Gag is diminished. Furthermore, overexpression of UBP in virus-producing cells resulted in a significant reduction in HIV-1 virion release. Taken together, these data indicate that UBP plays a role in Vpu-mediated enhancement of particle release.

Human immunodeficiency virus type 1 Vpu has been shown to facilitate virus release from HeLa cells. We demonstrated that Vpu expression is not required for efficient virus release from Cos 1 and CV-1 cells. A yeast GAL4 transcriptional activation system was used to screen for cellular proteins that may interact with Vpu. One such protein was identified which we provisionally designate "Vpu interactive protein" or VIP.

Viral protein U (Vpu) is a protein encoded by human immunodeficiency virus type 1 (HIV-1) that promotes the degradation of the virus receptor, CD4, and enhances the release of virus particles from cells. We isolated a cDNA that encodes a novel cellular protein that interacts with Vpu in vitro, in vivo, and in yeast cells. This Vpu-binding protein (UBP) has a molecular mass of 41 kDa and is expressed ubiquitously in human tissues at the RNA level. UBP is a novel member of the tetratricopeptide repeat (TPR) protein family containing four copies of the 34-amino-acid TPR motif. Other proteins that contain TPR motifs include members of the immunophilin superfamily, organelle-targeting proteins, and a protein phosphatase. UBP also interacts directly with HIV-1 Gag protein, the principal structural component of the viral capsid. However, when Vpu and Gag are coexpressed, stable interaction between UBP and Gag is diminished. Furthermore, overexpression of UBP in virus-producing cells resulted in a significant reduction in HIV-1 virion release. Taken together, these data indicate that UBP plays a role in Vpu-mediated enhancement of particle release.

MATERIALS AND METHODSA procedwe has been developed using Percoll density gradients for the isolation and purification of nuclei from germinated conidia of wild-type Neurospora crassa St. Lawrence strain 74A. Crude nuclei were purified isopycnically in gradients of Percoll, which is silica coated with polyvinylpyrrolidone. A DNA:RNA:protein ratio of 1:3.5:6.5 was found in purified nuclei. Cytoplasmic contamination was found to be neglgible in the nuclear preparations, as determined by electron microscopy and by folowing a radioactively-labeled ribosome tag during the isolation procedure. A small amount of endogenous ribonuclease activity was detected in the crude nuclear preparations, but not in suspensions of nuclei purified In the PercoU gradients. Ribosomal RNA was extracted from the nuclei in good yields, and electrophoretic analysis indicated the presence of precursor rRNA molecules, as well as the mature 17S and 25S rRNA species. Using the Percoll gradient system, the buoyant density of purified Neurospora nuclei was detennined to be 1.08 grams per milliliter based on refractive index measurements.The availability of purified nuclei greatly facilitates such studies as the synthesis and processing of precursor ribosomal RNA (prerRNA) and of precursor nucleoprotein particles. Although few problems are encountered with nuclei extraction in animal systems, the thick cell wall and prevalent endogenous nucleases in plants and fungi (3, 5), which are released upon cell disruption, make it a much more difficult task in these systems. In the fungal system Neurospora crassa, there has been only one report of the successful isolation of nuclei (3). This paper describes a procedure for the isolation and purification of Neurospora nuclei which uses Percoll as the gradient medium. Using this method, we have been able to determine accurately the buoyant density of the nuclei, since Percoll consists of colloidal silica particles coated with polyvinylpyrrolidone, to which the nuclear membrane is imper- [v/v] glycerol, 5 mM MgCl2, 10 mm CaCl2, 1% [v/v] Triton X-100, adjusted to pH 7.5). The resultant homogeneous slurry was poured into a French pressure cell (precooled at -700C overnight), and placed at -700C for 45 min. A maximum pressure of 20,000 p.s.i. was applied to disrupt the cells. Three volumes of ice-cold buffer A were added to the frozen eluant, and the mixture was vortexed. The solution was homogenized in a sterilized Omnimixer bucket at a setting of 55 for 20 min at 40C.To remove the bulk cellular debris, the homogenate was centrifuged at 2,400g for 10 min at 4°C in a Dupont-Sorvall RC5 centrifuge, SS34 rotor. A 10-cm 14-gauge cannula was used to remove the supernatant liquid. The pellets were resuspended in the same volume of buffer A as removed, and were centrifuged above. Two repetitions ofthese steps generated three supernatants. The pooled supernatants were centrifuged for 50 min at 9,000g and the crude nuclear pellets were obtained after careful removal of the supernatant liquid. The crude nuc...

Nuclear density determination and the purification of wild type Neurospora nuclei Nuclear density determination and the purification of wild type Neurospora nuclei using Percoll gradients. using Percoll gradients.