The general transcription factor P-TEFb stimulates RNA polymerase II elongation and cotranscriptional processing of pre-mRNA. Contributing to a functional equilibrium important for growth control, a reservoir of P-TEFb is maintained in an inactive snRNP where 7SK snRNA is a central scaffold. Here, we identify PIP7S as a La-related protein stably associated with and required for 7SK snRNP integrity. PIP7S binds and stabilizes nearly all the nuclear 7SK via 3' -UUU-OH, leading to the sequestration and inactivation of P-TEFb. This function requires its La domain and intact C terminus. The latter is frequently deleted in human tumors due to microsatellite instability-associated mutations. Consistent with the tumor suppressor role of a Drosophila homolog of PIP7S, loss of PIP7S function shifts the P-TEFb equilibrium toward the active state, disrupts epithelial differentiation, and causes P-TEFb-dependent malignant transformation. Through PIP7S modulation of P-TEFb, our data thus link a general elongation factor to growth control and tumorigenesis.

In vitro-assembled core-like particles produced from alphavirus capsid protein and nucleic acid were studied by cryoelectron microscopy. These particles were found to have a diameter of 420 Å with 240 copies of the capsid protein arranged in a T‫4؍‬ icosahedral surface lattice, similar to the nucleocapsid core in mature virions. However, when the particles were subjected to gentle purification procedures, they were damaged, preventing generation of reliable structural information. Similarly, purified nucleocapsid cores isolated from virus-infected cells or from mature virus particles were also of poor quality. This suggested that in the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.

No abstract

The assembly of the alphavirus nucleocapsid core has been investigated using an in vitro assembly system. The C-terminal two-thirds of capsid protein (CP), residues 81 to 264 in Sindbis virus (SINV), have been previously shown to have all the RNA-CP and CP-CP contacts required for core assembly in vitro. Helix I, which is located in the N-terminal dispensable region of the CP, has been proposed to stabilize the core by forming a coiled coil in the CP dimer formed by the interaction of residues 81 to 264. We examined the ability of heterologous alphavirus CPs to dimerize and form phenotypically mixed core-like particles (CLPs) using an in vitro assembly system. The CPs of SINV and Ross River virus (RRV) do not form phenotypically mixed CLPs, but SINV and Western equine encephalitis virus CPs do form mixed cores. In addition, CP dimers do not form between SINV and RRV in these assembly reactions. In contrast, an N-terminal truncated SINV CP (residues 81 to 264) forms phenotypically mixed CLPs when it is assembled with full-length heterologous CPs, suggesting that the region that controls the mixing is present in the N-terminal 80 residues. Furthermore, this result suggests that the dimeric interaction, which was absent between SINV and RRV CPs, can be restored by the removal of the N-terminal 80 residues of the SINV CP. We mapped the determinant that is responsible for phenotypic mixing onto helix I by using domain swapping experiments. Thus, discrimination of the CP partner in alphavirus core assembly appears to be dependent on helix I sequence compatibility. These results suggest that helix I provides one of the important interactions during nucleocapsid core formation and may play a regulatory role during the early steps of the assembly process.