Human Vgamma2Vdelta2 T cells contain cytoplasmic RANTES.

The adult human Vgamma2Vdelta2 T cell repertoire is a product of chronic selection in the periphery. Endogenous antigens drive the expansion of cells expressing the Vgamma2Vdelta2 TCR. Thus, we would expect the majority of circulating Vgamma2Vdelta2 T cells to be antigen experienced and to have memory phenotype, in contrast to the alpha/beta TCR+ subsets that include a substantial fraction of naive cells. We sought to characterize functional aspects of Vgamma2Vdelta2 T cells that might show whether circulating cells are memory or naive. For these studies, we focus on the expression of the CC chemokine regulated upon activation normal T cell expressed and secreted (RANTES). In naive alphabeta T cells, an initial stimulus triggers the onset of RANTES transcription followed later by protein expression. In memory CD8+ alphabeta T cells, RANTES mRNA is already present in unstimulated cells and protein expression is triggered immediately by TCR signaling; some cells may also contain RANTES protein in cytoplasmic stores. We show here that the vast majority of circulating human T cells contain RANTES protein in cytoplasmic stores and the chemokine is secreted rapidly after TCR signaling. Primary Vgamma2Vdelta2 T cell lines obtained after in vitro stimulation with phosphoantigens behaved similarly to circulating Vgamma2Vdelta2 T cells and contained both RANTES mRNA and protein, but only very low levels of mRNA or protein for macrophage inflammatory protein (MIP)-1alpha or MIP-1beta. The presence of stored RANTES shows that circulating Vgamma2Vdelta2 T cells are mostly memory phenotype and capable of rapid chemokine responses to phosphoantigen stimulation. Considering that one of 40 circulating CD3+ lymphocytes is Vgamma2Vdelta2+, they comprise the largest circulating memory population against a single antigen, and phosphoantigen stimulation will trigger a rapid activation with immediate release of RANTES.

Retroviral splicing suppressor requires three nonconsensus uridines in a 5' splice site-like sequence.

Rous sarcoma virus RNA contains a negative regulator of splicing (NRS) element that aids in maintenance of unspliced RNA. The NRS binds U1 snRNA at a sequence that deviates from the 5' splice site consensus by substitution of U's for A's at three positions: -2, +3, and +4. All three of these U's are important for NRS-mediated splicing suppression. Substitution of a single nonconsensus C or G at any of these sites diminished NRS activity, whereas substitution of a single A generated a preferred 5' splice site within the NRS.

Rous sarcoma virus DR posttranscriptional elements use a novel RNA export pathway.

Rous sarcoma virus (RSV), a simple retrovirus, needs to export unspliced viral RNA from the nucleus to the cytoplasm, circumventing the host cell restriction on cytoplasmic expression of intron-containing RNA. The cytoplasmic accumulation of full-length viral RNA is promoted by two cis-acting direct repeat (DR) elements that flank the src gene; at least one copy of the DR sequence is necessary for viral replication. We show here that the DR mediates export of a reporter construct from the nucleus, suggesting it is a constitutive transport element (CTE). In contrast, human immunodeficiency virus type 1 (HIV-1) and other complex retroviruses encode accessory proteins, Rev or Rex, which promote export of incompletely spliced viral transcripts. This RNA export pathway is CRM1 dependent and can be blocked by the cytotoxic agent leptomycin B. We show here that DR-mediated export is CRM1 independent, suggesting that RSV uses a different export pathway from that of HIV-1 and other complex retroviruses. The simian retroviruses have a CTE which interacts with the cellular Tap export protein. However, we were unable to detect binding of the RSV DR RNA to Tap, suggesting it may use a different export pathway from that of the simian retroviruses. These data suggest that the RSV DR element uses a novel nucleocytoplasmic export pathway.