Martin Braddock

HIV expression strategies: Ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems

The pol gene of the human immunodeficiency virus (HIV-1) is expressed as a gag:pol fusion, arising from a ribosomal frameshift that brings the overlapping, out-of-phase gag and pol genes into translational phase. In this study, we show that HIV frameshifting is mediated by a very short sequence in the viral RNA. We demonstrate the importance of a homopolymeric run within this sequence and conclude that HIV frameshifting is not dependent on stem-loop structures downstream from the frameshift site. Our analysis also indicates that the sequence requirements are identical in mammalian and yeast systems.

HIV-1 TAR RNA-binding proteins control TAT activation of translation in Xenopus oocytes.

Human immunodeficiency virus (HIV‐1) gene expression is activated by the viral TAT protein that interacts with an RNA sequence, TAR, located at the 5′ end of all viral mRNAs. TAT functions primarily as a transcriptional activator in mammalian cells. However, in Xenopus oocytes TAT functions primarily as a translational activator. TAR is an RNA structure comprising a partially base‐paired stem, a tripyrimidine bulge in the upper stem, and an unpaired six‐nucleotide loop. In vitro, TAT binds directly to the bulge with no requirement for the loop. In vivo, however, mutations in the loop abolish TAT activation of transcription and translation, implying a requirement for TAR‐binding cellular factors. We now provide genetic evidence for the presence of two TAR‐specific cellular factors in Xenopus oocytes. These factors display independent and mutually exclusive interactions with either the loop or the bulge region of TAR. Furthermore, by using in vivo RNA competition assays we show that the cellular factors regulate the accessibility of the TAT binding site. The fact that Xenopus oocytes contain factors that specifically interact with a human viral RNA sequence might indicate that the TAT/TAR interaction is subverting a conserved pathway in the cell.— Braddock, M., Powell, R., Blanchard, A. D., Kingsman, A. J., and Kingsman, S. M. HIV‐1 TAR RNA binding proteins control TAT activation of translation in Xenopus oocytes. FASEB J. 7: 214‐222; 1993.

Application of the Firefly Luciferase Reporter Gene to Microinjection Experiments in Xenopus Oocytes

The human immunodeficiency virus (HIV-1) is the aetiological agent of the acquired immune-deficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983; Gallo et al., 1984). HIV-1 has the typical genetic organisation of retro viruses, with three major genes gag, pol and env. In addition, HIV-1 has additional short open reading frames that encode various regulatory proteins making it one of the most complex retroviruses that has been described (Varmus et al., 1984). The regulation of gene expression in HIV-1 has been shown to be critically dependent on the virally-encoded TAT protein (Dayton et al., 1986).

Quantitative Localisation of HIV Proteins in Mammalian Cells

The human immunodeficiency virus (HIV-1) has the typical genetic organisation of retroviruses, with three major genes, namely gag, pol and env . In addition, the virus encodes several regulatory proteins. The regulation of gene expression in HIV-1 is critically dependent on the virally encoded protein (Dayton et al., 1986; Fisher et al., 1986). The TAT protein is essential for viral replication. TAT is a positive feedback transactivator that increases the level of gene expression from the HIV-1 long terminal repeat (LTR) and, therefore, increases the rate of its own synthesis and the synthesis of all viral proteins. The exact mechanism of TAT action has been the subject of much controversy and to date is not fully understood. The TAT transactivator protein interacts with a cis-acting element called TAR which is located immediately downstream of the transcription start-site and is, therefore, present in the 5′ untranslated regions of all HIV mRNAs (Rosen et al., 1985).