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The human immunodeficiency virus type 1 (HIV-1) Vpr protein is an attractive target for antiretroviral drug development. The conservation both of the structure along virus evolution and the amino acid sequence in viral isolates from patients underlines the importance of Vpr for the establishment and progression of HIV-1 disease. While its contribution to virus replication in dividing and non-dividing cells and to the pathogenesis of HIV-1 in many different cell types, both extracellular and intracellular forms, have been extensively studied, its precise mechanism of action nevertheless remains enigmatic. The present review discusses how the apparently multifaceted interplay between Vpr and host cells may be due to the impairment of basic metabolic pathways. Vpr protein modifies host cell energy metabolism, oxidative status, and proteasome function, all of which are likely conditioned by the concentration and multimerization of the protein. The characterization of Vpr domains along with new laboratory tools for the assessment of their function has become increasingly relevant in recent years. With these advances, it is conceivable that drug discovery efforts involving Vpr-targeted antiretrovirals will experience substantial growth in the coming years., Competing Interests: The author declares no conflict of interest.

Viral protein U (Vpu) is a lentiviral viroporin encoded by human immunodeficiency virus type 1 (HIV-1) and some simian immunodeficiency virus (SIV) strains. This small protein of 81 amino acids contains a single transmembrane domain that allows for supramolecular organization via homoligomerization or interaction with other proteins. The topology and trafficking of Vpu through subcellular compartments result in pleiotropic effects in host cells. Notwithstanding the high variability of its amino acid sequence, the functionality of Vpu is well conserved in pandemic virus isolates. This review outlines our current knowledge on the interactions of Vpu with the host cell. The regulation of cellular physiology by Vpu and the validity of this viroporin as a therapeutic target are also discussed.

Human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that belongs to the viroporin family. Viroporins interact with cell membranes, triggering membrane permeabilization and promoting release of viral particles. In vitro electrophysiological methods have revealed changes in membrane ion currents when Vpu is present; however, in vivo the molecular mechanism of Vpu at the plasma membrane is still uncertain. We used the yeast Saccharomyces cerevisiae as a genetic model system to analyze how Vpu ion channel impacts cellular homeostasis. Inducible expression of Vpu impaired cell growth, suggesting that this viral protein is toxic to yeast cultures. This toxicity decreased with extracellular acidic pH. Also, Vpu toxicity diminished as the extracellular K(+) concentration was increased. However, expression of the Vpu protein suppresses the growth defect of K(+) uptake-deficient yeast (Δtrk1,2). The phenotype rescue of these highly hyperpolarized cells was almost total when they were grown in medium supplemented with high concentrations of KCl (100 mM) at pH 7.0 but was significantly reduced when the extracellular K(+) concentration or pH was decreased. These results indicate that Vpu has the ability to modify K(+) transport in both yeast strains. Here, we show also that Vpu confers tolerance to the aminoglycoside antibiotic hygromycin B in Δtrk1,2 yeast. Our results suggest that Vpu interferes with cell growth of wild-type yeast but improves proliferation of the hyperpolarized trk1,2 mutant by inducing plasma membrane depolarization. Furthermore, evaluation of the ion channel activity of the Vpu protein in Δtrk1,2 yeast could aid in the development of a high-throughput screening assay for molecules that target the retroviral protein.

We have examined the effect of Rev on the regulation of the expression of RRE containing mRNAs when they were synthesised in the nucleus or directly in the cytoplasm. In the nuclear expression system, Rev enhanced env mRNA transport by about 1.6-fold, while translation of this mRNA was increased more than a 100-fold. These findings indicate that the target of Rev activity is located mainly at the translational level. Synthesis of Env using a recombinant vaccinia virus system, which synthesised env mRNA directly in the cytoplasm, is also enhanced by Rev. Finally, RRE functioning was examined using a luciferase mRNA bearing this element. Rev stimulated the synthesis of Luciferase both when the luc mRNA was made in the nucleus or in cytoplasm. Our results indicate that the effect of Rev on env mRNA transport is low compared with the enhancement of translation of this mRNA.

The human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that forms oligomeric structures in membranes. Expression of vpu using Sindbis virus (SV) as a vector leads to permeabilization of plasma membrane to hydrophilic molecules and impaired maturation of wild type SV glycoproteins in BHK cells. The 6K protein is a membrane protein encoded in the SV genome that facilitates budding of virus particles and regulates transport of viral glycoproteins through the secretory pathway. Some of these functions were assayed with a SV mutant containing a partially deleted 6K gene. Transfection of BHK cells with pSVDelta6K vector rendered defective SVDelta6K virus, which had lower membrane permeabilization, impaired glycoprotein processing, and deficient virion budding. Replacement of 6K function by HIV-1 Vpu in SVDelta6K was tested by cloning the vpu gene under a duplicated late promoter (pSVDelta6KVpu). The presence of the vpu gene in the 6K-deleted virus enhances membrane permeability, modifies glycoprotein precursor processing, and facilitates infectious virus particle production. Restoration of infectivity of 6K-deleted SV by Vpu was evidenced by increased PFU production and cytopathic effect on infected cells. The modification of SVDelta6K glycoprotein maturation by Vpu was reflected in augmented processing of B precursor and impairment of PE2 cleavage. Taken together, our data support the notion that HIV-1 Vpu and SV 6K proteins share some analogous functions., (Copyright 2001 Academic Press.)

Infection of T lymphocytes by the human immunodeficiency virus causes drastic alterations in the intracellular cation content of the infected cells. The human immunodeficiency virus type 1 genome encodes several accessory proteins, including Vpu, an integral membrane protein that forms ion channels in planar lipid bilayers. The effect of Vpu on the permeability of the plasma membrane to several molecules has been analyzed. Expression of vpu in Escherichia coli cells increases membrane permeability to a number of molecules such as 2-nitrophenyl beta-D-galactopyranoside, uridine, the impermeable translation inhibitor hygromycin B, and lysozyme. In addition, transient expression of Vpu in eukaryotic COS cells enhances entry of charged molecules such as hygromycin B and neurobiotin into these cells. The effect of Vpu on cell membrane permeability resembles that reported for other membrane-active proteins from different animal viruses, including influenza M2, Semliki Forest virus 6K, and poliovirus 2B and 3A proteins.

Comparison of the amino acid sequences of human immunodeficiency virus (HIV) Nef protein and several RNA-binding proteins shows similarities in some regions of these proteins. Thus, poliovirus protein 2C, an RNA-binding protein, shares with Nef the sequence YXQQ...MDD...DXXD. In addition, both proteins contain an Arg-rich motif that, in the case of poliovirus 2C, is involved in RNA-binding activity. Moreover, the RNA-binding, anti-terminator N proteins of lambda, phi21 and P22 phages show sequence similarities with HIV Nef at the Arg-rich motif. To assess the significance of this motif, native and deletion variants of Nef protein were assayed for RNA-binding activity. The N-terminal 35 amino acids of HIV-1 Nef that comprise the Arg-rich motif are sufficient for RNA binding. Point mutations engineered at the Arg-rich motif of HIV-1 Nef revealed that basic amino acid residues are essential for RNA-binding activity. The Nef proteins from HIV-2 and SIV can also interact with RNA, while the same proteins with the N-terminal Arg-rich domain truncated fail to interact with RNA. These findings indicate that all three Nef proteins from HIV-1, HIV-2 and simian immunodeficiency virus belong to the RNA-binding family of proteins. The three proteins contain an Arg-rich region at the N-terminus which is necessary to interact with RNA.

The function of human immunodeficiency virus nef gene product has been much debated but the precise activity of this protein in the HIV replication cycle remains unknown. HIV-1 Nef was obtained as a fusion protein with maltose binding protein (MBF), purified by amylose column chromatography and separated from MBP by cleavage with factor Xa. Purified HIV-1 Nef protein, but not the fusion protein MBP-Nef, binds to RNA in vitro as tested by three different assays, radioactive or non-radioactive. North-western analysis, UV cross-linking or band-shift analysis. This activity was lost in a deletion mutant lacking 22 amino acids from the amino terminus of HIV-1 Nef, while a deletion of 44 residues from the carboxy terminus of the protein does not impair the RNA binding activity. Moreover, a single amino acid replacement, Arg to Gly at position 22 produces a Nef variant deficient in its ability to interact with RNA. Different Nef proteins from HIV-1, HIV-2 or SIV were fused to MBP and cleaved with factor Xa. The different Nef proteins were all endowed with RNA-binding capacity. Sequence similarities between several RNA binding proteins, including picornavirus 2C and different Nef proteins are observed. The function of Nef during the HIV replication cycle is discussed on the basis of the present findings.

The transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1) has been implicated in the cytopathology observed during HIV infection. The first amino acids located at the amino terminus are involved in membrane fusion and syncytium formation, while sequences located at the carboxy terminus have been predicted to interact with membranes and modify membrane permeability. The HIV-1 gp41 gene has been cloned and expressed in Escherichia coli cells by using pET vectors to analyze changes in membrane permeability produced by this protein. This system is well suited for expressing toxic genes in an inducible manner and for analyzing the function of proteins that modify membrane permeability. gp41 enhances the permeability of the bacterial membrane to hygromycin B despite the low level of expression of this protein. To localize the regions of gp41 responsible for these effects, a number of fragments spanning different portions of gp41 were inducibly expressed in E. coli. Two regions of gp41 were shown to increase membrane permeability: one located at the carboxy terminus, where two highly amphipathic helices have been predicted, and another one corresponding to the membrane-spanning domain. Expression of the central region of gp41 comprising this domain was highly lytic for E. coli cells and increased membrane permeability to a number of compounds. These findings are discussed in the light of HIV-induced cytopathology and gp41 structure.