CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

Declining home range area predicts reduced late‐life survival in two wild ungulate populations

Demographic senescence is increasingly recognised as an important force shaping the dynamics of wild vertebrate populations. However, our understanding of the processes that underpin these declines in survival and fertility in old age remains limited. Evidence for age‐related changes in foraging behaviour and habitat use is emerging from wild vertebrate studies, but the extent to which these are driven by within‐individual changes, and the consequences for fitness, remain unclear. Using longitudinal census observations collected over four decades from two long‐term individual‐based studies of unmanaged ungulates, we demonstrate consistent within‐individual declines in home range area with age in adult females. In both systems, we found that within‐individual decreases in home range area were associated with increased risk of mortality the following year. Our results provide the first evidence from the wild that age‐related changes in space use are predictive of adult mortality.     

Are Archaea inherently less diverse than Bacteria in the same environments?

Like Bacteria, Archaea occur in a wide variety of environments, only some of which can be considered 'extreme'. We compare archaeal diversity, as represented by 173 16S rRNA gene libraries described in published reports, to bacterial diversity in 79 libraries from the same source environments. An objective assessment indicated that 114 archaeal libraries and 45 bacterial libraries were large enough to yield stable estimates of total phylotype richness. Archaeal libraries were seldom as large or diverse as bacterial libraries from the same environments. However, a relatively larger proportion of libraries were large enough to effectively capture rare as well as dominant phylotypes in archaeal communities. In contrast to bacterial libraries, the number of phylotypes did not correlate with library size; thus, 'larger' may not necessarily be 'better' for determining diversity in archaeal libraries. Differences in diversity suggest possible differences in ecological roles of Archaea and Bacteria; however, information is lacking on relative abundances and metabolic activities within the sampled communities, as well as the possible existence of microhabitats. The significance of phylogenetic diversity as opposed to functional diversity remains unclear, and should be a high priority for continuing research.     

pH-stability patterns of some strains of Newcastle disease and fowl-plague viruses

Summary The strains of fowl-plague virus being tested completely lost their infectivity to chick embryos when stored at p_H 4 for one hour or more while those of the virus of Newcastle disease were all infective to chick embryos when stored at this p_H-value for periods up to 7 days.     

Effect of some chemicals on the hemagglutination activities and infectivity to chick embryos of different strains of Newcastle disease and fowl-plague viruses

Summary Crystal violet in concentrations above 100 p.p.m. induced appreciable inhibition of the hemagglutination and infectivity powers of the various strains of Newcastle disease and fowl-plague viruses being tested.     

Effect of some chemicals on the hemagglutination activities and infectivity to chick embryos of different strains of Newcastle disease and fowl-plague viruses

Summary Ascorbic acid reduced the agglutination activities of the various strains of Newcastle disease and fowl-plague viruses used but was in-effective with respects to their infectivity powers.