Biomedical subjects

John R Day

Publications and source records attributed to John R Day.

4 recordsLinked to original sources

PubMed2006

The effect of the membrane-proximal tyrosine-based sorting signal of HIV-1 gp41 on viral infectivity depends on sequences within gp120.

The cytoplasmic domain of the HIV-1 Env glycoprotein (gp41) contains sequences that affect the trafficking of Env within the host cell. We previously showed that the membrane-proximal tyrosine-based adaptor protein (AP)-binding signal of gp41 (Y712XXL) is required for optimal viral infectivity and entry into target cells. Because these effects were not attributable to an effect on the incorporation of Env into virions, we hypothesized that they involved targeting of viral assembly to specific endosomal membranes that conferred greater fusogenicity. To further elaborate this hypothesis, we mutated the C-terminal leucine-based AP-binding signal of gp41 (LL855/856). In contrast to Env Y712, the leucine signal was dispensable for viral infectivity in both single cycle assays and during spreading infections within cultures of peripheral blood mononuclear cells (PBMCs). To test the hypothesis that these AP-binding motifs target Env to endosomes during viral morphogenesis, we compared the subcellular localization of wild-type Env to mutants of the Y712 and LL855/856 signals. The results failed to support the hypothesis that these signals target viral assembly to specific endosomal membranes. Strikingly, in the context of a C2-V3 region that confers macrophage-tropism, mutation of Y712 no longer markedly affected viral infectivity in either single cycle assays or during spreading infection within PBMCs, and it did not impair viral entry. These data indicate that the importance of the tyrosine-based sorting signal in gp41 for optimal viral infectivity depends on sequences in gp120. This observation is consistent with the hypothesis that the Y712 residue is part of the ectodomain of gp41 in virion-associated Env. We speculate that as part of the ectodomain, Y712 could affect specifically the conformation of the more positively charged CXCR4-tropic V3 loop in a manner that augments viral fusogenicity and infectivity.

Amino Acid Motifs↗

PubMed2006

A computer-based, image-analysis method to quantify HIV-1 infection in a single-cycle infectious center assay.

Rapid and precise quantitation of the infectivity of HIV is important for molecular virologic studies as well as for measuring the activities of antiviral drugs and neutralizing antibodies. In the present study, an indicator cell line and image-analysis software were used to quantify HIV infectivity. Cells of the P4R5 line, which express the receptors for HIV infection as well as beta-galactosidase under the control of the HIV-1 long terminal repeat, were infected either with CXCR4- or CCR5-using viruses, including primary isolates, then stained 2 days later with X-gal to turn infected cells blue. Digital images of monolayers of the infected cells were captured using a high resolution CCD video camera and a macro video zoom lens. A software program was developed to process the images and to count the blue-stained foci of infection. The assay was applied to assess the infectivity of site-directed viral mutants, and to measure the activity of antiviral drugs and neutralizing antibody. The results indicate that the described method allows for the rapid quantitation of infected cells over a wide range of viral inocula with reproducibility, accuracy and relatively low cost.

Anti-HIV Agents↗

PubMed2005

Leucine-specific, functional interactions between human immunodeficiency virus type 1 Nef and adaptor protein complexes.

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the mu subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the sigma1 plus gamma subunits of AP-1 or the sigma3 plus delta subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the mu subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The mu and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.

Adaptor Protein Complex mu Subunits↗

PubMed2004

The membrane-proximal tyrosine-based sorting signal of human immunodeficiency virus type 1 gp41 is required for optimal viral infectivity.

The membrane-proximal tyrosine-based sorting motif in the cytoplasmic domain of the human immunodeficiency virus type 1 Env glycoprotein is important for endocytosis from the plasma membrane, basolateral targeting of viral budding in polarized epithelial cells, and polarized budding from a localized region of the lymphocyte plasma membrane. To study the role of the Env sorting motif (Y712XXL) in infectivity, the incorporation of Env into virions, and viral entry, we disrupted the motif with a tyrosine-to-alanine substitution. To investigate the relationship between the Env sorting motif and the enhancement of infectivity by Nef, the EnvY712A substitution was made in both Nef-positive and Nef-negative backgrounds. In spreading infections, including those using primary lymphocytes, the growth of the Y712A mutant was as impaired as Nef-negative virus, and the EnvY712A/Delta-Nef combination mutant was almost completely defective. In single-round infections using CD4-positive HeLa cells, the EnvY712A mutation impaired infectivity, and Nef retained the ability to enhance the infectivity in the context of EnvY712A. EnvY712 and Nef were required for the optimal infectivity of virions produced from either HEK293T or MT4 cells, but these sequences were required for the optimal incorporation of Env only when virions were produced from MT4 cells. Despite the wild-type levels of Env in viruses produced from 293T cells, the entry of the EnvY712A and Delta-Nef mutants into target cells was impaired. We conclude that the membrane-proximal tyrosine-based sorting motif of gp41 Env is, like Nef, important for optimal viral infectivity and, in the case of MT4 T cells, virion incorporation of Env. Nef does not require the Y712XXL motif to enhance viral infectivity. The finding that EnvY712 and Nef each affect the efficiency of viral entry independently of the Env content of virions suggests that both viral proteins are involved in trafficking events that influence morphogenesis to produce maximally fusogenic virus.

Amino Acid Motifs↗