Biomedical subjects

John C Guatelli

Publications and source records attributed to John C Guatelli.

7 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↗

PubMed2006

Modulation of cellular protein trafficking by human immunodeficiency virus type 1 Nef: role of the acidic residue in the ExxxLL motif.

The nef gene contributes to the replication of primate lentiviruses by altering the trafficking of cellular proteins involved in adaptive immunity (class I and II major histocompatibility complex [MHC]) and viral transmission (CD4 and DC-SIGN). A conserved acidic leucine-based sequence (E(160)xxxLL) within human immunodeficiency virus type 1 (HIV-1) Nef binds to the cellular adaptor protein (AP) complexes, which mediate protein sorting into endosomal vesicles. The leucine residues in this motif are required for the down-regulation of CD4 and for the up-regulation of DC-SIGN and the invariant chain of MHC class II, but the role of the acidic residue is unclear. Here, substitution of E160 with uncharged residues impaired the ability of Nef to up-regulate the expression of the invariant chain and DC-SIGN at the cell surface, whereas substitution with a basic residue was required for a similar effect on the down-regulation of CD4. All substitutions of E160 relieved the Nef-mediated block to transferrin uptake. E160 was required for the efficient interaction of Nef with AP-1 and AP-3 and for the stabilization of these complexes on endosomal membranes in living cells. Systematic mutation of the ExxxLL sequence together with correlation of binding and functional data leads to the hypotheses that AP-1 and AP-3 are major cofactors for the effect of Nef on the trafficking of transferrin, are less important but contribute to the modulation of the invariant chain and DC-SIGN, and are least critical for the modulation of CD4. The data suggest that the E160 residue plays a differential role in the modulation of leucine-dependent Nef-targets and support a model in which distinct AP complexes are used by Nef to modulate different cellular proteins.

Adaptor Protein Complex 1↗

PubMed2005

HIV-1 Nef stabilizes AP-1 on membranes without inducing ARF1-independent de novo attachment.

HIV-1 Nef affects the trafficking of numerous cellular proteins to optimize viral replication and evade host defenses. The adaptor protein (AP) complexes, which form part of the cytoplasmic coat of endosomal vesicles, are key cellular co-factors for Nef. Nef binds these complexes and alters their physiologic cycle of attachment and release from membranes. Specifically, while AP-1 normally becomes cytosolic when attachment events are blocked by inhibition of the GTPase cycle of ADP-ribosylation factor-1 (ARF1), the complex remains membrane-associated in Nef-expressing cells. To investigate the mechanism of this effect, we used a permeabilized cell system to detect the de novo attachment of exogenous AP-1 to endosomal membranes. Nef did not mediate de novo attachment independently of ARF1, despite its ability to maintain the association of AP-1 with endosomal membranes when the activity of ARF1 was blocked. We conclude that Nef stabilizes AP complexes on endosomal membranes after ARF1-dependent attachment. This stabilization may facilitate coat formation and stimulate the trafficking of multiple cellular proteins.

ADP-Ribosylation Factor 1↗

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↗

PubMed2003

Broadly increased sensitivity to cytotoxic T lymphocytes resulting from Nef epitope escape mutations.

Nef is an HIV-1 protein that is absent in most retroviruses, yet its reading frame is highly maintained despite frequent targeting by CD8(+) CTL in vivo. Because Nef is not necessarily required for viral replication, this consistent maintenance suggests that Nef plays an important role(s) and substantial fitness constraints prevent its loss in vivo. The ability of Nef to down-regulate cell surface MHC class I (MHC-I) molecules and render infected cells resistant to CTL in general is likely to be an important contributing function. We demonstrate that mutational escape of HIV-1 from Nef-specific CTL in vitro leads to progeny virions that are increased in their susceptibility to CTL of specificities for proteins other than Nef. The escape mutants contain multiple nef mutations that impair the ability of the virus to down-regulate MHC-I through disruption of its reading frame as well as epitope point mutations. Given the rarity of nef frameshifts in vivo, these data support the concept that the ability to down-regulate MHC-I could be a key constraint for preservation of Nef in vivo.

Amino Acid Sequence↗