Extracellularly truncated desmoglein 1 compromises desmosomes in MDCK cells.

The formation and stability of epithelial tissue involves cell adhesion and the connection of the intermediate filaments of contiguous cells, mediated by desmosomes. The cadherin family members Desmocollins (Dsc) and Desmogleins (Dsg) mediate desmosome extracellular adhesion. The main intracellular molecules identified linking Dscs and Dsgs with the intermediate filament network are Plakoglobin (PG), Plakophilins (PPs) and Desmoplakin (DP). Previous studies on desmosome-mediated adhesion have focused on the intracellular domains of Dsc and Dsg because of their capacity to interact with PG, PPs and DP. This study examines the role of the extracellular domain of Dsg1 upon desmosome stability in MDCK cells. Dsg1 was constructed containing an extracellular deletion (Dsg delta 1EC) and was expressed in MDCK cells. A high expressor Dsg delta 1EC/MDCK clone was obtained and analysed for its capacity to form desmosomes in cell monolayers and when growing under mechanical stress in three-dimensional collagen cultures. Phenotypic changes associated with the ectopic expression of Dsg1 delta EC in MDCK cells were: disturbance of the cytokeratin network, a change in the quality and number of desmosomes and impairment of the formation of cysts in suspension cultures. Interestingly, Dsg1 delta EC was not localized in desmosomes, but was still able to maintain its intracytoplasmic interaction with PG, suggesting that the disruptive effects were largely due to PG and/or PP sequestration.

The regulation of the expression, phosphorylation, and protein associations of pp125FAK during rat brain development.

We have studied both the expression and the interactions of focal adhesion kinase (FAK) during brain development. We have discovered that during different periods of development, FAK apparently has different properties. During the early stage of neurogenesis, FAK is phosphorylated, shows multiple isoforms, and interacts with the proto-oncogenes, src, fyn, and lyn. At this stage, FAK also interacts with both the N- and C-terminal SH2 domains of GAP, a negative regulator of the ras pathway. During later embryonic development, none of these protein interactions are apparent even though FAK is still predominantly phosphorylated. By adulthood FAK is largely unphosphorylated and migrates as a single protein species on SDS--PAGE. We discuss these results in terms of the dynamic cell movements that occur during embryonic brain development.

HIV infection of choriocarcinoma cell lines derived from human placenta: the role of membrane CD4 and Fc-Rs into HIV entry.

We have shown previously that trophoblast cells from human placenta can be infected with HIV-1 and a productive infection established. Recently, (1991, J. Virol. 65, 2102-2107) Zachar et al. and D. M. Phillips and X. Tan (1992, AIDS Res. Hum. Retroviruses 8, 1697-1705) have described in vitro infection of choriocarcinoma cell lines. Using choriocarcinoma cell lines (JAR, BeWo, and FD25, a trophoblast-derived cell line) we have infected these cells with several laboratory strains of virus and have shown that this can be prevented either by sCD4 or by antibodies to CD4. This provides strong evidence that the infection may be through CD4. In addition, we have found that infection of JAR and FD25 cells by HIV-1/Lai was enhanced in the presence of human antisera to HIV-1. This supports an additional role for immunoglobulin receptors (Fc-R) in the entry of virus into the cell. We report here evidence that CD4 and Fc-R on the cell surface play crucial roles in the entry of HIV into such placenta-derived cell lines.

A natural antisense RNA derived from the HIV-1 env gene encodes a protein which is recognized by circulating antibodies of HIV+ individuals.

A naturally occurring antisense RNA, transcribed in the opposite direction and complementary to the envelope transcript, was identified in various cell lines chronically infected with HIV-1. In T cells, the antisense transcript is constitutively expressed and enhanced by activation with phorbol myristate acetate. The open reading frame corresponding to the antisense transcript, when expressed in vitro, encodes a protein with an apparent molecular mass of 19 kDa. Antibodies against this protein have been detected in several sera of HIV+ individuals and not in any of the noninfected control sera. These results indicate, for the first time, that expression of an antisense open reading frame most likely accompanies the HIV infection cycle in humans.

Sequences in the rev-responsive element responsible for premature translational arrest in the human-immunodeficiency-virus-type-1 envelope.

Cell-free translation in the presence of pancreatic microsomal membranes of the full-length envelope transcript of the human immunodeficiency virus type 1 (HIV-1) yielded the expected extensively glycosylated and immunologically reactive gp160 envelope-protein precursor. In addition to this gp160, a shorter glycoprotein, which we designated gp120*, was produced due to a premature translation arrest. Utilizing kinetic experiments, pulse-chase analyses and various gp160 envelope RNA mutants, we demonstrated that the in-vitro-produced gp120* was not formed by cleavage of the gp160 precursor or by internal initiation of translation. A gp120 produced before gp160 synthesis was completed, and, independent of the gp160 proteolytic processing, has been shown to be produced and sequestered in the endoplasmic reticulum of HIV-1-infected cells [Willey, R. L., Klimkait, T., Frucht, D. M., Bonifacino, J. S. & Martin, M. A. (1991) Virology 184, 319-329]. The specific translational arrest shown to occur in vitro was found to be dependent on the Rev-responsive element, since deletion of this highly structured sequence abolished the production of gp120*. We found that the combination of two contiguous putative stem loops of the Rev-responsive element, located at nucleotides 7494-7522 and 7525-7550 of the HIV-1 Rev-responsive-element sequence, was responsible for the production of this truncated protein. To our knowledge, these stem-loop structures, distinct from that known to bind the Rev protein, represent the first example responsible for the production of alternative products by premature translational arrest in higher eukaryotes.

Transcriptional and post-transcriptional mechanisms are involved in the absence of CD4 surface expression in two HIV-1 chronically infected T cell lines.

In vivo infection of human T cell lymphocytes by HIV-1 is mediated by the specific binding of the HIV-1 envelope glycoprotein gp120 to the T cell CD4 receptor. One of the post-infection events observed in vivo is the progressive loss of CD4+ T cells. One possible mechanism is the production of infected T cells which are lacking in surface expression of the CD4 receptor protein. We have analysed this possibility utilizing the two HIV-1 chronically-infected CD4- cell lines, 8E5 and ACH-2, both of which are derived from a CD4+ parental strain (A3.01) after HIV-1 infection. In each cell (8E5 and ACH-2) the loss of CD4 surface expression was found to occur by different mechanisms. In ACH-2 cells, neither CD4 protein nor the 3 kb CD4 RNA transcript could be detected. However, treatment of ACH-2 cells with cycloheximide elicited production of the 3 kb transcript suggesting the possibility for a repressor protein(s) to act at the level of transcription and/or stability of the 3 kb mRNA. In contrast, in 8E5 cells the level of the 3 kb CD4 RNA was comparable with that found in the CD4+ A3.01 parental strain. Analysis of the 8E5 strain revealed the presence of a CD4- gp160 bimolecular protein complex sequestered internally in the rough endoplasmic reticulum (RER). Finally, the protein tyrosine kinase p56lck, normally associated with the cellular membrane, appeared to be linked to the RER and bound to the CD4- gp160 proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Internalization of HIV glycoprotein gp120 is associated with down-modulation of membrane CD4 and p56lck together with impairment of T cell activation.

We previously demonstrated that long term treatment of the Ag-specific CD4+ T cell clone P28D with soluble HIV envelope glycoprotein gp120 results in a marked impairment of CD3/TCR-mediated responses. In this report, to further understand these inhibitory effects, the binding properties and internalization of gp120 have been investigated, in parallel with functional studies, in long term incubations of P28D cells with gp120. Immunofluorescence studies show that surface-bound gp120 level is maximal within 1 h of incubation at 37 degrees C and then gradually decreases. This decrease is accompanied by a progressive down-modulation of membrane CD4 (30-35% loss over a 18-h incubation period) without concomitant alteration of the CD4 mRNA steady-state level. Similar experiments performed with 125I-labeled gp120 demonstrate that the glycoprotein is progressively internalized (up to 35% internalized material after 18 h) and that it accumulates inside the cells. Confocal microscopy studies show that internalized gp120 is concentrated in localized intracellular compartments. CD4 also accumulates in compartments with a similar localization and is stained with mAb OKT4 but not with mAb OKT4a. Concomitantly to internalization of gp120 and disappearance of membrane CD4, a correlated loss of the CD4-associated tyrosine kinase p56lck is evidenced. Interestingly, a progressive impairment of the P28D responses to specific Ag or to anti-CD3 mAb is also observed. Inhibitions of T cell proliferation increase with the degree of both CD4 and p56lck down-modulation. Removal of exogenous gp120 results in a rapid and spontaneous release of internalized gp120 into a degraded form. A progressive restoration of CD4 and p56lck levels is also noticed. In parallel, CD3/TCR-mediated responses of clone P28D are fully recovered. Altogether, our results suggest that HIV-1 glycoprotein gp120 is able to down-modulate membrane CD4 presumably by a cointernalization process and to further down-modulate the associated p56lck. This dual phenomenon is presumably involved in the direct immunosuppressive effect of gp120 on the CD3/TCR-mediated activation pathway.

Suboptimal and optimal activation signals modulate differently the expression of HIV-1 and cytokine genes.

Expression of HIV-1 and cytokine genes was investigated in chronically infected lymphocytic and promonocytic cell lines. As in normal human peripheral blood lymphocytes (PBL), a suboptimal activation signal with phorbol myristate acetate (PMA) did not trigger significant cytokine expression, whereas optimal activation signal (PMA + ionomycin) did. In contrast, a suboptimal activation was sufficient to up-regulate expression of HIV transcripts with kinetics similar to that observed in cells infected de novo by HIV. The level of HIV RNA in the promonocytic line was very low and markedly delayed when compared to the lymphocytic lines. We concluded that HIV induction required weaker activation signals than cytokine induction and that kinetics and level of HIV expression were not modified by induction of these cytokines. However, HIV expression appeared to alter the regulation of genes involved in proliferation and functional differentiation such as a decreased expression of IL-2 and IL-2R alpha and an increased expression of IFN gamma and TNF alpha mRNA.