The role of the gp41 membrane-proximal tyrosine motif in simian immunodeficiency virus pathogenesis / Samra E. Elser.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Elser, Samra E., author.

Contributor:

Hoxie, James A., degree supervisor.

Betts, Michael R., degree committee member.

Collman, Ronald G., degree committee member.

Lackner, Andrew A., degree committee member.

Marks, Michael S., degree committee member.

University of Pennsylvania. Department of Cell and Molecular Biology, degree granting institution.

Language:

English

Subjects (All):

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Local Subjects:

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Physical Description:

xii, 202 leaves : illustrations (chiefly color) ; 29 cm

Production:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2016.

Summary:

A tyrosine-dependent sorting signal in membrane-proximal region of the envelope protein cytoplasmic tail domain is conserved across all primate lentiviruses. It is known to be involved in mediating endocytosis of envelope from the cell surface and in directing sorting of envelope to the basolateral membrane in polarized cells, and its strict conservation implies that this motif is critical for viral function. Our laboratory has previously shown that ablation of two amino acids from this motif produces a virus, SIVmac239ΔGY, that replicates well in rhesus (RhM) and pig-tailed macaques (PTM), but fails to infect macrophages in vivo and spares CD4+ T cells in gastrointestinal lamina propria. Further, while 90% of SIVmac239ΔGY-infected PTM control the virus to undetectable levels, a small subset of PTM and all RhM progressed to AIDS without evidence of increased gut damage and microbial translocation, uncoupling this source of immune activation from development of AIDS. Additional mutations in the gp41 cytoplasmic tail in progressors were correlated with development of disease.

In this thesis, we identify in vitro properties that distinguish SIVmac239ΔGY from its parent and evaluate a number of putative compensatory mutations for their ability to restore a wildtype phenotype to SIVmac239ΔGY in these assays. We determined that SIVmac239ΔGY virions have reduced envelope levels on virions that are partially rescued by addition of one of two compensatory mutations, S727P or R722G. Growth of SIVmac239ΔGY was reduced in macaque PBMCs and modulated by mutations that arose in vivo during chronic SIVmac239ΔGY infection. We also identified differences in Env cellular localization and impaired endocytosis and basolateral sorting resulting from the ΔGY mutation. A novel ΔQTH mutation that arose in vivo produced functional tyrosine-dependent motifs that restored basolateral sorting and endocytic function to SIVmac239ΔGY, the latter in an AP-2 dependent manner. Compared to SIVmac239, SIVmac239ΔGY-containing mutants also exhibited differences in cell surface distribution of envelope that may contribute to reduction in envelope content of virions.

To test if putative compensatory mutations were sufficient to reconstitute an SIVmac239-like phenotype in animals, we then inoculated naïve PTM with SIVmac239ΔGY R722G or SIVmac239ΔGY R722G + ΔQTH (YFQL). We assessed key differentiators of SIVmac239 and SIVmac239ΔGY, including the ability to infect macrophages and to deplete gut CD4+ T cells. Although full pathogenicity and macrophage infection were not restored, 3/6 animals became progressors and developed high peak viral loads, with trends toward increased depletion of gut CD4+ T cells, and two died. Engineered mutations were stable throughout infection, and in progressing SIVmac239ΔGY + R722G infected animals, additional mutations arose that appear to reconstitute endocytosis signals. These findings serve to further highlight the exceptional importance of the membrane-proximal tyrosine motif to optimal function of SIV in vivo.

Notes:

Ph. D. University of Pennsylvania 2016.

Department: Cell and Molecular Biology.

Supervisor: James A. Hoxie.

Includes bibliographical references.