Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex.

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag-containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo--namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.

Inhibitors of STAT3, ß-catenin, and IGF-1R sensitize mouse PIK3CA-mutant breast cancer to PI3K inhibitors.

Although mutations in the phosphoinositide 3-kinase catalytic subunit (PIK3CA) are common in breast cancer, PI3K inhibitors alone have shown modest efficacy. We sought to identify additional pathways altered in PIK3CA-mutant tumors that might be targeted in combination with PI3K inhibitors. We generated two transgenic mouse models expressing the human PIK3CA-H1047R- and the -E545K hotspot-mutant genes in the mammary gland and evaluated their effects on development and tumor formation. Molecular analysis identified pathways altered in these mutant tumors, which were also targeted in multiple cell lines derived from the PIK3CA tumors. Finally, public databases were analyzed to determine whether novel pathways identified in the mouse tumors were altered in human tumors harboring mutant PIK3CA. Mutant mice showed increased branching and delayed involution of the mammary gland compared to parental FVB/N mice. Mammary tumors arose in 30% of the MMTV-PIK3CA-H1047R and in 13% of -E545K mice. Compared to MMTV-Her-2 transgenic mouse mammary tumors, H1047R tumors showed increased upregulation of Wnt/ß-catenin/Axin2, hepatocyte growth factor (Hgf)/Stat3, insulin-like growth factor 2 (Igf-2), and Igf-1R pathways. Inhibitors of STAT3, ß-catenin, and IGF-1R sensitized H1047R-derived mouse tumor cells and PIK3CA-H1047R overexpressing human HS578T breast cancer cells to the cytotoxic effects of PI3K inhibitors. Analysis of The Cancer Genome Atlas database showed that, unlike primary PIK3CA-wild-type and HER-2+ breast carcinomas, PIK3CA-mutant tumors display increased expression of AXIN2, HGF, STAT3, IGF-1, and IGF-2 mRNA and activation of AKT, IGF1-MTOR, and WNT canonical signaling pathways. Drugs targeting additional pathways that are altered in PIK3CA-mutant tumors may improve treatment regimens using PI3K inhibitors alone.

Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4.

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.