Full-Length Glycosylated Gag of Murine Leukemia Virus Can Associate with the Viral Envelope as a Type I Integral Membrane Protein.

The glycosylated Gag protein (gPr80) of murine leukemia viruses (MLVs) has been shown to exhibit multiple roles in facilitating retrovirus release, infection, and resistance to host-encoded retroviral restriction factors, such as APOBEC3, SERINC3, and SERINC5. One way in which gPr80 helps MLVs to escape host innate immune restriction is by increasing capsid stability, a feature that protects viral replication intermediates from being detected by cytosolic DNA sensors. gPr80 also increases the resistance of MLVs to deamination and restriction by mouse APOBEC3 (mA3). How the gPr80 accessory protein, with its three N-linked glycosylation sites, contributes to these resistance mechanisms is still not fully understood. Here we further characterized the function of gPr80 and, more specifically, revealed that the asparagines targeted for glycosylation in gPr80 also contribute to capsid stability through their parallel involvement in the Pr65 Gag structural polyprotein. In fact, we demonstrate that sensitivity to deamination by the mA3 and human A3 proteins is directly linked to capsid stability. We also show that full-length gPr80 is detected in purified viruses. However, our results suggest that gPr80 is inserted in the NexoCcyto orientation of a type I integral membrane protein. Additionally, our experiments have revealed the existence of a large population of Env-deficient virus-like particles (VLPs) harboring gPr80 inserted in the opposite (NcytoCexo) polarity, which is typical of type II integral membrane proteins. Overall this study provides new insight into the complex nature of the MLV gPr80 accessory protein.IMPORTANCE Viruses have evolved numerous strategies to infect, spread in, and persist in their hosts. Here we analyze the details of how the MLV-encoded glycosylated Gag (gPr80) protein protects the virus from being restricted by host innate immune defenses. gPr80 is a variant of the structural Pr65 Gag protein with an 88-amino-acid extended leader sequence that directs the protein for translation and glycosylation in the endoplasmic reticulum. This study dissects the specific contributions of gPr80 glycans and capsid stability in helping the virus to infect cells, spread, and counteract the effects of the host intrinsic restriction factor APOBEC3. Overall this study provides further insight into the elusive role of the gPr80 protein.

Effect of phorbol esters on the macrophage-mediated biodegradation of polyurethanes via protein kinase C activation and other pathways.

It was previously found that re-seeding monocyte-derived macrophages (MDM) on polycarbonate-based polyurethanes (PCNUs) in the presence of the protein kinase C (PKC) activator phorbol myristate acetate (PMA) inhibited MDM-mediated degradation of PCNUs synthesized with 1,6-hexane diisocyanate (HDI), as well as esterase activity and monocyte-specific esterase (MSE) protein. However, no effect on the degradation of a 4,4'-methylene bisphenyl (MDI)-derived PCNU (MDI321) occurred. This finding suggested that oxidation, a process linked to the PKC pathway, was not activated in the same manner for all PCNUs. In the current study MDM were re-seeded onto the above PCNU surfaces with PMA, PKC-inactive 4alphaPMA and the PKC inhibitor bisindolylmaleimide I hydrochloride (BIM) for 48 h before assaying for PCNU degradation, esterase activity, MSE protein, DNA, cell viability and cell morphology. 4alphaPMA did not alter MDM-mediated HDI PCNU degradation but MDI321 degradation increased in this condition. BIM alone had no effect on any parameter; however, when BIM and PMA were added together, the PMA inhibition of biodegradation, esterase activity and MSE protein was partially reversed for MDM on HDI PCNUs only. Adding PMA to MDM on HDI PCNUs increased intercellular connections, whereas 4alphaPMA or BIM+PMA increased cell size. Although this study demonstrated a role for oxidation via a PKC-activated pathway in MDM-mediated PCNU degradation, phorbol esters appear to also activate non-PKC pathways that have roles in biodegradation. Moreover, the sensitivity to material surface chemistry in the MDM response to each PCNU dictates a multi-factorial degradative process involving alternate material specific oxidative and hydrolytic mechanisms.