Microsoft Word - ASLV fusion_JBC(Rev2)

Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell typespecific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of aciddependent fusion of the Avian Sarcoma and Leukosis Virus, ASLV, with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate, when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells. Infectivity of enveloped viruses often varies depending on the cell type, even for cells expressing comparable levels of cognate receptors. This is largely due to the varied efficiency of multiple steps of entry leading to productive infection. Whereas multiple host factors are involved in late post-fusion steps of virus entry (19), the effects of intrinsic, cell type-dependent factors and of extrinsic factors on viral fusion is poorly characterized. After the initial interaction of viruses with cellular receptors or attachment factors, low endosomal pH is required to trigger fusion-inducing conformational changes in most viral proteins (reviewed in (10,11)). The low pH requirement of virus entry could also stem from the need for optimal milieu (e.g., endosomal protease activity) for priming the viral glycoproteins for the fusion reaction (10). It is thus likely that cell typedependent regulation of endosomal pH modulates the efficiency and kinetics of virus fusion. To date, however, only a few studies have directly examined the link between the pH in virus-carrying endosomes and the efficiency/kinetics of subsequent viral fusion (12-15). Endosome-resident lipids and proteins have been implicated in the completion of virus fusion and/or the nucleocapsid release into the cytoplasm (8,1622). Cells can also express restriction factors that interfere with the viral fusion step. Recent studies have shown that expression of interferon-inducible transmembrane proteins (IFITMs) inhibits fusion of unrelated enveloped viruses in vitro and in vivo (2325). Using single virus imaging, we have shown that human IFITM3 blocks the formation of small fusion pores without inhibiting the influenza virus hemifusion with endosomes (26). Different models for the IFITMs’ antiviral activity have been proposed, including the “stiffening” of cell membranes that disfavors viral fusion (24). However, direct evidence supporting the current