Receptor-Mediated Entry by Equine Infectious Anemia Virus Utilizes a pH-Dependent Endocytic Pathway

Abstract

Previous studies of human and nonhuman primate lentiviral entry mechanisms indicate a predominant use of pH-independent pathways, although more recent studies of human immunodeficiency virus type 1 entry appear to reveal the use of a low-pH-dependent entry pathway in certain target cells. To expand the characterization of the specificity of lentiviral entry mechanisms, we have in the current study examined the entry pathway of equine infectious anemia virus (EIAV) during infection of its natural target, equine macrophages, permissive equine fibroblastic cell lines, and an engineered mouse cell line expressing the recently defined equine lentivirus receptor-1. The specificity of EIAV entry into these various cells was determined by assaying the effects of specific drug treatments on the level of virus entry as measured by quantitative real-time PCR assay of early reverse transcripts or by measurements of virion production. The results of these studies demonstrated that EIAV entry into all cell types was substantially inhibited in a dose-dependent manner by treatment with the vacuolar H ؉ -ATPase inhibitors concanamycin A and bafilomycin A1 or the lysosomotropic weak base ammonium chloride. In contrast, treatments with sucrose to block clathrin-mediated endocytosis or with chloroquine to block organelle acidification failed to inhibit EIAV entry into the same target cells. The observed inhibition of EIAV entry was shown not to be related to cytotoxicity. Taken together, these experiments reveal for the first time that EIAV receptor-mediated entry into target cells is via a low-pH-dependent endocytic pathway.

Endocytosis and a Low-pH Step Are Required for Productive Entry of Equine Infectious Anemia Virus

“…Our results provide strong evidence that EIAV enters cells through a low-pH-dependent mechanism and are consistent with studies performed by Jin and Montelaro (14). Several lysosomotropic agents that prevent acidification of endosomal compartments through different mechanisms, such as the weak base ammonium chloride, the carboxylic ionophore monensin, and the H ϩ -ATPase inhibitor bafilomycin A, inhibited EIAV infection.…”

“…However, EIAV particles were more infectious at low pH, suggesting the EIAV envelope conformation in the viral particle is not the same as that found on the surface of infected cells. These results are in general agreement with the conclusions drawn by Jin et al using independent experiments with a different strain of EIAV, as described in the companion paper appearing in this issue (14).…”

S2 from equine infectious anemia virus is an infectivity factor which counteracts the retroviral inhibitors SERINC5 and SERINC3

“…Such increased resistance to the host factor depends on the EIAV env, reminiscent of the glycoproteins derived from some HIV-1 isolates and RNA viruses such as VSV and Ebola. EIAV env has been reported to promote virus entry via a pH-dependent pathway (22), thus reinforcing the hypothesis that the endosomal uptake could provide a port of entry that is less sensitive to SERINC5 inhibition (4). It remains to be established whether resistance to SERINC5 is a general feature shared by all EIAV isolates.…”

Rhesus Rotavirus Entry into a Polarized Epithelium Is Endocytosis Dependent and Involves Sequential VP4 Conformational Changes

“…BafA1 and concanamycin A are more specific and efficient than chloroquine or ammonium chloride in blocking endocytic acidification, and a cell type-specific action of these drugs was suggested previously (38). The fact that ammonium chloride and chloroquine have no effect on RRV infectivity combined with the substantial effects of BafA1 and concanamycin A suggested that although RRV entry is likely to involve endocytosis, the direct role of endosomal acidification was less clear.…”