Differentiation of acid-ph-dependent and -nondependent entry pathways for mouse hepatitis virus

Abstract

Early events of infection of MHV were studied in comparison with those of VSV, which is known to enter cells by an endocytic pathway. Treatment of mouse L-2 fibroblasts with ammonium chloride, chloroquine, or dansylcadaverine inhibited infection of MHV to a much lesser degree than that of VSV, suggesting a relatively minor role for the endocytic pathway and functional endosomes in MHV infection. Endocytosis of MHV and VSV into L-2 cells was assayed by the recovery of infectious (i.e., not uncoated) viruses from homogenates of cells harvested within the first few minutes of infection (and treated with protease to remove surface-bound virus). The results thus suggest that while a small proportion of the MHV inoculum is internalized by endocytosis, productive infection does not depend on functional endocytosis as utilized by VSV. Studies on direct virion-mediated cell fusion showed that MHV can induce fusion at pH 7.4, whereas VSV causes fusion at pH 5.0. Taken together, the above results suggest that MHV enters L-2 cells predominantly by membrane fusion with a non-acidified compartment such as the plasma membrane, endocytic vesicles, or endosomes (prior to their acidification). Results obtained from cell lines which differed in permissiveness to MHV infection suggested that the ability to support MHV infection does not correlate with endocytosis. Rather, nonpermissive cells, such as rat astrocytoma (C-6) and Vero cells, showed higher levels of recoverable internalized MHV than did fully permissive L-2 cells. Cells which are normally nonpermissive to MHV, could be rendered MHV-susceptible by PEG-induced fusion of cell surface-bound virus. Such PEG-mediated susceptibility to MHV infection was insensitive to inhibition by ammonium chloride, supporting the idea that host cell restriction of MHV infection in C-6 and Vero cells may be due to a block in nonendosomal membrane fusion. Thus endocytic internalization of MHV, which clearly occurs in a variety of cells, does not guarantee productive infection.

Inhibition of Endosomal/Lysosomal Degradation Increases the Infectivity of Human Immunodeficiency Virus

“…Such agents are nonspecific weak bases that diffuse across membranes in a concentration-dependent manner and thereby neutralize the pH of endocytic vesicles (16,43). Viruses unaffected by lysosomotropic agents are said to enter by fusing in a pH-independent manner with the plasma membrane, and viruses whose infectivity was decreased or blocked by these agents are said to enter the host cell by fusing with endocytic vesicles in a pHdependent manner (25,38). However, these agents are nonspecific and have other side effects on the cell.…”

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

“…Many coronaviruses mediate cell-cell fusion at neutral pH (28). However, inhibitors of endosomal acidification can block coronavirus replication, either because of effects on coronaviral glycoproteins or possibly effects on postentry steps of the viral life cycle (29).…”

Establishment of persistent hepatitis C virus infection and replication in vitro.

“…virus-cell) interactions. PEG is known to be a membrane-fusing agent (Ahkong et al, 1987) and increases the efficiency of infections in a number of virus systems and cell lines (Hoekstra et al, 1989 ;Kooi et al, 1991 ;Subramanian et al, 1995) by fusing viral and cellular membranes and therefore increasing penetration rates. DMSO is used for vesicular fusion reactions (Geron & Meiri, 1985 ;Varga & Martonosi, 1992) and stabilizes cell culture systems (Isom et al, 1985), presumably by initiating formation of gap junctions (T. Yoshizawa, personal communication).…”

“…Successful infection and propagation of the virus was assessed by detection of HCV plus-and minus-strand RNA using nested RT-PCR with specific primers. To improve in vitro infections, the effects of polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were investigated, as these substances have been shown to increase the efficiency of in vitro infection with other viruses such as hepatitis B virus (Gripon et al, 1988(Gripon et al, , 1993, Sendai virus (Hoekstra et al, 1989), herpes simplex virus types 1 and 2 (Subramanian et al, 1995) and mouse hepatitis virus (Kooi et al, 1991). Since HCV was reported to be associated with very low density lipoprotein (VLDL) fractions (Thomssen et al, 1992), cell culture methods stimulating expression of LDL receptors (Havekes et al, 1983 ;Sviridov et al, 1990 ;Kostner, 1993) were examined as well.…”