Requirements for entry of poliovirus RNA into cells at low pH.

HeLa S3 cells were protected against infection by poliovirus type I by the presence of monensin and N,N'-dicyclohexylcarbodiimide (DCCD), compounds elevating the pH of acidic intracellular compartments. The protection was fully overcome by exposing the cells to pH 5.5 and lower, and at approximately pH 6.1 it was reduced by half. Measurements of the ability of the virus to enter the detergent phase under conditions where Triton X-114 was separated from water indicated that the virus is hydrophilic at neutral pH, and that it exposes hydrophobic regions at low pH. When the cells were pretreated with acetic acid, which reduces the intracellular pH, virus entry was inhibited, indicating that a pH gradient across the membrane is necessary for infection. Under all conditions which induced infection, the virus particles were altered to more slowly sedimenting material. Also, virus bound to aldehyde-fixed cells was altered when exposed to low pH at 37 degrees C. The data indicate that poliovirus bound to receptors on cells exposes hydrophobic regions at low pH, and that at physiological temperature it undergoes alteration. This alteration may be a necessary, but not sufficient requirement for infection.     

Entry of poliovirus into cells does not require a low-pH step.

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.     

Endocytosis and a low-pH step are required for productive entry of equine infectious anemia virus

Recently, it has become evident that entry of some retroviruses into host cells is dependent upon a vesicle-localized, low-pH step. The entry mechanism of equine infectious anemia virus (EIAV) has yet to be examined. Here, we demonstrate that wild-type strains of EIAV require a low-pH step for productive entry. Lysosomotropic agents that inhibit the acidification of internal vesicles inhibited productive entry of EIAV. The presence of ammonium chloride (30 mM), monensin (30 microM), or bafilomycin A (50 nM) in the medium dramatically decreased the number of EIAV antigen-positive cells. We found that a low-pH step was required for EIAV infection of tissue culture cell lines as well as primary cells, such as endothelial cells and monocyte-derived macrophages. The ammonium chloride treatment did not reduce virion stability, nor did the treatment prevent virion binding to cells. Consistent with a requirement for a low-pH step, virion infectivity was enhanced more than threefold by brief low-pH treatment following binding of viral particles to permissive cells. A superinfecting variant strain of EIAV, vMA-1c, did not require a low-pH step for productive infection of fibroblasts. However, lysosomotropic agents were inhibitory to vMA-1c infection in the other cell types that vMA-1c infected but did not superinfect, indicating that the entry pathway used by vMA-1c for superinfection abrogates the need for the low-pH step.     

Role of a low-pH environment in adenovirus enhancement of the toxicity of a Pseudomonas exotoxin-epidermal growth factor conjugate

A conjugate of Pseudomonas exotoxin and epidermal growth factor (PE-EGF) inhibits proteins synthesis in KB cells, and this inhibition is increased by adenovirus. Protein synthesis inhibition is dependent on the amount of adenovirus and PE-EGF used and the time of incubation of cells with these agents. With 1 microgram of adenovirus and 0.5 micrograms of PE-EGF per ml, protein synthesis is inhibited about 80% in a 60-min experiment. Under these conditions neither adenovirus nor PE-EGF alone has any effect. In the presence of several weak bases or monensin, the enhancement of toxicity was substantially inhibited; half-maximal inhibition was achieved with 40 microM chloroquine, 10 mM ammonium chloride, 5 mM methylamine, 0.1 mM N-hexylamine and 1 microM monensin. At the concentrations employed, none of the inhibitors affected the amount of virus taken up or bound to the cell surface, and chloroquine had no effect on the amount of EGF taken up in 60 min. Chloroquine did not prevent the toxicity of the PE-EGF (5 micrograms/ml) alone. Because these compounds are known to elevate the pH in receptosomes, it seems likely that the acidification of the receptosome either enhances the lysis of the membrane by adenovirus or enhances some other step in the release of PE-EGF.     

Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells.

The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.     

Effect of monensin on the assembly of Uukuniemi virus in the Golgi complex.

The effect of the carboxylic ionophore monensin on the maturation of Uukuniemi virus, a bunyavirus, and the transport of its two membrane glycoproteins, G1 and G2, were studied in chicken embryo fibroblasts and baby hamster kidney cells. Virus maturation, which occurs in the Golgi complex (E. Kuismanen, K. Hedman, J. Saraste, and R. F. Pettersson, Mol. Cell. Biol. 2:1444-1458, 1982; E. Kuismanen, B. B?ng, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984), was effectively inhibited by the drug (1 or 10 microM) as studied by electron microscopy and by assaying the release of infectious or radiolabeled virus. Immunoelectron microscopy showed that association of viral nucleocapsids with the cytoplasmic surface of glycoprotein-containing Golgi membranes, a prerequisite for virus budding, was unaffected by monensin. In the presence of the drug, the virus glycoproteins assembled into long, tubular structures extending into the lumen of Golgi-derived vacuoles, suggesting that monensin inhibited a terminal step in the assembly of the virus. Intracellular transport and expression of the virus membrane glycoproteins G1 and G2 at the cell surface were not inhibited by monensin as studied by immunocytochemical and radiolabeling techniques. Pulse-chase experiments in the presence of monensin showed that intracellular G1 acquired only partially endo-H-resistant glycans. The sialylation of G1 appearing on the cell surface in the presence of the drug was decreased, whereas sialylation of G2 apparently was inhibited to a lesser extent, as shown by external labeling of the cells with the periodate-boro[3H]hydride method. Thus, monensin exerted a differential effect on the terminal glycosylation of G1 and G2. Unlike several membrane and secretory glycoproteins, both G1 and G2 could enter a functional transport pathway in the presence of monensin and become expressed at the cell surface.     

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells.

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.     

Vaccinia virus entry into cells via a low-pH-dependent endosomal pathway

Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.     

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.     

Na entry and Na-K pump activity in murine,hamster, and human cells - effect of monensin,serum, platelet extract,and viral transformation

The relationship between Na entry and the activity of the Na-K pump has been investigated in a variety of cell types by testing the effect of the Naionophore monensin, mitogenic stimulation with serum and oncogenic transformation by SV₄₀ and polyoma virus. We found that addition of monensin increases intracellular Na in quiescent cultures of murine, hamster, and human cells. In each case, the rise in intracellular Na by monensin is associated with an increase in the activity of the Na-K pump, which was measured as ouabain-inhibitable ⁸⁶Rb uptake. The addition of serum to quiescent cultures stimulates ⁸⁶Rb uptake in all cell types studied. Serum alone causes an increase in intracellular potassium with no consistent change in intracellular Na. In the presence of the Na-K pump inhibitor ouabain, serum causes a marked increase in intracellular Na, with little change in intracellular K. This pattern is interpreted as indicating that the primary effect of serum is to increase Na entry into the cells. A low concentration of monensin (0.2 μg/ml) mimics the effect of serum on ion fluxes and content, which supports the conclusion that serum and monensin stimulate ⁸⁶Rb uptake in the same manner, namely by increasing Na entry into the cells. In addition, a partially purified platelet extract stimulates Na entry and ⁸⁶Rb uptake in quiescent 3T3 cells. Finally 3T3 cells transformed by SV₄₀ or polyoma virus exhibit a higher rate of Na entry and of Na-K pump activity than their untransformed 3T3 counterparts. All these results indicate that the rate of Na entry plays an important role in the regulation of the activity of the Na-K pump and that an increase in Na and K movements is a rapid response elicited by serum in a variety of cell types.     

Effects of monensin on morphogenesis and infectivity of Friend murine leukemia virus.

The transport of the gp70 glycoprotein to the cell surface and concomitant release of infectious virus was inhibited by treatment of Friend murine leukemia virus-infected Eveline cells with the sodium ionophore monensin. Virus yields were reduced more than 50-fold by 10(-5) M monensin, whereas particle production was reduced by 50% in monensin-treated cells. The resulting particles failed to incorporate newly synthesized gp70 and p15(E), whereas the other structural proteins, p30, p15, p12, and p10, were incorporated into virions. However, monensin did not inhibit the incorporation into virions of preformed gp70. A reduction in the efficiency of cleavage of the PrENV glycoprotein precursor and a defect in the processing of simple endo-H-sensitive to complex endo-H-resistant oligosaccharides suggest that intracellular transport of gp70 may be blocked before its entry into the Golgi apparatus. Fewer particles were found to bud from the cell surface, but intracellular vacuoles with budding virions were detected. Ferritin labeling and pulse-chase studies suggested a cell surface origin for these vacuoles. These experiments indicate that monensin inhibits the transport of Friend murine leukemia virus glycoproteins at an early stage, with a resultant block in the assembly and release of infectious virus.     

Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore

Analysis of viral glycoprotein expression on surfaces of monensin- treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin- Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance was not altered by the ionophore. Viral protein synthesis was not inhibited by monensin in either VSV- or influenza virus-infected cells. However, the electrophoretic mobilities of viral glycoproteins were altered, and analysis of pronase-derived glycopeptides by gel filtration indicated that the addition of sialic acid residues to the VSV G protein was impaired in monensin-treated cells. Reduced incorporation of fucose and galactose into influenza virus HA was observed in the presence of the ionophore, but the incompletely processed HA protein was cleaved, transported to the cell surface, and incorporated into budding virus particles. In contrast to the differential effects of monensin on VSV and influenza virus replication previously observed in monolayer cultures of MDCK cells, yields of both viruses were found to be significantly reduced by high concentrations of monensin in suspension cultures, indicating that cellular architecture may play a role in determining the sensitivity of virus replication to the drug. Nigericin, an ionophore that facilitates transport of potassium ions across membranes, blocked the replication of both influenza virus and VSV in MDCK cell monolayers, indicating that the ion specificity of ionophores influences their effect on the replication of enveloped viruses.     

The proteolytic cleavage of PE2 to envelope glycoprotein E2 is not strictly required for the maturation of Sindbis virus.

The ionophore monensin has been shown previously to block the maturation of Sindbis virus as well as prevent the cleavage of pE2 to E2 when applied to cells in high concentration. We found that a moderate dose of monensin reduced virus titer and inhibited the cleavage of pE2 to E2. Under these conditions, pE2 appeared on the cell surface in a form susceptible to lactoperoxidase-mediated iodination. This pE2 was incorporated into virions, replacing E2. PE2-containing virions had a normal PFU-to-particle ratio, cosedimented with normal virus, and retained a normal morphology when negatively stained preparations were examined by electron microscopy. We conclude that the cleavage of pE2 to form E2 is not an absolute prerequisite for virus maturation. Recently, Russell et al. have reached a similar conclusion (D. L. Russell, J. M. Dalrymple, and R. E. Johnston, J. Virol. 63:1619-1629, 1989).     

Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.     

Effect of monensin on Mason-Pfizer monkey virus glycoprotein synthesis.

The effect of the monovalent carboxylic ionophore monensin on the biosynthesis, intracellular transport, and surface expression of the glycoproteins of Mason-Pfizer monkey virus was examined. Cells treated with monensin at concentrations of 10(-7) or 10(-6) M continued to synthesize virus particles, which from electron microscopic studies appeared to bud normally from the plasma membrane of the cells. However, the particles released had an altered buoyant density in sucrose gradients and were noninfectious. These noninfectious virions had a normal complement of non-glycosylated polypeptides but showed a significantly reduced amount of glycosylated proteins. The gp70 and gp20 polypeptides appeared to be completely absent, and a heterogeneous, higher-molecular-weight protein was observed on the virions instead. Studies on intracellular protein synthesis indicated that the precursor (Pr86env) to gp70 and gp20 is synthesized normally but is not cleaved to the mature proteins. Immunofluorescence studies showed, however, that the uncleaved molecule is expressed on the cell surface. In this system, therefore, Mason-Pfizer monkey virus glycoprotein migration appears to occur in the presence of monensin, whereas the cleavage and insertion of the glycoproteins into virions are inhibited.     

Action of monensin, a monovalent cationophore, on cultured human fibroblasts: evidence that it induces high cellular accumulation of glucosyl- and lactosylceramide (gluco- and lactocerebroside)

We have exposed cultured human fibroblasts to micromolar concentrations of the ionophore monensin. A salient result was a rapid accumulation in these cells of glucosylceramide (glucocerebroside, GlcCer) and lactosylceramide (lactocerebroside, LacCer). When we incubated these cells with radioactively labeled galactose, GlcCer and LacCer became highly labeled. These results indicate that monensin greatly increases these simplest glycosphingolipids that are the precursor to the major plasma membrane glycosphingolipids. We observed, simultaneously, a decreased incorporation of labeled galactose into some more highly glycosylated neutral glycosphingolipids and sialoglycosphingolipids (gangliosides), and unlike GlcCer and LacCer, the cellular content of these more highly glycosylated compounds remained the same in the presence or absence of monensin. We have found that cultured Gaucher disease fibroblasts, with genetically impaired lysosomal glucocerebrosidase activity, accumulated even more GlcCer and LacCer than normal cells upon exposure to monensin. This finding shows that monensin affects biosynthesis rather than merely disrupting lysosomal degradation that is already deleted with respect to GlcCer in Gaucher disease cells. These results represent the first indication of an apparently remarkable effect of the monovalent ionophore, monensin, on plasma membrane glycosphingolipid biosynthesis. The evidence suggests a regulatory distinction between initial and higher intracellular glycosylation steps. Monensin does not diminish and may augment initial anabolic mono- and diglycosylations and also appears to inhibit higher glycosylations of glycosphingolipids.     

Entry of Rice dwarf virus into cultured cells of its insect vector involves clathrin-mediated endocytosis

Electron microscopy revealed that the entry of Rice dwarf virus (RDV) into insect vector cells involved endocytosis via coated pits. The treatment of cells with drugs that block receptor-mediated or clathrin-mediated endocytosis significantly reduced RDV infectivity. However, the drug that blocks caveola-mediated endocytosis had a negligible effect on such infection. Infection was also inhibited when cells had been pretreated with bafilomycin A1, which interferes with acidification of endosomes. Moreover, immunofluorescence staining indicated that the virus is internalized into early endosomes. Together, our data indicate that RDV enters insect vector cells through receptor-mediated, clathrin-dependent endocytosis and is sequestered in early endosomes.     

Adenovirus-induced leakage of co-endocytosed macromolecules into the cytosol

The early interaction between KB cells and adenovirus was studied by examining the uptake of an extracellular fluorescent macromolecule, FITC (fluorescein isothiocyanate)-labeled dextran (FD). When cells in suspension were incubated with both adenovirus and FD, cell-associated FD increased 2- to 3-fold the value obtained without adenovirus. Under fluorescence microscopy, cells incubated with adenovirus showed bright, whole-cellular fluorescence; whereas, those incubated without adenovirus, or with heat-inactivated virus, showed weaker fluorescence, mainly of the pinocytic vesicles. The increased uptake of the FD by adenovirus was inhibited by treating KB cells with the drugs chloroquine, ammonium chloride and monensin that raise the pH of the acidic compartment. Entry of adenovirus into the KB cell's nucleus also was inhibited by these drugs. The conclusion is that entry of adenovirus into the cell involves its passage of an acidic compartment (probably the endocytic vesicle) and that co-endocytosed macromolecules are released into the cytosol on entry.     

Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.