Jaagsiekte sheep retrovirus utilizes a pH-dependent endocytosis pathway for entry

Abstract

Using Moloney murine leukemia virus pseudovirions bearing the envelope protein of Jaagsiekte sheep retrovirus (JSRV), we report here that entry was weakly inhibited by lysosomotropic agents but was profoundly blocked by bafilomycin A1 (BafA1). Kinetics studies revealed that JSRV entry is a slow process and was substantially blocked by a dominant-negative mutant of dynamin. Interestingly, a low-pH pulse overcame the BafA1 block to JSRV infection, although this occurred only if virus-bound cells were preincubated at 37 degrees C, consistent with a very early entry event such as endocytosis being required before the low-pH-dependent step occurs. Moreover, JSRV pseudovirions were resistant to low-pH inactivation. Altogether, this study reveals that JSRV utilizes a pH-dependent, dynamin-associated endocytosis pathway for entry that differs from the classical pH-dependent entry pathway of vesicular stomatitis virus.

FIG. 1.

JSRV entry is profoundly blocked by BafA1 but is weakly inhibited by lysosomotropic agents. Mouse NIH 3T3 cells overexpressing human Hyal2 (NIH 3T3/LH2SN) were pretreated with NH₄Cl (A), chloroquine (B), or BafA1 (C) for 2 h at the indicated concentrations and were exposed to GFP-encoding MoMLV pseudovirions bearing JSRV Env, G protein of VSV (VSV-G), or 10A1 MLV Env in the presence of the agents for 4 h. Noninternalized viral particles were inactivated by citrate buffer (pH 3.0) (JSRV and 10A1) or by 0.25% trypsin (VSV), and cells were incubated for an additional 2 h in the presence of the agents. Following several washes with phosphate-buffered saline, cells were incubated in regular medium for 48 h, and percentages of GFP-positive cells were determined by flow cytometry. The percent infection was calculated relative to results for dimethylsulfoxide-treated cells, and values are the means for two to five independent experiments performed in duplicate ± standard deviations. Experiments were also performed with human HTX and 293 cells expressing endogenous Hyal2, with similar results, except that no inhibitory effect on JSRV by chloroquine was found in HTX cells. Note that comparable multiplicities of infection, typically 0.1 to 0.5, of different pseudovirions were used in a single experiment. The Student t test was used for statistical analysis in these and other experiments throughout this study.

FIG. 2.

Kinetics of JSRV entry is relatively slow. (A) NIH 3T3/LH2SN cells were bound by JSRV, 10A1, or VSV pseudovirions (MOIs of 0.1 and 0.5, respectively) for 2 h at 4°C to prevent internalization, unbound viral particles were removed with cold phosphate-buffered saline, and the virus-cell complexes were warmed to 37°C (t = 0) to initiate viral internalization and infection. Noninternalized viruses were inactivated at the indicated times after the temperature shift, and viral infectivity was measured 48 h postinfection by flow cytometry. The percent infection was calculated relative to the maximal infection at 4 h following the temperature shift to 37°C. (B) Virus-cell complexes were formed as described for panel A, but 25 nM BafA1 was added instead of inactivating noninternalized virus at 10 min, 30 min, 1 h, 2 h, 3 h, and 4 h after initiation of infection. Note that the total infection period was 6 h in all cases. The percent infection was calculated relative to results for mock-treated cells, and values are averages of two-independent experiments performed in duplicate ± standard deviations.

FIG. 3.

Low pH conditionally overcomes a BafA1 block on JSRV infection. NIH 3T3/LH2SN cells were given a mock pretreatment (left columns) or pretreated with 25 nM BafA1 for 2 h (middle and right columns) and then were prebound with MoMLV pseudovirions (MOI = 0.1 to 0.5) bearing JSRV Env (A), VSV-G (B), or 10A1 MLV Env (C) for 1 h at 4°C in the absence or presence of the drug. Unbound viruses were removed by cold phosphate-buffered saline, and virion-cell complexes were treated with a pH 7.0 or pH 5.0 pulse at 37°C for 10 min, followed by a continued incubation at 37°C for 4 h in the presence of 25 nM BafA1 (middle columns). Alternatively, virion-bound cells were preincubated at 37°C for 1 h prior to the pH 7.0 or 5.0 pulse, followed by an additional 3-h incubation in the presence of 25 nM BafA1 (right columns). In both cases, the total infection period was 4 h. After 4 h, noninternalized viral particles were inactivated, and viral infectivity was determined 48 h later. The percent infection was calculated relative to results for the DMSO/pH 7.0-treated cells, and values are means of three independent experiments performed in duplicate ± standard deviations.

FIG. 4.

JSRV pseudovirions are resistant to low-pH inactivation. MoMLV pseudovirion stocks bearing JSRV Env, VSV-G, or SFV E1 were warmed at 37°C for 5 min, and then PBS-NaOH or PBS-HCl (pH 7.6, 7.0, 5.6, 5.0, 3.6, or 3.0) was added and virions were incubated for an additional 30 min at 37°C, after which samples were neutralized by rapid addition of equal volumes of regular medium containing 25 mM HEPES in phosphate-buffered saline (pH 7.4). NIH 3T3/LH2SN cells were exposed to serial dilutions of each sample for 8 h, and viral infectivity was determined by flow cytometry 48 h later. The values are calculated relative to the pH 7.6-treated viral stocks and are averages for two to four independent experiments performed in duplicate ± standard deviations. The P values (calculated using Student's t test) for VSV-G pseudovirions were 0.259 at pH 5.6, 0.303 at pH 5.0, and 0.052 at pH 3.0, respectively.

FIG. 5.

JSRV entry is blocked by a dominant-negative mutant of dynamin. Human 293 cells were transiently transfected with plasmids encoding amino-terminally hemagglutinin epitope-tagged dynamin wild type (Dyn^-WT) or a dominant-negative mutant (Dyn^-K44A) (kind gifts of Eric Cohen, Institut de Recherches Cliniques de Montréal, Montréal, Canada) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). (A) Expression of Dyn^-WT and Dyn^-K44A. Cell lysates were harvested from a portion of the transfected cells and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using our previously published methods (19), and expression of dynamin was detected by Western blotting using an anti-hemagglutinin antibody (Sigma, St. Louis, MO) to detect the HA-dynamin fusion protein (upper panel). Similar amounts of cell lysates were loaded onto a sodium dodecyl sulfate-polyacrylamide gel and were analyzed for β-actin using an antibody against β-actin (Sigma) (lower panel). (B) Relative infectivity of JRSV pseudovirions in cells expressing Dyn^-WT or Dyn^-K44A. Twenty-four to forty-eight hours posttransfection, transfected cells were exposed to serial dilutions of alkaline phosphatase (AP)-encoding MoMLV pseudovirions bearing JSRV Env, VSV-G, or 10A1 MLV Env. Viral titers were determined by counting AP-positive foci 48 h postinfection. Values are calculated relative to viral titers obtained from the empty vector-transfected cells and are means for three independent experiments ± standard deviations.