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Cleavage of the flavivirus prM protein by a cellular furin-like protease is a hallmark of virion maturation. While this cleavage is a required step in the viral life cycle, it can be inefficient. Virions that retain uncleaved prM may be infectious. We investigated whether cleavage by furin of prM on partially mature West Nile virus (WNV) during virus entry contributes to infectivity. Using quantitative assays of WNV infection, we found that virions incorporating considerable amounts of uncleaved prM protein were insensitive to treatment of cells with a potent inhibitor of furin activity. Thus, partially mature WNV does not require furin-like proteases for infectivity.
Flaviviruses are a group of positive-stranded RNA viruses responsible for considerable global morbidity and mortality. West Nile virus (WNV) is a mosquito-borne neurotropic member of this genus that is an emerging pathogen (16). Flaviviruses are small spherical particles that incorporate two viral glycoproteins that orchestrate virus entry into cells (reviewed in reference 18). The envelope (E) protein is an approximately 53-kDa protein that mediates virus attachment to cells and drives the pH-dependent fusion of viral and cellular membranes in the endosomes of infected cells. On immature virions, the 20-kDa premembrane (prM) protein interacts with the E protein to form 60 heterotrimeric spikes and functions to prevent adventitious fusion between viral and cellular membranes during egress through acidic compartments of the secretory pathway (9, 30, 31). Transit through these compartments catalyzes a pH-dependent rearrangement of prM and E proteins on the virion that exposes a recognition motif on prM for cellular furin-like serine proteases (15, 27, 28). Cleavage of prM results in the formation of an approximately 14-kDa “pr” protein and a small membrane-anchored M peptide. Release of the virion into the neutral environment of the extracellular space promotes the release of the pr protein and the formation of mature virus particles (28).
While the cleavage of prM is a critical step in the flavivirus life cycle (5), the extent of cleavage required for virus infectivity is not clear. Biochemical studies of the prM content of mosquito-borne flaviviruses released from cells suggest that cleavage can be inefficient (6, 9, 10, 13, 14, 17). The demonstration that more than 90% of dengue virus (DENV) particles can be precipitated with a prM-reactive antibody suggests that “partially mature” virions may be a significant component of the population of virus particles released from cells (12). Several lines of evidence support the idea that uncleaved prM is present on infectious virions (3, 8). The presence of prM on virions has been shown to increase the sensitivity of virus particles to neutralization by some E-specific antibodies (1, 19). Furthermore, antibodies specific for prM enhance virus infectivity in vitro and in vivo (2, 4, 11, 25, 29). Enhancing understanding of the stoichiometry of prM cleavage required for infectivity would facilitate a deeper conception of the complexity arising from heterogeneity in populations of virions released from infected cells. Complicating this analysis is the intriguing possibility that prM on virus particles is also cleaved by furin in acidic compartments of the endosome during virus entry. In support of this possibility, antibody-dependent enhancement (ADE) of prM-containing “immature” DENV virions (with no detectable cleavage of prM) produced in furin-deficient Lovo cells was found to be blocked by treatment with an inhibitor of furin-like proteases (25).
To investigate the requirement for cleavage of prM by a furin-like protease on WNV during the virus entry process, we performed a series of infection-inhibition studies using commercially available inhibitors of furin. Control experiments performed using recombinant furin and a well-characterized fluorogenic substrate revealed dose-dependent furin-like protease activity that could be inhibited by the inhibitor Dec-RVKR-CMK (Fig. 1A and B) (7). Furin-like protease activity was also measured in a Raji B cell line that stably expresses the C-type lectin DC-SIGNR (Fig. 1C); these cells are highly permissive to WNV infection (3). Furin-like protease activity was also measured in a variety of other cell lines (Vero, HEK-293T, BHK-21, and K562) (data not shown). In each instance, treatment of 5 × 104 cells, corresponding to the number of cells used in the infectivity studies described below, with furin inhibitor (FI) resulted in a reduction of substrate cleavage to background levels (Fig. 1D). Two additional inhibitors tested (hexa-d-arginine and anthrax lethal factor protease inhibitor) were capable of inhibiting the activity of recombinant furin to various degrees but were considerably less potent in assays with cells and were not used in subsequent studies (data not shown).
We next investigated the impact of inhibition of the activity of furin-like proteases on the infectivity of WNV. These studies were performed using an infectious clone of WNV lineage I (NY99 strain) engineered to express green fluorescent protein (GFP) by the use of a modification of a previously described molecular clone strategy (T. Y. Lin and T. Pierson, submitted for publication) (26). Raji-DC-SIGNR cells were preincubated with FI (50 μM) and infected with serial 2-fold dilutions of virus stocks produced in mammalian (HEK-293T, Vero, or BHK) or insect (C6/36) cells. Virus infectivity was measured 16 h postinfection using flow cytometry; this time point was selected because it reflects a single round of infection with the GFP-expressing virus (Fig. 2A to D). Overall, the efficiencies of infection in the presence and absence of FI at each dilution of virus were similar; the largest mean reduction in infection of FI-treated cells was approximately 13% (n = 3). While FI treatment was not overtly toxic to cells (data not shown), it did result in a reduction of approximately 10% in GFP expression, as measured by mean channel fluorescence of GFP in infected Raji-DC-SIGNR cells, identifying a potential indirect effect of inhibiting furin-like enzymes on viral replication. Because prM-containing virions may represent a modest proportion of infectious WNV virions released from cells, analysis of the infectivity of the entire population of virions by the use of an inhibitor of furin may be an insensitive test. Therefore, we used two additional approaches to focus our analysis on those virions that exhibited incorporation of prM. In this context, a significant impact of FI would be expected if cleavage of prM during virus entry were a required event in the life cycle of partially mature viruses.
First, we measured the infectivity of WNV lineage II strain 956 in the presence or absence of FI. This WNV strain lacks an asparagine-linked carbohydrate on the E protein but is glycosylated on prM (10). Interactions between WNV and Raji-DC-SIGNR cells are mediated by carbohydrates and therefore require the presence of prM on the WNV 956 virion; use of Raji-DC-SIGNR cells in infectivity studies preferentially selects for WNV 956 virions that retain uncleaved prM (3). A variant of WNV 956 that encodes GFP was produced from a molecular clone by the use of HEK-293T cells as described previously (23). Raji-DC-SIGNR cells were incubated in the presence or absence of FI and infected with serial 2-fold dilutions of virus. Infectivity was monitored at 16 h postinfection using flow cytometry. Treatment of cells with FI did not significantly reduce the infectivity of WNV 956 (Fig. 2E; n = 2). Because attachment to and infection of Raji-DC-SIGNR cells by this strain is restricted to the subset of virus particles that retain uncleaved prM, these results demonstrate that complete cleavage of prM is not an absolute requirement for infectivity.
We next performed similar studies using populations of virions that vary markedly with respect to the efficiency of prM cleavage. WNV reporter virus particles (RVPs) are pseudoinfectious virions produced by genetic complementation of a subgenomic replicon that carries a reporter gene (24). Approaches to modulate the efficiency of maturation of WNV RVPs have been previously described (19); ectopic expression of human furin in RVP-producing cells markedly increases the efficiency of prM cleavage, whereas treatment of producer cells with NH4Cl reduces the extent of maturation. WNV RVPs were produced in HEK-293T cells in the presence of furin overexpression or NH4Cl. A marked difference in the efficiencies of prM cleavage when RVPs were produced using these methods was verified by Western blot analysis (Fig. 3A). Similar results were obtained using WNV RVPs produced using a variant of the prM protein that encodes a V5 tag (V5 RVP) (Fig. 3C). Both stocks of RVPs were subjected to titration on Raji-DC-SIGNR cells in the presence or absence of FI (Fig. 3B and D). As expected, the specific infectivity of RVPs produced in the presence of NH4Cl was markedly reduced in comparison to that seen with the more mature preparation (19). Notably, no significant reduction of infectivity was observed when target Raji-DC-SIGNR cells were incubated in the presence of FI, despite the considerable prM content of each NH4Cl preparation (n = 4 and 6 for WNV RVP and WNV VS RVP, respectively).
The neutralization potency of many E protein-specific antibodies is strongly influenced by the maturation state of the virion. Several E protein-specific monoclonal antibodies (MAbs) bind virions more efficiently in the presence of uncleaved prM (1, 19) and are useful functional probes for detection of the presence of prM on infectious virions. To confirm that the significant uncleaved prM detected in NH4Cl preparations was present on infectious virions, neutralization studies were performed with maturation state-sensitive antibodies. MAb E16 (domain III) was capable of equivalently neutralizing furin and NH4Cl RVPs (Fig. 3E), as expected (P = 0.17; n = 7) (19–21), whereas furin RVPs were considerably more resistant to neutralization by the maturation-sensitive MAbs E53 (domain II) and E121 (domain I) than the NH4Cl RVP preparations (19, 22) (Fig. 3F and G). The neutralization potency (i.e., the 50% effective concentration [EC50]) of E16 (P = 0.41; n = 7), E53 (P = 0.87; n = 7), and E121 (P = 0.41; n = 3) was not impacted significantly by FI treatment. Altogether, these studies demonstrate that infectivity of RVPs that retain significant uncleaved prM does not require processing by furin during virus entry.
Antibody-dependent enhancement (ADE) of immature DENV infection has been shown to be sensitive to treatment with FI (25). To investigate whether the activity of a furin-like protease is required during Fc-γ receptor-mediated entry of WNV into cells, ADE experiments were performed using furin and NH4Cl RVPs and a panel of six maturation-sensitive antibodies. RVPs were incubated with serial dilutions of antibody for 1 h and then added to Fc-γRIIA-expressing K562 cells in the presence or absence of FI. All MAbs were capable of enhancing infection of both furin and NH4Cl RVPs. The levels of impact of FI treatment were minimal and equivalent for the prM-containing (NH4Cl) and prM-deficient (furin) RVP preparations (Fig. 4A to F). To explore whether the enhancement mediated by antibodies that recognize prM was sensitive to FI treatment, experiments were performed using V5 RVPs and a commercial anti-V5 antibody. Significant enhancement was observed with RVPs produced in the presence of NH4Cl but not with exogenously expressed furin, as expected, and was not sensitive to the presence of FI in K562 target cells (Fig. 4G).
Flavivirus-infected cells release a heterogeneous population of virions that differ with respect to the extent of virion maturation. While uncleaved prM is readily apparent in biochemical studies of flaviviruses, the distribution of uncleaved prM molecules among infectious virions is not known. In this study, we investigated whether the infectivity of incompletely cleaved virions requires processing of prM by furin-like proteases during entry. Our studies failed to identify a subset of virions whose infectivity was sensitive to inhibition of furin-like proteases, even when the efficiency of virion maturation was significantly reduced. A previous study of immature DENV produced in Lovo cells suggested a requirement for cleavage of prM during antibody-mediated entry of virus into K562 cells that was not apparent in our studies of WNV (25). Several factors may account for the differences between these two studies. One interesting possibility is that the stoichiometric requirements for cleavage of prM may differ between DENV and WNV. While inhibitors of furin markedly decrease cleavage of WNV prM during virus egress (data not shown), it is possible that as-yet-unidentified proteases that are not sensitive to inhibitors of furin-like proteases may cleave partially mature WNV during entry. The sensitivity of DENV infectivity to FI was established principally by using experiments that measured virus release (25). A quantitative assessment of the size and contribution of the FI-sensitive population of DENV by the use of indirect assays may be difficult, as small changes in the number of infection events could translate into large differences in virus output. This is particularly true if FI-treatment has a subtle impact on the viral life cycle that does not relate directly to prM cleavage. While we cannot rule out the possibility that virions containing essentially unprocessed prM may be cleaved by furin during virus entry, our data suggest that particles at this extreme of the maturation spectrum represent a very small fraction of the prM-containing partially mature WNV released from cells. Taken together, our studies suggest that the infectivity of prM-containing partially mature WNV is not dependent on cleavage of prM during virus entry.
This work was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (NIAID).
We are grateful to Michael Diamond for his thoughtful comments on our manuscript. We thank members of our laboratory for useful discussions and their comments on the manuscript.
Published ahead of print on 31 August 2011.