To test the inhibitory potential of S1P-specific a 1 -antitrypsins on proteolytic processing of LASV GP, stably transfected CHO-K1 Tet-On cells, and non-transfected wild-type CHO-K1 Tet-On cells as well as SRD-12B cells were infected with VSVDG/ LASVGP at an MOI of 0.2 in the presence or absence of doxycycline. To allow only one replication cycle, cell lysates were analyzed 10 h post-infection for detection of LASV GP cleavage by Western blot analysis using a GP-specific antiserum that recognizes both the precursor GP-C and the cleaved subunit GP-2. In CHO-K1 Tet-On cells LASV GP was efficiently cleaved, regardless of whether doxycycline was present or not. In contrast, virtually no detectable cleavage of GP was observed in SRD-12B cells that are deficient in S1P (Fig. 3A, lanes 1-4) . Without expression of the various antitrypsins efficient cleavage was detected in these stably transfected cell lines, similar to the processing of GP in wild-type CHO-K1 Tet-On cells (Fig. 3A , lanes 1, 5, 7, 9, 11, and 13) . In contrast, cells expressing the S1Padapted a 1 -antitrypsins inhibited proteolytic maturation of LASV GP (Fig. 3A, lanes 6, 8, 10, and 12) . Furthermore, our results show Effect of S1P Inhibition on LASV Replication www.plosntds.org that the presence of a furin-specific a 1 -AT did not influence LASV GP-C processing, demonstrating the specificity of the generated S1P-adapted a 1 -antitrypsins (Fig. 3A, lanes 13 and 14) . Quantification of GP-2 cleavage revealed that the a 1 -AT variant RRIL exhibited the greatest inhibitory effect on GP processing (.80% inhibition) followed by a 1 -AT RRLL (.60% inhibition), which possesses the amino acid cleavage motif of the LASV GP-C. Also a 1 -AT variants RRVL and RRYL were found to be inhibitory, although to a lesser extent (inhibition less than 50%) than the variants RRIL and RRLL (Fig. 3B) . Taken together, these data clearly demonstrate that S1P-specific a 1 -antitrypsins efficiently block the maturation cleavage of LASV GP, however, they differ in regard to their inhibitory potential.