Nelfinavir Impairs Glycosylation of Herpes Simplex Virus 1 Envelope Proteins and Blocks Virus Maturation

Figure 2

NFV does not inhibit activity of the HSV-1 protease. The coding sequence of the full-length protein product of UL26 ORF (pUL26) and the expression proteins employed are diagramed in panel (a). Transfection vectors were constructed to express the VP24 protease with an N-terminal HA tag (“protease”), the pUL26.5 with an N-terminal FLAG tag (Substrate 1), or the S129A pUL26 mutant inactive protease-scaffold protein with an N-terminal FLAG tag (Substrate 2). Tags are depicted in grey at the left end of each construct. The release (R) and maturation (M) cleavage sites are shown. After transfection into HEK293-T cells, construct expression and substrate cleavage were evaluated by Western blot using anti-HA and FLAG antibodies. Each protein expressed alone was of the expected size (28.8, 37.9, and 70.6 kD, resp., including tag). Similarly, coexpression of protease with each of the 2 substrates resulted in the N-terminal cleavage products of the expected sizes. The generation of substrate cleavage products was not affected by the addition of NFV at concentrations that inhibit HSV-1 replication in vitro.

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