L-domain flanking sequences are important for host interactions and efficient budding of vesicular stomatitis virus recombinants

Abstract

Vesicular stomatitis virus (VSV) possesses a PPPY and a PSAP motif within the matrix (M) protein. The PPPY motif has significant L-domain activity in BHK-21 cells, whereas the PSAP motif does not. Since the core PSAP motif alone is insufficient to provide L-domain activity, we modified upstream or downstream amino acids flanking the PSAP core motif to determine their effect on L-domain activity. VSV recombinants were recovered that contained single or multiple amino acid mutations in upstream or downstream sequences flanking the PSAP core. Recombinant viruses were examined for growth kinetics, budding efficiency, and functional interactions with host proteins. We demonstrate that the composition of amino acids surrounding the L-domain core motifs are critical for efficient L-domain activity and for interactions with host proteins in the context of a VSV infection.

FIG. 1.

Growth kinetics of the M6, M6PY>A4, and recombinant viruses, which possess single amino acid substitutions at positions 34 and 41 of the M protein of the PY>A4 virus. (A) Diagram of the VSV genome with the M gene highlighted in gray. The VSV M protein (amino acids 1 to 229) is expanded, and the sequences of amino acids 20 to 31 and amino acids 33 to 44 are indicated. The dotted line indicates that the VSV-WT M sequence is maintained. The amino acid sequence within the L domain region (amino acids 20 to 31) is shown for VSV-WT, PY>A4, M6, M6PY>A4, PY>A4-E34R, and PY>A4-I41P. (B) Graph of growth kinetics and titers of the viruses shown above at the indicated times p.i. of BHK-21 cells. Each titer represents the average of at least two independent experiments.

FIG. 2.

Growth kinetics of M6PY>A4 recombinant viruses, which possess single or double amino acid substitution at positions 33 and 34 of the M protein of the M6PY>A4 virus. (A) The amino acid sequence within the L domain region (amino acids 20 to 31) is shown for VSV-WT, M6PY>A4, M6PY>A4-S33A, -S33M, -R34A, -R34E, and -SR>ME as shown in Fig. 1. (B) Graph of growth kinetics and titers of the viruses shown above at the indicated times p.i. of BHK-21 cells. Each titer represents the average of at least two independent experiments.

FIG. 3.

Growth kinetics of the recombinant viruses, which possess either the upstream four or the downstream three amino acids of the PTAP motif from HIV-1 p6Gag in place of those of the PSAP motif of VSV. (A) The amino acid sequence within the L-domain region (amino acids 20 to 31) is shown for VSV-WT, PY>A4, PY>A4-MEYA>SRLE, and PY>A4-IDK>PEE as shown in Fig. 1. (B) Graph of the growth kinetics and titers of the viruses shown above at the indicated times p.i. of BHK-21 cells. Each titer represents the average of at least two independent experiments.

FIG. 4.

Characterization of virion protein profiles and M protein synthesis in infected BHK-21 cells. Virions released from BHK-21 cells at 8 h p.i. were purified and subjected to SDS-PAGE analysis. The G, N/P, and M proteins are indicated for VSV-WT (lane 1), PY>A4 (lane 2), M6 (lane 3), M6PY>A4 (lane 4), PY>A4-E34R (lane 5), PY>A4-I41P (lane 6), M6PY>A4-S33M (lane 7), M6PY>A4-S33A (lane 8), M6PY>A4-R34E (lane 9), M6PY>A4-R34A (lane 10), M6PY>A4-SR>ME (lane 11), PY>A4-MEYA>SRLE (lane 12), and PY>A4-IDK>PEE (lane 13). The L protein is not shown. Infected cell lysates were analyzed by Western blotting using anti-M MAb (lanes 14 to 26).

FIG. 5.

(A) Packaging of endogenous tsg101 into virions. Western blotting using anti-tsg101 antiserum to detect endogenous tsg101 packaged into VSV-WT (lane 1), PY>A4-MEYA>SRLE (lane 2), PY>A4-IDK>PEE (lane 3), M6 (lane 4), M6PY>A4 (lane 5), and endogenous tsg101 in uninfected BHK-21 cells is also shown as a control (lane 6). Equivalent amounts of virion proteins were shown to be loaded onto the gel (data not shown). (B) tsg101 siRNA inhibits the budding of active PT/SAP motif-containing recombinants. Human 293T cells were first transfected with tsg101-specific siRNA or an NS siRNA and then infected with PS>A4, M6, M6PY>A4, PY>A4-MEYA>SRLE, or PY>A4-IDK>PEE. Virion release in the presence of NS siRNA was set at 1.0 for all viruses. Each white bar represents an average of at least three independent experiments.