Identification of domains in gag important for prototypic foamy virus egress

Abstract

Sequence motifs (L domains) have been described in viral structural proteins. Mutations in these lead to a defect at a late stage in virus assembly and budding. For several viruses, recruitment of an endosomal sorting complexes required for transport 1 subunit (Tsg101), a component of the class E vacuolar protein sorting (EVPS) machinery, is a prerequisite for virion budding. To effect this, Tsg101 interacts with the PT/SAP L domain. We have identified candidate L-domain motifs, PSAP, PPPI, and YEIL, in the prototypic foamy virus (PFV) Gag protein, based on their homology to known viral L domains. Mutation of the PSAP and PPPI motifs individually reduced PFV egress, and their combined mutation had an additive effect. When PSAP was mutated, residual infectious PFV release was unaffected by dominant negative Vps4 (an ATPase involved in the final stages of budding), and sensitivity to dominant negative Tsg101 was dramatically reduced, suggesting that the PSAP motif functions as a conventional class E VPS-dependent L domain. Consistent with this notion, yeast two-hybrid analysis showed a PSAP motif-dependent interaction between PFV Gag and Tsg101. Surprisingly, PFV release which is dependent on the PPPI motif was Vps4-independent and was partially inhibited by dominant negative Tsg101, suggesting that PPPI functions by an unconventional mechanism to facilitate PFV egress. Mutation of the YEIL sequence completely abolished particle formation and also reduced the rate of Gag processing by the viral protease, suggesting that the integrity of YEIL is required at an assembly step prior to budding and YEIL is not acting as an L domain.

FIG. 1.

Schematic representation of the PFV Gag protein. Possible positions of the matrix (MA), capsid (CA), and nucleocapsid (NC) regions are indicated in brackets. The positions of putative L domains PSAP, PPPI, and YEIL are underlined along with the mutations made. The dotted line indicates the major proteolytic cleavage site for the viral protease.

FIG. 2.

The PSAP and PPPI motifs are required for optimal release of PFV vector and wild-type virus. The solid columns show titers of vector released from 293T cells transfected with either the wild-type (MH71), PSAP-mutated (MH71 L1), PPPI-mutated (MH71 L2), PSAP/PPPI dual-mutated (MH71 L1/L2), or YEIL-mutated (MH71 L3) vector constructs, all cotransfected with the pczHFVenv construct. Vector was titrated on D17 cells. The hashed columns show titers of virus released from 293T cells transfected with either the wild-type (pczHSRV2), PSAP-mutated (pczHSRV2 L1), PPPI-mutated (pczHSRV2 L2), PSAP/PPPI dual-mutated (pczHSRV2 L1/L2), or YEIL-mutated (pczHSRV2 L3) virus constructs. Virus is titrated on BHLL cells. Results shown are the means and standard errors of three independent experiments.

FIG. 3.

Western blots showing the PFV Gag proteins for vector and L-domain mutants. (A) Gag proteins from lysates of 293T cells transfected with the pczHFVenv construct and either the wild-type (MH71), PSAP/PPPI dual-mutated (MH71 L1/L2), PPPI-mutated (MH71 L2), or PSAP-mutated (MH71 L1) vector constructs and harvested at day 3 posttransfection. Normal Gag processing is seen for the L1, L2, and L1/L2 double mutants. (B) Gag in lysates from cells 3 days posttransfection with the pczHFVenv construct and either the wild-type (MH71) or YEIL mutation (MH71 L3) where little of the 68-kDa cleavage product is observed. In this case, the 293T cells were transfected with half the normal amount of MH71 or MH71 L3 construct. For panels A and B, identical results were obtained when lysates were prepared on days 1 and 2 posttransfection. (C) Gag proteins of sucrose-cushion purified vector particles from the wild-type and L-domain mutant vector preparations. A reduced amount of Gag is seen in the mutant vector preparations consistent with the reduced titer. In the case of the L3 mutant, only unprocessed Gag is detected in the culture supernatant.

FIG. 4.

Effect of the dominant negative Vps4, Tsg101 or AIP-1/ALIX proteins on release of PFV virus or vector. Hatched bars indicate titers of virus produced from 293T cells transfected with pczHSRV2 (wild type or mutant), solid bars show titers of vector from cells transfected with MH71 (wild type or mutant). All vector constructs were cotransfected with pczHFVenv. In all cases, negative controls were also cotransfected with pCR3.1GFP or pDSRED-C1. (A) Effect of expression of dominant negative Vps4 (VPS4-223 M-GFP). (B) Expression of dominant negative Vps4B/SKD1 E228Q. (C) Expression of dominant negative truncated Tsg101 (pCR3.1-YFP-Tsg101 [1-157]). (D) Expression of dominant negative truncated AIP-1/ALIX [pCR3.1-YFP-ALIX(d1-176)].

FIG. 5.

EM analysis of 293T cells transfected with PFV vector constructs. (A) Wild-type PFV vector constructs. (B) Wild-type PFV vector constructs cotransfected with dominant negative Vps4.