The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies

Abstract

By using a reverse genetics system that is based on the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we have identified the arenavirus small RING finger Z protein as the main driving force of virus budding. Both LCMV and Lassa fever virus (LFV) Z proteins exhibited self-budding activity, and both substituted efficiently for the late domain that is present in the Gag protein of Rous sarcoma virus. LCMV and LFV Z proteins contain proline-rich motifs that are characteristic of late domains. Mutations in the PPPY motif of LCMV Z severely impaired the formation of virus-like particles. LFV Z contains two different proline-rich motifs, PPPY and PTAP, which are separated by eight amino acids. Mutational analysis revealed that both motifs are required for efficient LFV Z-mediated budding. Both LCMV and LFV Z proteins recruited to the plasma membrane Tsg101, which is a component of the class E vacuolar protein sorting machinery that has been implicated in budding of HIV and Ebola virus. Targeting of Tsg101 by RNA interference caused a strong reduction in Z-mediated budding. These results indicate that Z is the arenavirus functional counterpart of the matrix proteins found in other negative strand enveloped RNA viruses. Moreover, members of the vacuolar protein sorting pathway appear to play an important role in arena-virus budding. These findings open possibilities for antiviral strategies to combat LFV and other hemorrhagic fever arenaviruses.

Fig. 1.

LCMV Z is strictly required for production of VLP. (A) CAT expression by an LCMV MG in the presence of different combinations of LCMV proteins. Nonacetylated (NAc) and monoacetylated (MAc) chloramphenicol. (B) Release of vesicles containing Z protein. VLPs present in SP of transfected cells were harvested by ultracentrifugation through a 20% sucrose cushion and were analyzed by Western blotting. (C) Detection by RT-PCR of MG RNA associated with VLPs, which are present in the SP of transfected cells.

Fig. 2.

Z has features of a bona fide budding protein. (A) Z has self-budding activity. Z was immunoprecipitated from the SP of transfected cells, and was metabolically labeled with [³⁵S]Met/[³⁵S]Cys. As a control, cells were transfected with a plasmid encoding for LCMV NPs. (B) Z can substitute for the RSV L domain. Proteins present in lysates and SP of cells transfected with the indicated construct and metabolically labeled with [³⁵S]Met/[³⁵S]Cys were immunoprecipitated to detect Gag cleavage products. (C) Comparison of the proline-rich motifs present in arenavirus Z and in different viral Gag and M proteins.

Fig. 3.

The PPPY motif is responsible for the budding activity of LCMV Z. (A) Sequence and expression of Z proteins with mutated PPPY motifs is shown. Identity is represented by a dashed line. Proteins were detected by Western blot by using an α-HA antibody. (B) Mutant proteins had no effect on MG-derived CAT activity. (C) Mutations in the PPPY motif caused a dramatic reduction in VLP production. MG RNA associated with VLP was amplified by RT-PCR. (D) Z-containing mutations in the PPPY failed to restore the budding properties of RSV Gag protein lacking its L domain. Proteins present in the lysates and SP of transfected cells were analyzed by IPP. CA, capsid protein; PR, protease.

Fig. 4.

Both PTAP and PPPY motifs are required for efficient budding mediated by LFV Z. (A) LFV Z has self-budding activity. IPP of LFV Z from the SP of transfected cells labeled with [³⁵S]Met/[³⁵S]Cys. (B) LFV Z can functionally substitute for the L domain of RSV Gag. IPP of proteins present in the lysates and SP of transfected cells revealed the presence of Gag cleavage products. (C) LFV Z can efficiently substitute for LCMV Z in formation of infectious VLPs. (D) The sequence and expression of LFV Z with mutated proline-rich motifs. Mutated residues are gray. Intact motifs are represented by a box. Proteins were detected by Western blot by using an α-HA antibody. (E) Mutations in both the PTAP and the PPPY motif impaired production of VLP. MG RNA associated with VLPs was amplified by RT-PCR.

Fig. 5.

Wild-type and mutant LCMV and LFV Z proteins have the same pattern of intracellular distribution when expressed in BHK-21 cells. AAPA, Z proteins whose PPPY domain has been mutated to AAPA; LTAL, Z proteins whose PTAP motif has been mutated to LTAL.

Fig. 6.

(A) Z coexpression of LCMV Z-HA (red) and Tsg101-myc (green) in BHK-21 cells results in colocalization of both proteins. (B) Transfection of a plasmid that directs the synthesis of siRNA specific for EGFP (pS-EGFP) resulted in decreased levels of EGFP protein, as detected by Western blot or direct fluorescence. (C) Cotransfection of cells with plasmids Tsg101-myc and pS-Tsg101 causes a severe reduction in the expression levels of Tsg101-myc protein. (D) Intracellular expression of siRNA specific for Tsg101 results in impaired VLP formation.