A GFP-tagged version of the pseudorabies virus protein UL56 localizes to the Golgi and trans-Golgi network through a predicted C-terminal leucine-rich helix in transfected cells

Abstract

Background Pseudorabies virus (PRV) protein UL56 (pUL56) has been implicated in viral dissemination and virulence in vivo. However, the properties of PRV pUL56 remain largely unknown. In the present study, we aim to investigate the subcellular localization of pUL56 and the underlying molecular basis in transfected cells. Methods Constructs of N-terminal green fluorescent protein (GFP) fused pUL56 and its truncations were employed for investigating subcellular localization and further identifying amino acids crucial for pUL56 localization in transfected Vero cells. Finally, the identified amino acids were replaced with alanine for confirming if these mutations could impair the specific localization of pUL56. Results The pUL56 predominantly localized at the Golgi and trans -Golgi network (TGN) through its predicted C-terminal transmembrane helix in transfected Vero cells. A Golgi-associated protein Rab6a, independent of interaction with pUL56, was significantly downregulated by pUL56. Further, we found three truncated pUL56 C-terminal fragments (174–184, 175–185 and 191–195) could restrict GFP in the perinuclear region, respectively. Within these truncations, the 174 proline (P), 181 leucine (L), 185 L and 191 L were essential for maintaining perinuclear accumulation, thus suggesting an important role of leucine. Alanine (A) mutagenesis assays were employed to generate a series of pUL56 C-terminal mutants on the basis of leucine. Finally, a pUL56 mutant M10 ( 174 P/A- 177 L/A- 181 L/A- 185 L/A- 191 L/A- 194 L/A- 195 I/A- 196-197 L/A- 200 L/A) lost Golgi-TGN localization. Thus, our data revealed that the leucine-rich transmembrane helix was responsible for pUL56 Golgi-TGN localization and retention, probably through specific intracellular membrane insertion. Conclusion Our data indicated that the C-terminal transmembrane helix was responsible for the Golgi-TGN localization of pUL56. In addition, the leucines within C-terminal transmembrane helix were essential for maintaining pUL56 Golgi-TGN retention in cells. Further, the pUL56 can induce downregulation of Golgi-associated protein Rab6a. Electronic supplementary material The online version of this article (10.1186/s12985-019-1191-z) contains supplementary material, which is available to authorized users.

Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection

“…We recently indicated that PRV pUL56 primarily localized to the Golgi complex and tans-Golgi network (TGN) [13]. Thus, we attempted to examine whether the interaction of pUL56 and BAG3 could specifically target BAG3 to the Golgi-TGN.…”

“…Given an important role of the pUL56 C-terminus in Golgi−TGN targeting and retention [13], it was essential to clarify the relationship between specific Golgi localization and BAG3 degradation induced by pUL56. Subsequently, the pCMV-HA-mBAG3 was co-transfected with pAcGFP, Figure 3.…”

“…The pUL56 was subsequently truncated into three segments S1 (1−67 aa), S2 (68−134 aa), and S3 (135−207 aa) throughout a full length sequence ( Figure 4C) [13]. The pCMV-HA-mBAG3 was co-transfected with pAcGFP, pAcGFP-UL56 S1, S2, or S3 (1 µg per plasmid) into HEK293T cells, respectively.…”

“…Given an important role of the pUL56 C-terminus in Golgi−TGN targeting and retention [13], it was essential to clarify the relationship between specific Golgi localization and BAG3 degradation induced by pUL56. Subsequently, the pCMV-HA-mBAG3 was co-transfected with pAcGFP, pAcGFP-pUL56, or pAcGFP-pUL56 M13 (encoding a pUL56 C-terminal mutant that has lost Golgi localization and retention), respectively ( Figure 4D) [13]. At 48 hpt, the relative levels of HA-mBAG3/β-actin were determined in each group.…”

“…PRV type II membrane protein UL56 (pUL56) has four PPxY motifs [13], which represents a potential BAG3 interactor. Recently, PRV pUL56 has been identified as a virulence-associated factor that contributes to viral dissemination in the rodent nervous system [14].…”