Late Budding Domains Research Articles

The YPLGVG sequence of the Nipah virus matrix protein is required for budding

BackgroundNipah virus (NiV) is a recently emerged paramyxovirus capable of causing fatal disease in a broad range of mammalian hosts, including humans. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the family Paramyxoviridae. Recombinant expression systems have played a crucial role in studying the cell biology of these Biosafety Level-4 restricted viruses. Henipavirus assembly and budding occurs at the plasma membrane, although the details of this process remain poorly understood. Multivesicular body (MVB) proteins have been found to play a role in the budding of several enveloped viruses, including some paramyxoviruses, and the recruitment of MVB proteins by viral proteins possessing late budding domains (L-domains) has become an important concept in the viral budding process. Previously we developed a system for producing NiV virus-like particles (VLPs) and demonstrated that the matrix (M) protein possessed an intrinsic budding ability and played a major role in assembly. Here, we have used this system to further explore the budding process by analyzing elements within the M protein that are critical for particle release.ResultsUsing rationally targeted site-directed mutagenesis we show that a NiV M sequence YPLGVG is required for M budding and that mutation or deletion of the sequence abrogates budding ability. Replacement of the native and overlapping Ebola VP40 L-domains with the NiV sequence failed to rescue VP40 budding; however, it did induce the cellular morphology of extensive filamentous projection consistent with wild-type VP40-expressing cells. Cells expressing wild-type NiV M also displayed this morphology, which was dependent on the YPLGVG sequence, and deletion of the sequence also resulted in nuclear localization of M. Dominant-negative VPS4 proteins had no effect on NiV M budding, suggesting that unlike other viruses such as Ebola, NiV M accomplishes budding independent of MVB cellular proteins.ConclusionThese data indicate that the YPLGVG motif within the NiV M protein plays an important role in M budding; however, involvement of any specific components of the cellular MVB sorting pathway in henipavirus budding remains to be demonstrated. Further investigation of henipavirus assembly and budding may yet reveal a novel mechanism(s) of viral assembly and release that could be applicable to other enveloped viruses or have therapeutic implications.

Refined study of the interaction between HIV-1 p6 late domain and ALIX

The interaction between the HIV-1 p6 late budding domain and ALIX, a class E vacuolar protein sorting factor, was explored by using the yeast two-hybrid approach. We refined the ALIX binding site of p6 as being the leucine triplet repeat sequence (Lxx)4 (LYPLTSLRSLFG). Intriguingly, the deletion of the C-terminal proline-rich region of ALIX prevented detectable binding to p6. In contrast, a four-amino acid deletion in the central hinge region of p6 increased its association with ALIX as shown by its ability to bind to ALIX lacking the proline rich domain. Finally, by using a random screening approach, the minimal ALIX391–510 fragment was found to specifically interact with this p6 deletion mutant. A parallel analysis of ALIX binding to the late domain p9 from EIAV revealed that p6 and p9, which exhibit distinct ALIX binding motives, likely bind differently to ALIX. Altogether, our data support a model where the C-terminal proline-rich domain of ALIX allows the access of its binding site to p6 by alleviating a conformational constraint resulting from the presence of the central p6 hinge.

The Role of Ubiquitin in Retroviral Egress

HIV and many other enveloped viruses encode a late budding domain (L-domain) that recruits the cellular machinery that mediates the separation of the nascent virion from the infected cell. The ubiquitin-proteasome system has been implicated in the L-domain activity, but the exact role of ubiquitin transfer and ubiquitin-binding proteins in the last step of viral replication remains elusive. It is now widely accepted that the class E vacuolar protein sorting pathway mediates both viral budding and vesicle budding into the multivesicular bodies and, remarkably, both budding events share the same topology and similar requirements for ubiquitin. In this review, the role of ubiquitin in viral budding is discussed in the light of recent advances in the understanding of the cellular mechanisms that assist the last step of HIV-1 release.

HIV-1 Vpu Promotes Release and Prevents Endocytosis of Nascent Retrovirus Particles from the Plasma Membrane

The human immunodeficiency virus (HIV) type-1 viral protein U (Vpu) protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV) Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP) were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes.

Functional Analysis of Late-Budding Domain Activity Associated with the PSAP Motif within the Vesicular Stomatitis Virus M Protein

A PPPY motif within the M protein of vesicular stomatitis virus (VSV) functions as a late-budding domain (L-domain); however, L-domain activity has yet to be associated with a downstream PSAP motif. VSV recombinants with mutations in the PPPY and/or PSAP motif were recovered by reverse genetics and examined for growth kinetics, plaque size, and budding efficiency by electron microscopy. Results indicate that unlike the PPPY motif, the PSAP motif alone does not possess L-domain activity. Finally, the insertion of the human immunodeficiency virus type 1 p6 L-domain and flanking sequences into the PSAP region of M protein rescued budding of a PPPY mutant of VSV to wild-type levels.

Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins.

The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs. PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I). However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I. Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein-protein interactions. In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1. Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner. Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function. Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery.

Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4.

The VP40 protein of Ebola virus can bud from mammalian cells in the form of lipid-bound, virus-like particles (VLPs), and late budding domains (L-domains) are conserved motifs (PTAP, PPxY, or YxxL; where "x" is any amino acid) that facilitate the budding of VP40-containing VLPs. VP40 is unique in that potential overlapping L-domains with the sequences PTAP and PPEY are present at amino acids 7 to 13 of VP40 (PTAPPEY). L-domains are thought to function by interacting with specific cellular proteins, such as the ubiquitin ligase Nedd4, and a component of the vacuolar protein sorting (vps) pathway, tsg101. Mutational analysis of the PTAPPEY sequence of VP40 was performed to understand further the contribution of each individual motif in promoting VP40 budding. In addition, the contribution of tsg101 and a second member of the vps pathway, vps4, in facilitating budding was addressed. Our results indicate that (i) both the PTAP and PPEY motifs contribute to efficient budding of VP40-containing VLPs; (ii) PTAP and PPEY can function as L-domains when separated and moved from the N terminus (amino acid position 7) to the C terminus (amino acid position 316) of full-length VP40; (iii) A VP40-PTAP/tsg101 interaction recruits tsg101 into budding VLPs; (iv) a VP40-PTAP/tsg101 interaction recruits VP40 into lipid raft microdomains; and (v) a dominant-negative mutant of vps4 (E228Q), but not wild-type vps4, significantly inhibited the budding of Ebola virus (Zaire). These results provide important insights into the complex interplay between viral and host proteins during the late stages of Ebola virus budding.

Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction.

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.