RetroCHMP3 blocks budding of enveloped viruses without blocking cytokinesis.

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.

Paramyxovirus-Like Particles as Protein Delivery Vehicles.

We have developed a flexible platform for delivery of proteins to target cell interiors using paramyxovirus-like particles. The key enabling feature is an appendage, 15 to 30 amino acid residues in length, that is added to cargo proteins and that induces them to bind to the viral matrix (M) protein during virus-like particle (VLP) assembly. The cargo is then incorporated within the VLPs as they bud, using the same interactions that normally direct viral genome packaging. The appendage can also serve as an epitope tag for cargo detection using a nucleocapsid (NP) protein-specific monoclonal antibody. Using this approach, we generated Renilla luciferase-loaded VLPs, green fluorescent protein-loaded VLPs, superoxide dismutase-loaded VLPs, and Cre recombinase-loaded VLPs. In each case, the VLPs could efficiently deliver their functional cargos to target cells and, in the case of Cre recombinase, to target cell nuclei. The strategy was employed using two different VLP production platforms, one based on parainfluenza virus 5 (PIV5) and the other based on Nipah virus, and in both cases efficient cargo packaging and delivery could be achieved. These findings provide a foundation for development of paramyxovirus-like particles as tools for safe and efficient delivery of therapeutic proteins to cells and tissues. IMPORTANCE Therapeutic proteins including transcription factors and genome editors have enormous clinical potential but are currently limited in part due to the challenges of safely and efficiently delivering these proteins to the interiors of target cells. Here, we have developed a new strategy for protein delivery based on manipulation of paramyxovirus genome packaging interactions.

Angiomotin-Like 1 Links Paramyxovirus M Proteins to NEDD4 Family Ubiquitin Ligases.

To define the links between paramyxovirus budding and cellular ESCRT machinery, we previously identified angiomotin-like 1 (AMOTL1) in a screen for host factors that bind to the matrix (M) protein of parainfluenza virus 5 (PIV5). This protein harbors three L/PPXY sequences, allowing it to interact with WW domain containing proteins including NEDD4 family members. We hypothesize that paramyxoviruses use AMOTL1 as a linker to indirectly recruit the same NEDD4 ubiquitin ligases for budding that other enveloped viruses recruit directly through their PPXY late domains. In support of this hypothesis, we found that AMOTL1 could link together M proteins and NEDD4 family proteins in three-way co-IP experiments. Both PIV5 and mumps virus M proteins could be linked to the NEDD4 family proteins NEDD4-1, NEDD4L, and NEDL1, provided that AMOTL1 was co-expressed as a bridging protein. AMOT and AMOTL2 could not substitute for AMOTL1, as they lacked the ability to bind with paramyxovirus M proteins. Attachment of a PPXY late domain sequence to PIV5 M protein obviated the need for AMOTL1 as a linker between M and NEDD4 proteins. Together, these results suggest a novel host factor recruitment strategy for paramyxoviruses to achieve particle release.

Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles.

UNLABELLED: Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE: Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools.

Role of ubiquitin in parainfluenza virus 5 particle formation.

Ubiquitin is important for the budding of many retroviruses and other enveloped viruses, but the precise role of ubiquitin in virus budding remains unclear. Here, we characterized the ubiquitination of the matrix (M) protein of a paramyxovirus, parainfluenza virus 5 (PIV5). The PIV5 M protein (but not the PIV5 nucleocapsid protein) was found to be targeted for monoubiquitination in transfected mammalian cells. Major sites of ubiquitin attachment identified by mass spectrometry analysis were lysine residues at amino acid positions 79/80, 130, and 247. The cumulative mutation of lysine residues 79, 80, and 130 to arginines led to an altered pattern of M protein ubiquitination and impaired viruslike particle (VLP) production. However, the cumulative mutation of lysine residues 79, 80, 130, and 247 to arginines restored M protein ubiquitination and VLP production, suggesting that ubiquitin is attached to alternative sites on the M protein when the primary ones have been removed. Additional lysine residues were targeted for mutagenesis based on the UbiPred algorithm. An M protein with seven lysine residues changed to arginines exhibited altered ubiquitination and poor VLP production. A recombinant virus encoding an M protein with seven lysines mutated was generated, and this virus exhibited a 6-fold-reduced maximum titer, with the defect being attributed mainly to the budding of noninfectious particles. The recombinant virus was assembly deficient, as judged by the redistribution of viral M and hemagglutinin-neuraminidase proteins in infected cells. Similar assembly defects were observed for the wild-type (wt) virus after treatment with a proteasome inhibitor. Collectively, these findings suggest that the monoubiquitination of the PIV5 M protein is important for proper virus assembly and for the budding of infectious particles.

The C-terminal end of parainfluenza virus 5 NP protein is important for virus-like particle production and M-NP protein interaction.

Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.

Mumps virus matrix, fusion, and nucleocapsid proteins cooperate for efficient production of virus-like particles.

Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells. To define mumps virus (MuV) proteins important for this process, viral proteins were expressed either singly or in combination in mammalian cells to produce virus-like particles (VLPs). Only the MuV matrix (M) protein when expressed by itself was capable of inducing particle release, but the quantity of these M-alone particles was very small. Efficient production of mumps VLPs occurred only when the M protein was coexpressed together with other viral proteins, with maximum production achieved upon coexpression of the viral M, nucleocapsid (NP), and fusion (F) proteins together. Electron microscopy analysis confirmed that VLPs were morphologically similar to MuV virions. The two MuV glycoproteins were not equal contributors to particle formation. The F protein was a major contributor to VLP production, while the hemagglutinin-neuraminidase protein made a smaller contribution. Evidence for the involvement of class E protein machinery in VLP budding was obtained, with mumps VLP production inhibited upon expression of dominant-negative versions of the class E proteins Vps4A and Chmp4b. Disruption of the sequence 24-FPVI-27 within the MuV M protein led to poor VLP production, consistent with findings of earlier studies of a related sequence, FPIV, important for the budding of parainfluenza virus 5. Together, these results demonstrate that different MuV structural proteins cooperate together for efficient particle production and that particle budding likely involves host class E protein machinery.

A single amino acid residue change in the P protein of parainfluenza virus 5 elevates viral gene expression.

Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA species through a process of pseudotemplated insertion of two G residues at a specific site during transcription, resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5 (rPIV5-CPI-) that exhibits elevated viral protein expression and induces production of cytokines, such as beta interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI- are due to mutations in the P protein and/or mutations in the V protein. To address this question, we used a minigenome system and recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that the P protein with six amino acid residue changes (Pcpi-) was more efficient than wild-type P in facilitating replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi-) still inhibits minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene expression in rPIV5-CPI- virus-infected cells can be attributed to a P protein with an increased ability to facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157 of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi-) is sufficient for elevated viral gene expression. Using mass spectrometry and (33)P labeling, we found that residue S157 of the P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at residue 157 plays an important role in regulating viral RNA replication.