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.

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.

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

UNLABELLED: Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE: Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections.

Paramyxovirus glycoprotein incorporation, assembly and budding: a three way dance for infectious particle production.

Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout.

Point mutations in the paramyxovirus F protein that enhance fusion activity shift the mechanism of complement-mediated virus neutralization.

Parainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.

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.

PIV5 M protein interaction with host protein angiomotin-like 1.

Paramyxovirus matrix (M) proteins organize virus assembly, functioning as adapters that link together viral ribonucleoprotein complexes and viral glycoproteins at infected cell plasma membranes. M proteins may also function to recruit and manipulate host factors to assist virus budding, similar to retroviral Gag proteins. By yeast two-hybrid screening, angiomotin-like 1 (AmotL1) was identified as a host factor that interacts with the M protein of parainfluenza virus 5 (PIV5). AmotL1-M protein interaction was observed in yeast, in transfected mammalian cells, and in virus-infected cells. Binding was mapped to a 83-amino acid region derived from the C-terminal portion of AmotL1. Overexpression of M-binding AmotL1-derived polypeptides potently inhibited production of PIV5 VLPs and impaired virus budding. Expression of these polypeptides moderately inhibited production of mumps VLPs, but had no effect on production of Nipah VLPs. siRNA-mediated depletion of AmotL1 protein reduced PIV5 budding, suggesting that this interaction is beneficial to paramyxovirus infection.

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.

Evidence for a new viral late-domain core sequence, FPIV, necessary for budding of a paramyxovirus.

Enveloped virus budding has been linked to both the ubiquitin-proteasome pathway and the vacuolar protein-sorting pathway of cells. We show here for the paramyxovirus SV5 that proteasome inhibitors and expression of dominant-negative VPS4(E228Q) ATPase blocks budding. The SV5 matrix (M) protein lacks previously defined late domains (e.g., P[T/S]AP, PPxY, YPDL) that recruit cellular factors. We identified a new motif for budding (core sequence FPIV) that can compensate functionally for lack of a PTAP late domain in budding human immunodeficiency virus type 1 virus-like particles (VLPs). Mutagenesis experiments suggest the more general sequence O-P-x-V. The proline residue was found to be critically important for function of this sequence, as substitution of this proline in the SV5 M protein resulted in poor budding of SV5 VLPs and failure of recombinant SV5 virus to replicate normally. Adaptation of mutant virus occurred rapidly, resulting in new proline residues elsewhere in the M protein. We hypothesize that these proline residues act to partially restore virus budding by generation of new motifs that act as suboptimal late domains.

Roles for the cytoplasmic tails of the fusion and hemagglutinin-neuraminidase proteins in budding of the paramyxovirus simian virus 5.

The efficient release of many enveloped viruses from cells involves the coalescence of viral components at sites of budding on the plasma membrane of infected cells. This coalescence is believed to require interactions between the cytoplasmic tails of surface glycoproteins and the matrix (M) protein. For the paramyxovirus simian virus 5 (SV5), the cytoplasmic tail of the hemagglutinin-neuraminidase (HN) protein has been shown previously to be important for normal virus budding. To investigate a role for the cytoplasmic tail of the fusion (F) protein in virus assembly and budding, we generated a series of F cytoplasmic tail-truncated recombinant viruses. Analysis of these viruses in tissue culture indicated that the cytoplasmic tail of the F protein was dispensable for normal virus replication and budding. To investigate further the requirements for assembly and budding of SV5, we generated two double-mutant recombinant viruses that lack 8 amino acids of the predicted 17-amino-acid HN protein cytoplasmic tail in combination with truncation of either 10 or 18 amino acids from the predicted 20-amino-acid F protein cytoplasmic tail. Both of the double mutant recombinant viruses displayed a replication defect in tissue culture and a budding defect, the extent of which was dependent on the length of the remaining F cytoplasmic tail. Taken together, this work and our earlier data on virus-like particle formation (A. P. Schmitt, G. P. Leser, D. L. Waning, and R. A. Lamb, J. Virol. 76:3953-3964, 2002) suggest a redundant role for the cytoplasmic tails of the HN and F proteins in virus assembly and budding.

Requirements for budding of paramyxovirus simian virus 5 virus-like particles.

Enveloped viruses are released from infected cells after coalescence of viral components at cellular membranes and budding of membranes to release particles. For some negative-strand RNA viruses (e.g., vesicular stomatitis virus and Ebola virus), the viral matrix (M) protein contains all of the information needed for budding, since virus-like particles (VLPs) are efficiently released from cells when the M protein is expressed from cDNA. To investigate the requirements for budding of the paramyxovirus simian virus 5 (SV5), its M protein was expressed in mammalian cells, and it was found that SV5 M protein alone could not induce vesicle budding and was not secreted from cells. Coexpression of M protein with the viral hemagglutinin-neuraminidase (HN) or fusion (F) glycoproteins also failed to result in significant VLP release. It was found that M protein in the form of VLPs was only secreted from cells, with an efficiency comparable to authentic virus budding, when M protein was coexpressed with one of the two glycoproteins, HN or F, together with the nucleocapsid (NP) protein. The VLPs appeared similar morphologically to authentic virions by electron microscopy. CsCl density gradient centrifugation indicated that almost all of the NP protein in the cells had assembled into nucleocapsid-like structures. Deletion of the F and HN cytoplasmic tails indicated an important role of these cytoplasmic tails in VLP budding. Furthermore, truncation of the HN cytoplasmic tail was found to be inhibitory toward budding, since it prevented coexpressed wild-type (wt) F protein from directing VLP budding. Conversely, truncation of the F protein cytoplasmic tail was not inhibitory and did not affect the ability of coexpressed wt HN protein to direct the budding of particles. Taken together, these data suggest that multiple viral components, including assembled nucleocapsids, have important roles in the paramyxovirus budding process.