Virus Maturation by Budding

Enveloped viruses mature by budding at cellular membranes. It has been generally thought that this process is driven by interactions between the viral transmembrane proteins and the internal virion components (core, capsid, or nucleocapsid). This model was particularly applicable to alphaviruses, which require both spike proteins and a nucleocapsid for budding. However, genetic studies have clearly shown that the retrovirus core protein, i.e., the Gag protein, is able to form enveloped particles by itself. Also, budding of negative-strand RNA viruses (rhabdoviruses, orthomyxoviruses, and paramyxoviruses) seems to be accomplished mainly by internal components, most probably the matrix protein, since the spike proteins are not absolutely required for budding of these viruses either. In contrast, budding of coronavirus particles can occur in the absence of the nucleocapsid and appears to require two membrane proteins only. Biochemical and structural data suggest that the proteins, which play a key role in budding, drive this process by forming a three-dimensional (cage-like) protein lattice at the surface of or within the membrane. Similarly, recent electron microscopic studies revealed that the alphavirus spike proteins are also engaged in extensive lateral interactions, forming a dense protein shell at the outer surface of the viral envelope. On the basis of these data, we propose that the budding of enveloped viruses in general is governed by lateral interactions between peripheral or integral membrane proteins. This new concept also provides answers to the question of how viral and cellular membrane proteins are sorted during budding. In addition, it has implications for the mechanism by which the virion is uncoated during virus entry.     

Spike protein-nucleocapsid interactions drive the budding of alphaviruses.

Semliki Forest virus (SFV) particles are released from infected cells by budding of nucleocapsids through plasma membrane regions that are modified by virus spike proteins. The budding process was studied with recombinant SFV genomes which lacked the nucleocapsid protein gene or, alternatively, the spike genes. No subviral particles were released from cells which expressed only the nucleocapsid protein or the spike proteins. Virus release was found to be strictly dependent on the coexpression of the nucleocapsid and the spike proteins. These results provide direct proof for the hypothesis that the alphavirus budding is driven by nucleocapsid-spike interactions. The importance of the viral 42S RNA for virus assembly and budding was investigated by using the heterologous vaccinia virus-T7 expression system for the synthesis of the SFV structural proteins. The results demonstrate that the viral genome is not absolutely required for formation of budding competent nucleocapsids, since small amounts of viruslike particles were assembled in the absence of 42S RNA.     

Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles

For influenza virus, we developed an efficient, noncytotoxic, plasmid-based virus-like particle (VLP) system to reflect authentic virus particles. This system was characterized biochemically by analysis of VLP protein composition, morphologically by electron microscopy, and functionally with a VLP infectivity assay. The VLP system was used to address the identity of the minimal set of viral proteins required for budding. Combinations of viral proteins were expressed in cells, and the polypeptide composition of the particles released into the culture media was analyzed. Contrary to previous findings in which matrix (M1) protein was considered to be the driving force of budding because M1 was found to be released copiously into the culture medium when M1 was expressed by using the vaccinia virus T7 RNA polymerase-driven overexpression system, in our noncytotoxic VLP system M1 was not released efficiently into the culture medium. Additionally, hemagglutinin (HA), when treated with exogenous neuraminidase (NA) or coexpressed with viral NA, could be released from cells independently of M1. Incorporation of M1 into VLPs required HA expression, although when M1 was omitted from VLPs, particles with morphologies similar to those of wild-type VLPs or viruses were observed. Furthermore, when HA and NA cytoplasmic tail mutants were included in the VLPs, M1 failed to be efficiently incorporated into VLPs, consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft microdomains and recruiting the internal viral core components. VLP formation also occurred independently of the function of Vps4 in the multivesicular body pathway, as dominant-negative Vps4 proteins failed to inhibit influenza VLP budding.     

A tyrosine-based motif in the cytoplasmic domain of the alphavirus envelope protein is essential for budding.

The budding of enveloped viruses from cellular membranes is believed to be dependent on the specific interaction between transmembrane spike proteins and cytoplasmic core components of the virus. We found that the cytoplasmic domain of the E2 transmembrane spike glycoprotein of Semliki Forest virus contains two essential determinants which are absolutely needed for budding. The first constitutes a single tyrosine residue in the context of a direct pentapeptide repeat. The tyrosine could only partially be substituted for other residues with aromatic or bulky hydrophobic side chains, although these immediately reverted to the original genotype. The second determinant involves palmitylated cysteine residues flanking the tyrosine repeat motif. The function of these is probably to anchor the tail against the inner surface of the membrane so that the tyrosine-containing motif is properly presented to the nucleocapsid. This is the first example where a membrane virus employs a tyrosine signal for the selective incorporation of spike proteins into budding structures.     

'Genome gating'; polarized intranuclear trafficking of influenza virus RNPs

Many viruses exploit cellular polarity to constrain the assembly and release of progeny virions to a desired surface. Influenza virus particles are released only from the apical surface of epithelial cells and this polarization is partly owing to specific targeting of the viral membrane proteins to the apical plasma membrane. The RNA genome of the virus is transcribed and replicated in the nucleus, necessitating nuclear export of the individual ribonucleoprotein (RNP) segments before they can be incorporated into budding virus particles. We show that the process of polarized virus assembly begins in the nucleus with the RNPs adopting a novel asymmetric distribution at the inner nuclear membrane prior to their export to the cytoplasm. The viral nucleoprotein, the major protein component of RNPs, displays the same polarized intranuclear distribution in the absence of other influenza virus components, suggesting the existence of a hitherto unrecognized polarity within the mammalian cell nucleus.     

Semliki forest virus budding: assay, mechanisms, and cholesterol requirement

All enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. We have developed a quantitative biochemical assay to monitor the budding of Semliki Forest virus (SFV), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. The assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using streptavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least 2 h, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continued exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed. The previously described cholesterol requirement in SFV exit was shown to be due to a block in budding in the absence of cholesterol and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells.     

Role of Matrix and Fusion Proteins in Budding of Sendai Virus

Paramyxoviruses are assembled at the surface of infected cells, where virions are formed by the process of budding. We investigated the roles of three Sendai virus (SV) membrane proteins in the production of virus-like particles. Expression of matrix (M) proteins from cDNA induced the budding and release of virus-like particles that contained M, as was previously observed with human parainfluenza virus type 1 (hPIV1). Expression of SV fusion (F) glycoprotein from cDNA caused the release of virus-like particles bearing surface F, although their release was less efficient than that of particles bearing M protein. Cells that expressed only hemagglutinin-neuraminidase (HN) released no HN-containing vesicles. Coexpression of M and F proteins enhanced the release of F protein by a factor greater than 4. The virus-like particles containing F and M were found in different density gradient fractions of the media of cells that coexpressed M and F, a finding that suggests that the two proteins formed separate vesicles and did not interact directly. Vesicles released by M or F proteins also contained cellular actin; therefore, actin may be involved in the budding process induced by viral M or F proteins. Deletion of C-terminal residues of M protein, which has a sequence similar to that of an actin-binding domain, significantly reduced release of the particles into medium. Site-directed mutagenesis of the cytoplasmic tail of F revealed two regions that affect the efficiency of budding: one domain comprising five consecutive amino acids conserved in SV and hPIV1 and one domain that is similar to the actin-binding domain required for budding induced by M protein. Our results indicate that both M and F proteins are able to drive the budding of SV and propose the possible role of actin in the budding process.     

Mechanisms for generating cell membrane antigens that are recognized by cytotoxic T lymphocytes

Studies with many viruses have revealed that viral specific protein synthesis is an obligatory step in generating antigens on target cells for antiviral cytotoxic T lymphocytes. This has been most clearly demonstrated with DI particles, virions that are structurally complete but lack infectious RNA. Adsorption of such particles onto target cell membranes does not render these cells susceptible to lytic attack by antiviral effector cells, unless some viral protein synthesis transpires. However, some viruses, such as Sendai virus, circumvent the requirement for viral protein synthesis via fusion of the viral envelope with the target cell membrane, a process mediated by a specialized fusion protein. Once inserted into the lipid bilayer, it is likely that viral components and self H-2 noncovalently associate so that the complex can be recognized by antiviral cytotoxic T cells. This idea is supported by the demonstration that viral proteins and H-2 containing membrane proteins, incorporated into reconstituted membrane vesicles or liposomes are recognized by cytotoxic T cells. These data further show that native rather than altered viral and H-2 molecules are the moieties recognized. Associations between antigen and H-2 have been detected by a variety of techniques and in some cases are not random but selective; that is, viral antigens perferentially associate with some H-2 alleles and not others. In summary, these findings indicate that although viral antigens are present in the mature virions, these components are not recognized by antiviral killer cells until integrated into the plasma membrane. This may be achieved either through direct fusion of the viral envelope with the target cell or following viral protein synthesis and insertion of viral antigens into the plasma membrane.     

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.     

Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes.

Budding of enveloped viruses has been shown to be driven by interactions between a nucleocapsid and a proteolipid membrane. By contrast, we here describe the assembly of viral envelopes independent of a nucleocapsid. Membrane particles containing coronaviral envelope proteins were assembled in and released from animal cells co-expressing these proteins' genes from transfected plasmids. Of the three viral membrane proteins only two were required for particle formation, the membrane glycoprotein (M) and the small envelope protein (E). The spike (S) protein was dispensable but was incorporated when present. Importantly, the nucleocapsid protein (N) was neither required not taken into the particles when present. The E protein, recently recognized to be a structural protein, was shown to be an integral membrane protein. The envelope vesicles were found by immunogold labelling and electron microscopy to form a homogeneous population of spherical particles indistinguishable from authentic coronavirions in size (approximately 100 nm in diameter) and shape. They were less dense than virions and sedimented slightly slower than virions in sucrose velocity gradients. The nucleocapsid-independent formation of apparently bona fide viral envelopes represents a novel mode of virus assembly.     

Intracellular hepadnavirus nucleocapsids are selected for secretion by envelope protein-independent membrane binding

Hepadnaviruses are DNA viruses but, as pararetroviruses, their morphogenesis initiates with the encapsidation of an RNA pregenome, and these viruses have therefore evolved mechanisms to exclude nucleocapsids that contain incompletely matured genomes from participating in budding and secretion. We provide here evidence that binding of hepadnavirus core particles from the cytosol to their target membranes is a distinct step in morphogenesis, discriminating among different populations of intracellular capsids. Using the duck hepatitis B virus (DHBV) and a flotation assay, we found about half of the intracellular capsids to be membrane associated due to an intrinsic membrane-binding affinity. In contrast to free cytosolic capsids, this subpopulation contained largely mature, double-stranded DNA genomes and lacked core protein hyperphosphorylation, both features characteristic for secreted virions. Against expectation, however, the selective membrane attachment observed did not require the presence of the large DHBV envelope protein, which has been considered to be crucial for nucleocapsid-membrane interaction. Furthermore, removal of surface-exposed phosphate residues from nonfloating capsids by itself did not suffice to confer membrane affinity and, finally, hyperphosphorylation was absent from nonenveloped nucleocapsids that were released from DHBV-transfected cells. Collectively, these observations argue for a model in which nucleocapsid maturation, involving the viral genome, capsid structure, and capsid dephosphorylation, leads to the exposure of a membrane-binding signal as a step crucial for selecting the matured nucleocapsid to be incorporated into the capsid-independent budding of virus particles.     

More than one door - Budding of enveloped viruses through cellular membranes

Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail.     

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.     

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.     

Mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus.

Pseudotypes of vesicular stomatitis virus (VSV) and Moloney murine leukemia virus (MuLV), defined by their resistance to neutralization by anti-VSV antiserum, are released preferentially at early times after infection of MuLV-producing cells with VSV. At later times, after synthesis of MuLV proteins has been inhibited by the VSV infection, neither MuLV virions nor the VSV (MuLV) pseudotypes are made. Infection of MuLV-producing cells with mutants of VSV having temperature-sensitive lesions in either G or M protein does not generate pseudotypes at nonpermissive temperature, indicating that both proteins are needed for pseudotypes to form. Although the pseudotypes resist neutralization by anti-VSV serum, they are inactivated by anti-VSV serum plus complement, and they can be precipitated by rabbit anti-VSV serum plus goat anti-rabbit IgG. These results, coupled with experiments using a temperature-sensitive mutant of VSV G protein grown at partly restrictive temperature, suggest that small numbers of VSV G protein are obligately incorporated into VSV(MuLV) pseudotypes. There appears to be a stringent requirement for recognition of the viral core by homologous envelope components as the nucleating step in the budding process. Only after such a nucleation can the envelope components of the second virus substitute into the membrane of the budding particle.     

Morphogenesis of hepatitis B virus and its subviral envelope particles

After cell hijacking and intracellular amplification, non-lytic enveloped viruses are usually released from the infected cell by budding across internal membranes or through the plasma membrane. The enveloped human hepatitis B virus (HBV) is an example of virus using an intracellular compartment to form new virions. Four decades after its discovery, HBV is still the primary cause of death by cancer due to a viral infection worldwide. Despite numerous studies on HBV genome replication little is known about its morphogenesis process. In addition to viral neogenesis, the HBV envelope proteins have the capability without any other viral component to form empty subviral envelope particles (SVPs), which are secreted into the blood of infected patients. A better knowledge of this process may be critical for future antiviral strategies. Previous studies have speculated that the morphogenesis of HBV and its SVPs occur through the same mechanisms. However, recent data clearly suggest that two different processes, including constitutive Golgi pathway or cellular machinery that generates internal vesicles of multivesicular bodies (MVB), independently form these two viral entities.     

M-X-I motif of semliki forest virus capsid protein affects nucleocapsid assembly

Alphavirus budding is driven by interactions between spike and nucleocapsid proteins at the plasma membrane. The binding motif, Y-X-L, on the spike protein E2 and the corresponding hydrophobic cavity on the capsid protein were described earlier. The spike-binding cavity has also been suggested to bind an internal hydrophobic motif, M113-X-I115, of the capsid protein. In this study we found that replacement of amino acids M113 and I115 with alanines, as single or double mutations, abolished formation of intracellular nucleocapsids. The mutants could still bud efficiently, but the NCs in the released virions were not stable after removal of the membrane and spike protein layer. In addition to wild-type spherical particles, elongated multicored particles were found at the plasma membrane and released from the host cell. We conclude that the internal capsid motif has a biological function in the viral life cycle, especially in assembly of nucleocapsids. We also provide further evidence that alphaviruses may assemble and bud from the plasma membrane in the absence of preformed nucleocapsids.     

Insertion of Capsid Proteins from Nonenveloped Viruses into the Retroviral Budding Pathway

Retroviral Gag proteins direct the assembly and release of virus particles from the plasma membrane. The budding machinery consists of three small domains, the M (membrane-binding), I (interaction), and L (late or "pinching-off") domains. In addition, Gag proteins contain sequences that control particle size. For Rous sarcoma virus (RSV), the size determinant maps to the capsid (CA)-spacer peptide (SP) sequence, but it functions only when I domains are present to enable particles of normal density to be produced. Small deletions throughout the CA-SP sequence result in the release of particles that are very large and heterogeneous, even when I domains are present. In this report, we show that particles of relatively uniform size and normal density are released by budding when the size determinant and I domains in RSV Gag are replaced with capsid proteins from two unrelated, nonenveloped viruses: simian virus 40 and satellite tobacco mosaic virus. These results indicate that capsid proteins of nonenveloped viruses can interact among themselves within the context of Gag and be inserted into the retroviral budding pathway merely by attaching the M and L domains to their amino termini. Thus, the differences in the assembly pathways of enveloped and nonenveloped viruses may be far simpler than previously thought.     

Overview of human endogenous retroviruses found in human genome assembly GRCh37/hg19

The late Nobel Laureate Sir Peter Medawar once memorably described viruses as ‘bad news wrapped in protein’. Virus assembly in HIV is a remarkably well coordinated process in which the virus achieves extracellular budding using primarily intracellular budding machinery and also the unusual phenomenon of export from the cell of an RNA. Recruitment of the ESCRT system by HIV is one of the best documented examples of the comprehensive way in which a virus hijacks a normal cellular process. This review is a summary of our current understanding of the budding process of HIV, from genomic RNA capture through budding and on to viral maturation, but centering on the proteins of the ESCRT pathway and highlighting some recent advances in our understanding of the cellular components involved and the complex interplay between the Gag protein and the genomic RNA.