Formation Of Virus-like Particles Research Articles

Investigation of Lipid‐Based Assembly of Viral Particles

Sustainment of viral infections from HIV, Ebola and the Human T‐Cell Leukemia Virus (HTLV) requires assembly at the host cell membrane to replicate via budding into new infectious viral particles. The assembly process requires lipid binding by viral matrix proteins as well as hijacking of host cell machinery to complete the budding and replication process. Currently, there is a paucity of information on the molecular architecture of this process including how lipids in the plasma membrane regulate the budding process. To analyze the lipids present when a virus buds from the plasma membrane we have begun a comprehensive and interdisciplinary investigation of the budding process. We have investigated the lipid binding and cellular targeting mechanisms of viral matrix proteins to understand their role in targeting the plasma membrane bud site. Next, we purified virus like particles (VLPs) from a number of infected human host cells to assess their lipid composition. The lipid compositions of these strains were then compared to the lipid composition of the plasma membrane of host cells via lipid mass spectrometry. Lastly, we performed a variety of mutagenic and lipid signaling assays to assess the role of different cofactors in VLP formation. Results elucidate the role of matrix proteins in viral budding and host cell lipid changes responsible for sustaining different types of viral infections.

Feline calicivirus VP2 is involved in the self-assembly of the capsid protein into virus-like particles

Feline calicivirus (FCV) is considered the most common upper respiratory tract disease (URTD) associated pathogen in cats. We previously expressed FCV VP1 capsid protein in insect cells by baculovirus system and we observed that this protein self-assemble into virus-like particles (VLPs) different in size and lacking the typical cup-like depressions of caliciviruses. In the present study, VP1 and the small basic structural protein VP2 of FCV were individually expressed by baculovirus system. Coinfection of insect cells with both recombinant viruses resulted in VP1 and VP2 self-assembly to form depressions similar to native capsids in size and appearance, demonstrating that VP2 interacts with the VP1 protein in the formation of VLPs.

A Role for the C Terminus of Mopeia Virus Nucleoprotein in Its Incorporation into Z Protein-Induced Virus-Like Particles

Arenaviruses are enveloped, negative-strand RNA viruses. For several arenaviruses, virus-like particle (VLP) formation requires the viral matrix Z protein. However, the mechanism by which viral ribonucleoprotein complexes are incorporated into virions is poorly understood. Here, we show that the expression of the Z protein and nucleoprotein (NP) of Mopeia virus, a close relative of the pathogenic Lassa virus, resulted in the highly selective incorporation of the NP protein into Z protein-induced VLPs. Moreover, the Z protein promoted the association of NP with cellular membranes, suggesting that the association of NP, Z, and the cellular membranes may facilitate the efficient incorporation of NP into VLPs. By employing a series of NP deletion constructs and testing their VLP incorporation, we further demonstrated an important role for the C-terminal half of NP in its incorporation into VLPs.

The Lack of an Inherent Membrane Targeting Signal Is Responsible for the Failure of the Matrix (M1) Protein of Influenza A Virus To Bud into Virus-Like Particles

The matrix protein (M1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M1 into virus particles. Here we explored the mechanisms that control M1 budding. Similarly to previous studies, we found that M1 by itself fails to form virus-like-particles (VLPs). We further demonstrated that M1, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane, where influenza virions bud. Remarkably, we showed that a 10-residue membrane targeting peptide from either the Fyn or Lck oncoprotein appended to M1 at the N terminus redirected M1 to the plasma membrane and allowed M1 particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 but not mutant M2 effectively targeted M1 to the plasma membrane and produced extracellular M1 VLPs. Our results suggest that influenza virus M1 may not possess an inherent membrane targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M1 in budding. This study highlights the fact that interactions of M1 with viral envelope proteins are essential to direct M1 to the plasma membrane for influenza virus particle release.

Efficient Budding of the Tacaribe Virus Matrix Protein Z Requires the Nucleoprotein

The Z protein has been shown for several arenaviruses to serve as the viral matrix protein. As such, Z provides the principal force for the budding of virus particles and is capable of forming virus-like particles (VLPs) when expressed alone. For most arenaviruses, this activity has been shown to be linked to the presence of proline-rich late-domain motifs in the C terminus; however, for the New World arenavirus Tacaribe virus (TCRV), no such motif exists within Z. It was recently demonstrated that while TCRV Z is still capable of functioning as a matrix protein to induce the formation of VLPs, neither its ASAP motif, which replaces a canonical PT/SAP motif in related viruses, nor its YxxL motif is involved in budding, leading to the suggestion that TCRV uses a novel budding mechanism. Here we show that in comparison to its closest relative, Junin virus (JUNV), TCRV Z buds only weakly when expressed in isolation. While this budding activity is independent of the ASAP or YxxL motif, it is significantly enhanced by coexpression with the nucleoprotein (NP), an effect not seen with JUNV Z. Interestingly, both the ASAP and YxxL motifs of Z appear to be critical for the recruitment of NP into VLPs, as well as for the enhancement of TCRV Z-mediated budding. While it is known that TCRV budding remains dependent on the endosomal sorting complex required for transport, our findings provide further evidence that TCRV uses a budding mechanism distinct from that of other known arenaviruses and suggest an essential role for NP in this process.

Viral Protein Determinants of Lassa Virus Entry and Release from Polarized Epithelial Cells

The epithelium plays a key role in the spread of Lassa virus. Transmission from rodents to humans occurs mainly via inhalation or ingestion of droplets, dust, or food contaminated with rodent urine. Here, we investigated Lassa virus infection in cultured epithelial cells and subsequent release of progeny viruses. We show that Lassa virus enters polarized Madin-Darby canine kidney (MDCK) cells mainly via the basolateral route, consistent with the basolateral localization of the cellular Lassa virus receptor alpha-dystroglycan. In contrast, progeny virus was efficiently released from the apical cell surface. Further, we determined the roles of the glycoprotein, matrix protein, and nucleoprotein in directed release of nascent virus. To do this, a virus-like-particle assay was developed in polarized MDCK cells based on the finding that, when expressed individually, both the glycoprotein GP and matrix protein Z form virus-like particles. We show that GP determines the apical release of Lassa virus from epithelial cells, presumably by recruiting the matrix protein Z to the site of virus assembly, which is in turn essential for nucleocapsid incorporation into virions.

P-body components are required for Ty1 retrotransposition during assembly of retrotransposition-competent virus-like particles.

Ty1 is a retrovirus-like retrotransposon whose replication is influenced by diverse cellular processes in Saccharomyces cerevisiae. We have identified cytoplasmic P-body components encoded by DHH1, KEM1, LSM1, and PAT1 as cofactors that posttranscriptionally enhance Ty1 retrotransposition. Using fluorescent in situ hybridization and immunofluorescence microscopy, we found that Ty1 mRNA and Gag colocalize to discrete cytoplasmic foci in wild-type cells. These foci, which are distinct from P-bodies, do not form in P-body component mutants or under conditions suboptimal for retrotransposition. Our immunoelectron microscopy (IEM) data suggest that mRNA/Gag foci are sites where virus-like particles (VLPs) cluster. Overexpression of Ty1 leads to a large increase in retrotransposition in wild-type cells, which allows VLPs to be detected by IEM. However, retrotransposition is still reduced in P-body component mutants under these conditions. Moreover, the percentage of Ty1 mRNA/Gag foci and VLP clusters and levels of integrase and reverse transcriptase are reduced in these mutants. Ty1 antisense RNAs, which have been reported to inhibit Ty1 transposition, are more abundant in the kem1Delta mutant and colocalize with Ty1 mRNA in the cytoplasm. Therefore, Kem1p may prevent the aggregation of Ty1 antisense and mRNAs. Overall, our results suggest that P-body components enhance the formation of retrotransposition-competent Ty1 VLPs.

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the (96)LPLGVA(101) sequence of eVP40 and the corresponding (84)LPLGIM(89) sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the (96)LPLGVA(101) motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the (96)LPLGVA(101) motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-DeltaLPLGVA failed to rescue the budding defective eVP40-DeltaLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-DeltaLPLGIM successfully rescued budding of mVP40-DeltaLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation

High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectious progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.

Efficient generation of cowpea mosaicvirus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants

To elucidate the mechanism of formation of cowpea mosaic virus (CPMV) particles, RNA-2-encoded precursor proteins were expressed in Spodoptera frugiperda cells. Processing of the 105K and 95K polyproteins in trans to give the mature Large (L) and Small (S) coat proteins required both the 32K proteinase cofactor and the 24K proteinase itself, while processing of VP60, consisting of the fused L-S protein, required only the 24K proteinase. Release of the L and S proteins resulted in the formation of virus-like particles (VLPs), showing that VP60 can act as a precursor of virus capsids. Processing of VP60 expressed in plants also led to efficient production of VLPs. Analysis of the VLPs produced by the action of the 24K proteinase on precursors showed that they were empty (RNA-free). This has important implications for the use of CPMV VLPs in biotechnology and nanotechnology as it will permit the use of noninfectious particles.

A catalytically and genetically optimized β-lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics

Nipah virus (NiV) and Hendra virus (HeV) are the only paramyxoviruses requiring Biosafety Level 4 (BSL-4) containment. Thus, study of henipavirus entry at less than BSL-4 conditions necessitates the use of cell-cell fusion or pseudotyped reporter virus assays. Yet, these surrogate assays may not fully emulate the biological properties unique to the virus being studied. Thus, we developed a henipaviral entry assay based on a β-lactamase-Nipah Matrix (βla-M) fusion protein. We first codon-optimized the bacterial βla and the NiV-M genes to ensure efficient expression in mammalian cells. The βla-M construct was able to bud and form virus-like particles (VLPs) that morphologically resembled paramyxoviruses. βla-M efficiently incorporated both NiV and HeV fusion and attachment glycoproteins. Entry of these VLPs was detected by cytosolic delivery of βla-M, resulting in enzymatic and fluorescent conversion of the pre-loaded CCF2-AM substrate. Soluble henipavirus receptors (ephrinB2) or antibodies against the F and/or G proteins blocked VLP entry. Additionally, a Y105W mutation engineered into the catalytic site of βla increased the sensitivity of our βla-M based infection assays by 2-fold. In toto, these methods will provide a more biologically relevant assay for studying henipavirus entry at less than BSL-4 conditions.

Functional analysis of the Bunyamwera orthobunyavirus Gc glycoprotein

The virion glycoproteins Gn and Gc of Bunyamwera orthobunyavirus (family Bunyaviridae) are encoded by the M RNA genome segment and have roles in both viral attachment and membrane fusion. To investigate further the structure and function of the Gc protein in viral replication, we generated 12 mutants that contain truncations from the N terminus. The effects of these deletions were analysed with regard to Golgi targeting, low pH-dependent membrane fusion, infectious virus-like particle (VLP) formation and virus infectivity. Our results show that the N-terminal half (453 residues) of the Gc ectodomain (909 residues in total) is dispensable for Golgi trafficking and cell fusion. However, deletions in this region resulted in a significant reduction in VLP formation. Four mutant viruses that contained N-terminal deletions in their Gc proteins were rescued, and found to be attenuated to different degrees in BHK-21 cells. Taken together, our data indicate that the N-terminal half of the Gc ectodomain is dispensable for replication in cell culture, whereas the C-terminal half is required to mediate cell fusion. A model for the domain structure of the Gc ectodomain is proposed.

Japanese encephalitis virus-based replicon RNAs/particles as an expression system for HIV-1 Pr55 Gag that is capable of producing virus-like particles

Ectopic expression of the structural protein Pr55 Gag of HIV-1 has been limited by the presence of inhibitory sequences in the gag coding region that must normally be counteracted by HIV-1 Rev and RRE. Here, we describe a cytoplasmic RNA replicon based on the RNA genome of Japanese encephalitis virus (JEV) that is capable of expressing HIV-1 gag without requiring Rev/RRE. This replicon system was constructed by deleting all three JEV structural protein-coding regions (C, prM, and E) from the 5′-proximal region of the genome and simultaneously inserting an HIV-1 gag expression cassette driven by the internal ribosome entry site of encephalomyocarditis virus into the 3′-proximal noncoding region of the genome. Transfection of this JEV replicon RNA led to expression of Pr55 Gag in the absence of Rev/RRE in the cytoplasm of hamster BHK-21, human HeLa, and mouse NIH/3T3 cells. Production of the Pr55 Gag derived from this JEV replicon RNA appeared to be increased by ∼3-fold when compared to that based on an alphavirus replicon RNA. Biochemical and morphological analyses demonstrated that the Pr55 Gag proteins were released into the culture medium in the form of virus-like particles. We also observed that the JEV replicon RNAs expressing the Pr55 Gag could be encapsidated into single-round infectious JEV replicon particles when transfected into a stable packaging cell line that provided the three JEV structural proteins in trans. This ectopic expression of the HIV-1 Pr55 Gag by JEV-based replicon RNAs/particles in diverse cell types may represent a useful molecular platform for various biological applications in medicine and industry.

Delivery of a foreign epitope by sharing amino acid residues with the carrier matrix

A broad range of structural viral proteins has the ability to assemble into virus-like particles (VLPs). Under the condition that modified subunits are still competent to assemble into VLPs, they are epitope delivery platforms suitable for vaccination purposes. The insertion of foreign sequences can be detrimental for the formation of chimeric VLPs as a result of misfolded subunit proteins. Hence, a strategy was adopted to screen for locations allowing the use of shared residues between the wildtype subunit sequence and the foreign insert. The insertion of a cysteine-containing sequence of hepatitis C virus (HCV) envelope protein 2 (E2) without adding an additional cysteine residue retained the ability of recombinant small hepatitis B surface antigen (HBsAg-S) to form secretion competent VLPs. A cysteine residue shared by the insert and the template protein avoided the formation of non-native disulfide bonds, and allowed the formation of VLPs. The chimeric HBsAg-S VLPs were similar to wildtype VLPs in density exposing the inserted foreign epitope and being immunogenic. Overall, the use of shared sequences between the insert and the subunit will facilitate the design of chimeric VLPs carrying multiple epitopes.

Assembly Properties of Human Immunodeficiency Virus Type 1 Gag-Leucine Zipper Chimeras: Implications for Retrovirus Assembly

Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant "Gag-Zipper" proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the "dimerizing" Gag-Zipper protein misassembles into very small particles, while the "trimerizing" protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the "dimerizing" Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.

CD4+T Helper Cell Responses against Human Bocavirus Viral Protein 2 Viruslike Particles in Healthy Adults

Background. Human bocavirus (HBoV) was recently described as a new member of the Parvoviridae family, and its possible association with respiratory illness in infants has been discussed. To date, HBoV genomes have been detected worldwide in respiratory tract samples obtained from children with pulmonary diseases, whereas only limited data on virus-specific immunity are available, mainly because of the lack of recombinant viral antigens. Methods. HBoV viruslike particles (VLPs) were produced in insect cells and characterized by electron microscopy and cesium chloride gradient centrifugation. HBoV viral protein 2 (VP2)-specific antibodies and CD4+ T helper cell responses were analyzed by enzyme-linked immunsorbent assay and enzyme-linked immunospot assay. Results. VP2 capsid proteins of HBoV were produced in insect cells infected with a recombinant baculovirus, and the formation of icosahedral VLPs (diameter, 21–25 nm; sedimentation density, 1.33 g/cm3) was demonstrated. A significant increase in secretion of VP2-specific interferon-γ was detected in cultures of peripheral blood mononuclear cells obtained from 69 healthy adults found to be positive for HBoV-specific immunoglobulin G antibodies, compared with control stimulations. In parallel, T cell responses against identically expressed parvovirus B19 VP2 VLPs were frequently observed in the individuals studied, without there being obvious cross-reactions between HBoV and parvovirus B19. Conclusions. Data suggest the presence of HBoV-specific immune responses in adults and strongly support a high prevalence of HBoV among humans.

BARE retrotransposons produce multiple groups of rarely polyadenylated transcripts from two differentially regulated promoters

The BARE retrotransposon family comprises more than 10(4) copies in the barley (Hordeum vulgare) genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing promoters and RNA-processing motifs required for retrotransposon replication. Members of the BARE1 subfamily are transcribed, translated, and form virus-like particles. Very similar retrotransposons are expressed as RNA and protein in other cereals and grasses. The BARE2 subfamily is, however, non-autonomous because it cannot produce the GAG capsid protein. The pattern of plant development implies that inheritance of integrated copies should critically depend, in the first instance, on cell-specific and tissue-specific expression patterns. We examined transcription of BARE within different barley tissues and analyzed the promoter function of the BARE LTR. The two promoters of the LTR vary independently in activity by tissue. In embryos TATA1 was almost inactive, whereas transcription in callus appears to be less tightly regulated than in other tissues. Deletion analyses of the LTR uncovered strong positive and negative regulatory elements. The promoters produce multiple groups of transcripts that are distinct by their start and stop points, by their sequences, and by whether they are polyadenylated. Some of these groups do not share the common end structures needed for template switching during replication. Only about 15% of BARE transcripts are polyadenylated. The data suggest that distinct subfamilies of transcripts may play independent roles in providing the proteins and replication templates for the BARE retrotransposon life cycle.

The M, E, and N Structural Proteins of the Severe Acute Respiratory Syndrome Coronavirus Are Required for Efficient Assembly, Trafficking, and Release of Virus-Like Particles

The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.

Plant-produced Hepatitis B Core Protein Chimera Carrying Anthrax Protective Antigen Domain-4

The hepatitis B core antigen (HBcAg) can generate a strong immune response and is recognized as an effective carrier for foreign epitopes. The domain-4 epitope of the anthrax protective antigen (PA-D4) plays an essential role in generating protective immunity against virulent Bacillus anthracis. Here we report the successful production of a recombinant protein comprised of the antigenic PA-D4 integrated into the c/e1 loop of HBcAg in transgenic low-alkaloid Nicotiana tabacum. Sera of mice injected with the plant-derived purified HB/PA-D4 protein exhibited significant anti-PA- and anti-HBcAg-specific IgG titers; however, formation of virus-like particles (VLP) was not observed. These data support the feasibility of producing complex protein chimeras in plants.

Role of the DxxDxD motif in the assembly and stability of betanodavirus particles

Piscine betanodavirus possesses a bipartite genome of single-stranded (+)RNAs. RNA2 cDNA of dragon grouper nervous necrosis virus (DGNNV) has been expressed previously to form virus-like particles (VLPs), which are highly similar to the native virion. Experiments with calcium-chelating or reducing/oxidizing reagents showed that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. The results of mutational analysis of DxxDxD, which is putatively involved in the coordination of calcium ions, showed that the D133N mutation significantly disrupted the assembly of VLPs, while D130N and D135N mutants produced heterogeneous VLPs with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of the D135N mutant were stable at a temperature of 85 degrees C, which is slightly higher than that for wild-type, whereas VLPs of the D130N mutant could not tolerate the thermal effects at a temperature higher than 60 degrees C. It was deduced that the three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and that, among them, the DxxD provides a more stable coordinate of calcium ligand than DxD.