Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Virion-incorporated PSGL-1 and CD43 inhibit both cell-free infection and transinfection of HIV-1 by preventing virus-cell binding.

HIV-1 particles incorporate various host transmembrane proteins in addition to viral Env glycoprotein during assembly at the plasma membrane. In polarized T cells, HIV-1 structural protein Gag localizes to the plasma membrane of uropod, a rear-end protrusion. Notably, uropod transmembrane proteins PSGL-1 and CD43 cocluster specifically with Gag assembling at the plasma membrane even in cells that do not form uropods. Recent reports have shown that expression of either PSGL-1 or CD43 in virus-producing cells reduces the infectivity of progeny virions and that HIV-1 infection reduces the cell surface expression of these proteins. However, the mechanisms for both processes remain to be determined. In this study, we found that virion incorporation of PSGL-1 and CD43 closely correlates with diminished virion infectivity. PSGL-1 and CD43 inhibited virus attachment to CD4+ cells irrespective of the presence of Env. These proteins also inhibited virion attachment to CD4- lymphoid organ fibroblastic reticular cells that mediate transinfection of CD4+ T cells. Consistent with the possibility that highly extended extracellular domains of these proteins physically block virus-cell attachment, the inhibitory effect of PSGL-1 required its full-length ectodomain. HIV-1 encoding Gag mutants that are defective in either coclustering with these host proteins or ESCRT-dependent particle release failed to reduce PSGL-1 on surface of infected cells. This study reveals an anti-HIV-1 mechanism that suppresses virus-cell attachment and a previously unappreciated process of HIV-1-mediated down-regulation of host antiviral proteins, both of which likely require virion incorporation of these proteins.

Development and characterization of a packaging cell line for pseudo-infectious yellow fever virus particle generation.

INTRODUCTION: Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. METHODS: HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. RESULTS: The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. CONCLUSIONS: HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.

Development and characterization of a packaging cell line for pseudo-infectious yellow fever virus particle generation

Abstract INTRODUCTION: Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. METHODS: HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. RESULTS: The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. CONCLUSIONS: HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.

Mapping out the intricate relationship of the HIV envelope protein and the membrane environment.

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications.

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against flaviviruses and coronavirus.

UNLABELLED: The tripartite motif-containing (TRIM) proteins have emerged as a new class of host antiviral restriction factors, with several demonstrating roles in regulating innate antiviral responses. Of >70 known TRIMs, TRIM56 inhibits replication of bovine viral diarrhea virus, a ruminant pestivirus of the family Flaviviridae, but has no appreciable effect on vesicular stomatitis virus (VSV), a rhabdovirus. Yet the antiviral spectrum of TRIM56 remains undefined. In particular, how TRIM56 impacts human-pathogenic viruses is unknown. Also unclear are the molecular determinants governing the antiviral activities of TRIM56. Herein, we show that TRIM56 poses a barrier to infections by yellow fever virus (YFV), dengue virus serotype 2 (DENV2), and human coronavirus virus (HCoV) OC43 but not encephalomyocarditis virus (EMCV). Moreover, by engineering cell lines conditionally expressing various TRIM56 mutants, we demonstrated that TRIM56's antiflavivirus effects required both the E3 ligase activity that lies in the N-terminal RING domain and the integrity of its C-terminal portion, while the restriction of HCoV-OC43 relied upon the TRIM56 E3 ligase activity alone. Furthermore, TRIM56 was revealed to impair YFV and DENV2 propagation by suppressing intracellular viral RNA accumulation but to compromise HCoV-OC43 infection at a later step in the viral life cycle, suggesting that distinct TRIM56 domains accommodate differing antiviral mechanisms. Altogether, TRIM56 is a versatile antiviral host factor that confers resistance to YFV, DENV2, and HCoV-OC43 through overlapping and distinct molecular determinants. IMPORTANCE: We previously reported tripartite motif protein 56 (TRIM56) as a host restriction factor of bovine viral diarrhea virus, a ruminant pathogen. However, the impact of TRIM56 on human-pathogenic RNA viruses is unknown. Herein, we demonstrate that TRIM56 restricts two medically important flaviviruses, yellow fever virus (YFV) and dengue virus serotype 2 (DENV2), and a human coronavirus, HCoV-OC43, but not encephalomyocarditis virus, a picornavirus. Further, we show that TRIM56-mediated inhibition of HCoV-OC43 multiplication depends solely on its E3 ligase activity, whereas its restriction of YFV and DENV2 requires both the E3 ligase activity and integrity of the C-terminal portion. The differing molecular determinants appear to accommodate distinct antiviral mechanisms TRIM56 adopts to target different families of viruses; while TRIM56 curbs intracellular YFV/DENV2 RNA replication, it acts at a later step in HCoV-OC43 life cycle. These novel findings illuminate the molecular basis of the versatility and specificity of TRIM56's antiviral activities against positive-strand RNA viruses.

Modification of the hepatitis B virus envelope protein glycosylation pattern interferes with secretion of viral particles, infectivity, and susceptibility to neutralizing antibodies.

UNLABELLED: The envelope proteins of hepatitis B virus (HBV) bear an N-linked glycosylation site at N146 within the immunodominant a-determinant in the antigenic loop (AGL) region. This glycosylation site is never fully functional, leading to a nearly 1/1 ratio of glycosylated/nonglycosylated isoforms in the viral envelope. Here we investigated the requirement for a precise positioning of N-linked glycan at amino acid 146 and the functions associated with the glycosylated and nonglycosylated isoforms. We observed that the removal of the N146 glycosylation site by mutagenesis was permissive to envelope protein synthesis and stability and to secretion of subviral particles (SVPs) and hepatitis delta virus (HDV) virions, but it was detrimental to HBV virion production. Several positions in the AGL could substitute for position 146 as the glycosylation acceptor site. At position 146, neither a glycan chain nor asparagine was absolutely required for infectivity, but there was a preference for a polar residue. Envelope proteins bearing 5 AGL glycosylation sites became hyperglycosylated, leading to an increased capacity for SVP secretion at the expense of HBV and HDV virion secretion. Infectivity-compatible N-glycosylation sites could be inserted at 3 positions (positions 115, 129, and 136), but when all three positions were glycosylated, the hyperglycosylated mutant was substantially attenuated at viral entry, while it acquired resistance to neutralizing antibodies. Taken together, these findings suggest that the nonglycosylated N146 is essential for infectivity, while the glycosylated form, in addition to its importance for HBV virion secretion, is instrumental in shielding the a-determinant from neutralizing antibodies. IMPORTANCE: At the surface of HBV particles, the immunodominant a-determinant is the main target of neutralizing antibodies and an essential determinant of infectivity. It contains an N-glycosylation site at position 146, which is functional on only half of the envelope proteins. Our data suggest that the coexistence of nonglycosylated and glycosylated N146 at the surface of HBV reflects the dual function of this determinant in infectivity and immune escape. Hence, a modification of the HBV glycosylation pattern affects not only virion assembly and infectivity but also immune escape.

Virus particle release from glycosphingolipid-enriched microdomains is essential for dendritic cell-mediated capture and transfer of HIV-1 and henipavirus.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DCs) to promote its transmission to T cells. We recently reported that the capture of HIV-1 by mature dendritic cells (MDCs) is mediated by an interaction between the glycosphingolipid (GSL) GM3 on virus particles and CD169/Siglec-1 on MDCs. Since HIV-1 preferentially buds from GSL-enriched lipid microdomains on the plasma membrane, we hypothesized that the virus assembly and budding site determines the ability of HIV-1 to interact with MDCs. In support of this hypothesis, mutations in the N-terminal basic domain (29/31KE) or deletion of the membrane-targeting domain of the HIV-1 matrix (MA) protein that altered the virus assembly and budding site to CD63(+)/Lamp-1-positive intracellular compartments resulted in lower levels of virion incorporation of GM3 and attenuation of virus capture by MDCs. Furthermore, MDC-mediated capture and transmission of MA mutant viruses to T cells were decreased, suggesting that HIV-1 acquires GSLs via budding from the plasma membrane to access the MDC-dependent trans infection pathway. Interestingly, MDC-mediated capture of Nipah and Hendra virus (recently emerged zoonotic paramyxoviruses) M (matrix) protein-derived virus-like particles that bud from GSL-enriched plasma membrane microdomains was also dependent on interactions between virion-incorporated GSLs and CD169. Moreover, capture and transfer of Nipah virus envelope glycoprotein-pseudotyped lentivirus particles by MDCs were severely attenuated upon depletion of GSLs from virus particles. These results suggest that GSL incorporation into virions is critical for the interaction of diverse enveloped RNA viruses with DCs and that the GSL-CD169 recognition nexus might be a conserved viral mechanism of parasitization of DC functions for systemic virus dissemination. IMPORTANCE: Dendritic cells (DCs) can capture HIV-1 particles and transfer captured virus particles to T cells without establishing productive infection in DCs, a mechanism of HIV-1 trans infection. We have recently identified CD169-mediated recognition of GM3, a host-derived glycosphingolipid (GSL) incorporated into the virus particle membrane, as the receptor and ligand for the DC-HIV trans infection pathway. In this study, we have identified the matrix (MA) domain of Gag to be the viral determinant that governs incorporation of GM3 into HIV-1 particles, a previously unappreciated function of the HIV-1 MA. In addition, we demonstrate that the GSL-CD169-dependent trans infection pathway is also utilized as a dissemination mechanism by henipaviruses. GSL incorporation in henipaviruses was also dependent on the viral capsid (M) protein-directed assembly and budding from GSL-enriched lipid microdomains. These findings provide evidence of a conserved mechanism of retrovirus and henipavirus parasitization of cell-to-cell recognition pathways for systemic virus dissemination.

CD36-specific antibodies block release of HIV-1 from infected primary macrophages and its transmission to T cells.

HIV-1-infected macrophages likely represent viral reservoirs, as they accumulate newly formed virions in internal virus-containing compartments (VCCs). However, the nature and biogenesis of VCCs remain poorly defined. We show that upon HIV-1 infection of primary human macrophages, Gag is recruited to preexisting compartments containing the scavenger receptor CD36, which then become VCCs. Silencing of CD36 in HIV-1-infected macrophages decreases the amount of virions released. Strikingly, soluble anti-CD36 antibodies, but not the natural ligands of CD36, inhibit release of virions from HIV-1-infected macrophages and the transmission of virus to CD4(+) T cells. The effect of the antibodies is potent, rapid, and induces the retention of virions within VCCs. Ectopic expression of CD36 in HeLa cells renders them susceptible to the inhibitory effect of the anti-CD36 mAb upon HIV-1 infection. We show that the anti-CD36 mAb inhibits HIV-1 release by clustering newly formed virions at their site of budding, and that signaling via CD36 is not required. Thus, HIV-1 reservoirs in macrophages may be tackled therapeutically using anti-CD36 antibodies to prevent viral dissemination.

Immunotherapy of allergic rhinitis: new therapeutic opportunities with virus-like particles filled with CpG motifs.

BACKGROUND: The incidence of allergic rhinitis (AR) has increased constantly over the last decades. The disease can significantly lower quality of life and subsequently might progress to allergic asthma. Allergen-specific immunotherapy is mostly used to cope with the cause of the disease. However, incidence of systemic reactions or limited compliance hampers the widespread use of this therapeutic approach. Therefore, new candidates are examined to improve immunotherapy of allergies. Recently, a new technology was developed with the aim to positively influence the immune system of allergic patients. Virus-like particles (VLPs) represent a potent vaccine platform that has been proven to be immunogenic and clinically effective. To enhance immune cell activation, addition of Toll-like receptor ligands and/or depot-forming adjuvants seems to be helpful. In this context, CpG motifs represent intensive investigated and potent stimulators of T cells. This article focuses on the function of VLPs and CpG motifs and their clinical experience for treatment of AR. METHODS: A literature review was performed. RESULTS: Several published studies showed a beneficial impact of the treatment on allergic symptoms. They tested VLPs filled with or without CpG motifs in combination with or without allergen. CONCLUSION: Results encourage further investigations of VLPs and CpG motifs as adjuncts to or even alternative candidates for immunotherapy of allergic disorders.

A novel immunocompetent murine tumor model for the evaluation of RCAd-enhanced RDAd transduction efficacy.

Low gene transfer rate in tumors, high dose-induced acute inflammatory response, and lack of an immunocompetent preclinical animal model to accurately reflect the therapeutic efficacy are prominent reasons for the lack of clinical success of adenoviral (Ad) vectors. In this study, we tested whether human replication-competent adenovirus (RCAd) can replicate in T739 mouse bladder transitional tumor cells (BTT) and lung adenocarcinoma cells (LA795), and whether RCAd can enhance the transduction rate and transgene expression of human replication defective adenoviruses (RDAd) in these tumor cells in vitro and in vivo. We demonstrated that human RCAd exhibited good infectability and cytopathologic effects in mouse BTT and LA795 cells, which was comparable to that in A549 and NCIH460 human tumor cells. In contrast, no infectability and cytopathologic effects were observed in other three mouse tumor cells such as 4T1, B16, and Lewis cells. The combined use of RCAd with RDAd significantly enhanced RDAd transduction efficiency in BTT and LA795 tumor cells in vitro and in vivo. When BTT and LA795 cells were co-infected with RDAd Ad-EGFP and RCAd, a large amount of E1a expression and 2-3 orders of increases in Ad-EGFP genomic DNA were observed. In contrast, the expression of the late gene Hexon is very low, which may explain ineffective packaging of viral particles. In conclusion, our study provided a novel immunocompetent animal model which is useful for evaluating RCAd infectability, cytopathy, and replication. The combined use of RCAd and RDAd provided a new solution for cancer gene therapy.

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity.

BACKGROUND: Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs). RESULTS: VLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. CONCLUSIONS: D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.

Evidence that productive human immunodeficiency virus type 1 assembly can occur in an intracellular compartment.

Human immunodeficiency virus type 1 (HIV-1) assembly occurs predominantly at the plasma membrane of infected cells. The targeting of assembly to intracellular compartments such as multivesicular bodies (MVBs) generally leads to a significant reduction in virus release efficiency, suggesting that MVBs are a nonproductive site for HIV-1 assembly. In the current study, we make use of an HIV-1 Gag-matrix mutant, 29/31KE, that is MVB targeted. We previously showed that this mutant is severely defective for virus particle production in HeLa cells but more modestly affected in primary macrophages. To more broadly examine the consequences of MVB targeting for virus production, we investigated 29/31KE particle production in a range of cell types. Surprisingly, this mutant supported highly efficient assembly and release in T cells despite its striking MVB Gag localization. Manipulation of cellular endocytic pathways revealed that unlike Vpu-defective HIV-1, which demonstrated intracellular Gag localization as a result of Gag endocytosis from the plasma membrane, 29/31KE mutant Gag was targeted directly to an MVB compartment. The 29/31KE mutant was unable to support multiple-round replication; however, this defect could be reversed by truncating the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 and by the acquisition of a 16EK change in matrix. The 16EK/29/31KE matrix mutant replicated efficiently in the MT-4 T-cell line despite maintaining an MVB-targeting phenotype. These results indicate that MVB-targeted Gag can be efficiently released from T cells and primary macrophages, suggesting that under some circumstances, late endosomal compartments can serve as productive sites for HIV-1 assembly in these physiologically relevant cell types.

Interaction with 7SL RNA but not with HIV-1 genomic RNA or P bodies is required for APOBEC3F virion packaging.

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F), like APOBEC3G, has broad antiviral activity against diverse retroelements, including Vif-deficient human immunodeficiency virus (HIV)-1. Its antiviral functions are known to rely on its virion encapsidation and be suppressed by HIV-1 Vif, which recruits Cullin5-based E3 ubiquitin ligases. However, the factors that mediate A3F virion packaging have not yet been identified. In this study, we demonstrate that A3F specifically interacts with cellular signal recognition particle (SRP) RNA (7SL RNA), which is selectively packaged into HIV-1 virions. Efficient packaging of 7SL RNA as well as A3F was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. Reducing 7SL RNA packaging by overexpression of SRP19 protein inhibited A3F virion packaging. Although A3F has been shown to interact with P bodies and viral genomic RNA, our data indicated that P bodies and HIV-1 genomic RNA were not required for A3F packaging. Thus, in addition to its well-known function in SRPs, 7SL RNA, which is encapsidated into diverse retroviruses, also participates in the innate antiviral function of host cytidine deaminases.

7SL RNA mediates virion packaging of the antiviral cytidine deaminase APOBEC3G.

Cytidine deaminase APOBEC3G (A3G) has broad antiviral activity against diverse retroviruses and/or retrotransposons, and its antiviral functions are believed to rely on its encapsidation into virions in an RNA-dependent fashion. However, the cofactors of A3G virion packaging have not yet been identified. We demonstrate here that A3G selectively interacts with certain polymerase III (Pol III)-derived RNAs, including Y3 and 7SL RNAs. Among A3G-binding Pol III-derived RNAs, 7SL RNA was preferentially packaged into human immunodeficiency virus type 1 (HIV-1) particles. Efficient packaging of 7SL RNA, as well as A3G, was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. A3G mutants that had reduced 7SL RNA binding but maintained wild-type levels of mRNA and tRNA binding were packaged poorly and had impaired antiviral activity. Reducing 7SL RNA packaging by overexpression of SRP19 proteins inhibited 7SL RNA and A3G virion packaging and impaired its antiviral function. Thus, 7SL RNA that is encapsidated into diverse retroviruses is a key cofactor of the antiviral A3G. This selective interaction of A3G with certain Pol III-derived RNAs raises the question of whether A3G and its cofactors may have as-yet-unidentified cellular functions.

Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response.

Bovine viral diarrhea virus (BVDV) replicons with deletions within the capsid, E(RNS) or E1 encoding region were constructed and efficiently packaged with a helper cell line. High titres of packaged replicons were observed as early as 24 h after transfection, whereas no virus progeny could be detected after transfection of non-complementing cells. Infection of bovine cell cultures with rescued viruses resulted in one cycle of replication without release of infectious virus particles, and no genetic reversion of the generated viruses was detected. Packaged replicons with a deletion within the capsid-coding region were characterized in vivo in immunization and challenge trials. Following immunization of calves with the replication-deficient virus, neither virus shedding nor viremia was detected. After challenge infection with virulent BVDV, all vaccinates were completely protected from disease as measured by the absence of viremia and shedding of challenge virus, which indicated that a 'sterilizing immunity' could be induced with the generated replication-deficient packaged replicons.

Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes.

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.

Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects.

DNA rich in nonmethylated CG motifs (CpGs) greatly facilitates induction of immune responses against coadministered Ags. CpGs are therefore among the most promising adjuvants known to date. Nevertheless, CpGs are characterized by two drawbacks. They have unfavorable pharmacokinetics and may exhibit systemic side effects, including splenomegaly. We show in this study that packaging CpGs into virus-like particles (VLPs) derived from the hepatitis B core Ag or the bacteriophage Qbeta is a simple and attractive method to reduce these two problems. CpGs packaged into VLPs are resistant to DNase I digestion, enhancing their stability. In addition, and in contrast to free CpGs, packaging CpGs prevents splenomegaly in mice, without affecting their immunostimulatory capacity. In fact, vaccination with CpG-loaded VLPs was able to induce high frequencies of peptide-specific CD8(+) T cells (4-14%), protected from infection with recombinant vaccinia viruses, and eradicated established solid fibrosarcoma tumors. Thus, packaging CpGs into VLPs improves both their immunogenicity and pharmacodynamics.

Papillomavirus-like particles induce acute activation of dendritic cells.

The role of viral structural proteins in the initiation of adaptive immune responses is poorly understood. To address this issue, we focused on the effect of noninfectious papillomavirus-like particles (VLPs) on dendritic cell (DC) activation. We found that murine bone marrow-derived dendritic cells (BMDCs) effectively bound and rapidly internalized bovine papillomavirus VLPS: Exposure to fully assembled VLPs of bovine papillomavirus, human papillomavirus (HPV)16 or HPV18, but not to predominately disordered HPV16 capsomers, induced acute phenotypic maturation of BMDCS: Structurally similar polyomavirus VLPs bound to the DC surface and were internalized, but failed to induce maturation. DCs that had incorporated HPV16 VLPs produced proinflammatory cytokines IL-6 and TNF-alpha; however, the release of these cytokines was delayed relative to LPS activation. Production of IL-12p70 by VLP-exposed DCs required the addition of syngeneic T cells or rIFN-gamma. Finally, BMDCs pulsed with HPV16 VLPs induced Th1-dominated primary T cell responses in vitro. Our data provide evidence that DCs respond to intact papillomavirus capsids and that they play a central role in VLP-induced immunity. These results offer a mechanistic explanation for the striking ability of papillomavirus VLP-based vaccines to induce potent T and B cell responses even in the absence of adjuvant.