CRISPR-Cas13b-mediated suppression of hepatitis B virus replication and protein expression.

BACKGROUND & AIMS: New antiviral approaches are urgently required that target multiple aspects of the hepatitis B virus (HBV) replication cycle to improve rates of functional cure. HBV RNA represents a novel therapeutic target. Here, we programmed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas13b endonuclease, to specifically target the HBV pregenomic RNA (pgRNA) and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pgRNA. Mammalian cells with replication competent wildtype HBV DNA of different genotypes, a HBV stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-blue fluorescent protein (BFP) and crRNAs plasmids and the impact on HBV replication and protein expression was measured. WT HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and sera HBsAg was measured. PspCas13b mRNA and crRNA were also delivered by lipid nanoparticles (LNP) in a HBsAg-expressing stable cell line and the impact on secreted HBsAg determined. RESULTS: Our HBV targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p<0.0001). HBV protein expression was also reduced in an HBV stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p<0.0001) in vivo. LNP-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p=0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: There is an urgent need for new treatments that target multiple aspects of the HBV replication cycle. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.

Comparative Longitudinal Serological Study of Anti-SARS-CoV-2 Antibody Profiles in People with COVID-19.

Serological diagnostic assays are essential tools for determining an individual's protection against viruses like SARS-CoV-2, tracking the spread of the virus in the community, and evaluating population immunity. To assess the diversity and quality of the anti-SARS-CoV-2 antibody response, we have compared the antibody profiles of people with mild, moderate, and severe COVID-19 using a dot blot assay. The test targeted the four major structural proteins of SARS-CoV-2, namely the nucleocapsid (N), spike (S) protein domains S1 and S2, and receptor-binding domain (RBD). Serum samples were collected from 63 participants at various time points for up to 300 days after disease onset. The dot blot assay revealed patient-specific differences in the anti-SARS-CoV-2 antibody profiles. Out of the 63 participants with confirmed SARS-CoV-2 infections and clinical COVID-19, 35/63 participants exhibited diverse and robust responses against the tested antigens, while 14/63 participants displayed either limited responses to a subset of antigens or no detectable antibody response to any of the antigens. Anti-N-specific antibody levels decreased within 300 days after disease onset, whereas anti-S-specific antibodies persisted. The dynamics of the antibody response did not change during the test period, indicating stable antibody profiles. Among the participants, 28/63 patients with restricted anti-S antibody profiles or undetectable anti-S antibody levels in the dot blot assay also exhibited weak neutralization activity, as measured by a surrogate virus neutralization test (sVNT) and a microneutralization test. These results indicate that in some cases, natural infections do not lead to the production of neutralizing antibodies. Furthermore, the study revealed significant serological variability among patients, regardless of the severity of their COVID-19 illness. These differences need to be carefully considered when evaluating the protective antibody status of individuals who have experienced primary SARS-CoV-2 infections.

Immunogenicity of Wild Type and Mutant Hepatitis B Surface Antigen Virus-like Particles (VLPs) in Mice with Pre-Existing Immunity against the Wild Type Vector.

Virus-like particles (VLPs), composed of the small hepatitis B virus surface antigen (HBsAgS), are the antigenic components of the hepatitis B virus (HBV) vaccine and represent the backbones for a chimeric anti-malaria vaccine and various vaccine candidates. Biological vectors have to face pre-existing anti-vector immune responses due to previous immune exposure. Vector recognition after natural infections or vaccinations can result in unwarranted outcomes, with compromising effects on clinical outcomes. In order to evaluate the impact of a pre-existing anti-HBsAgS immune response, we developed mutant VLPs composed of subunits with reduced HBsAgS-specific antigenicity. The insertion of a Plasmodium falciparum circumsporozoite protein (CSP)-derived epitope as a read-out allowed the assessment of wild type (wt) and mutant VLPs in the context of a pre-existing immune response. Mutant and wt VLP platforms with a CSP-epitope insert are immunogenic and have the ability to generate anti-CSP antibody responses in both naïve BALB/c mice and mice with a pre-existing anti-HBsAgS immune response, but with superior anti-CSP responses in mice with a pre-existing immunity. The data indicate that previous HBsAgS exposure facilitates enhanced antibody responses against foreign epitopes delivered by the HBsAgS platform, and, in this context, the state of immune sensitization alters the outcome of subsequent vaccinations.

Hepatitis Delta Virus (HDV) and Delta-Like Agents: Insights Into Their Origin.

Hepatitis delta virus (HDV) is a human pathogen, and the only known species in the genus Deltavirus. HDV is a satellite virus and depends on the hepatitis B virus (HBV) for packaging, release, and transmission. Extracellular HDV virions contain the genomic HDV RNA, a single-stranded negative-sense and covalently closed circular RNA molecule, which is associated with the HDV-encoded delta antigen forming a ribonucleoprotein complex, and enveloped by the HBV surface antigens. Replication occurs in the nucleus and is mediated by host enzymes and assisted by cis-acting ribozymes allowing the formation of monomer length molecules which are ligated by host ligases to form unbranched rod-like circles. Recently, meta-transcriptomic studies investigating various vertebrate and invertebrate samples identified RNA species with similarities to HDV RNA. The delta-like agents may be representatives of novel subviral agents or satellite viruses which share with HDV, the self-complementarity of the circular RNA genome, the ability to encode a protein, and the presence of ribozyme sequences. The widespread distribution of delta-like agents across different taxa with considerable phylogenetic distances may be instrumental in comprehending their evolutionary history by elucidating the transition from transcriptome to cellular circular RNAs to infectious subviral agents.

Clinical stage drugs targeting inhibitor of apoptosis proteins purge episomal Hepatitis B viral genome in preclinical models.

A major unmet clinical need is a therapeutic capable of removing hepatitis B virus (HBV) genome from the liver of infected individuals to reduce their risk of developing liver cancer. A strategy to deliver such a therapy could utilize the ability to target and promote apoptosis of infected hepatocytes. Presently there is no clinically relevant strategy that has been shown to effectively remove persistent episomal covalently closed circular HBV DNA (cccDNA) from the nucleus of hepatocytes. We used linearized single genome length HBV DNA of various genotypes to establish a cccDNA-like reservoir in immunocompetent mice and showed that clinical-stage orally administered drugs that antagonize the function of cellular inhibitor of apoptosis proteins can eliminate HBV replication and episomal HBV genome in the liver. Primary human liver organoid models were used to confirm the clinical relevance of these results. This study underscores a clinically tenable strategy for the potential elimination of chronic HBV reservoirs in patients.

The evolution and clinical impact of hepatitis B virus genome diversity.

The global burden of hepatitis B virus (HBV) is enormous, with 257 million persons chronically infected, resulting in more than 880,000 deaths per year worldwide. HBV exists as nine different genotypes, which differ in disease progression, natural history and response to therapy. HBV is an ancient virus, with the latest reports greatly expanding the host range of the Hepadnaviridae (to include fish and reptiles) and casting new light on the origins and evolution of this viral family. Although there is an effective preventive vaccine, there is no cure for chronic hepatitis B, largely owing to the persistence of a viral minichromosome that is not targeted by current therapies. HBV persistence is also facilitated through aberrant host immune responses, possibly due to the diverse intra-host viral populations that can respond to host-mounted and therapeutic selection pressures. This Review summarizes current knowledge on the influence of HBV diversity on disease progression and treatment response and the potential effect on new HBV therapies in the pipeline. The mechanisms by which HBV diversity can occur both within the individual host and at a population level are also discussed.

Glycoengineered hepatitis B virus-like particles with enhanced immunogenicity.

Virus-like particles (VLP) represent biological platforms for the development of novel products such as vaccines and delivery platforms for foreign antigenic sequences. VLPs composed of the small surface antigen (HBsAgS) derived from the hepatitis B virus (HBV) are the immunogenic components of a licensed, preventative vaccine, which contains aluminum hydroxide as adjuvant. Herein, we report that glycoengineering of N-glycosylated HBsAgS to generate hyper-glycosylated VLPs display an enhanced immunogenicity relative to the wild type (WT) HBsAgS VLPs when expressed in FreeStyle HEK 293F cells. Comparative mass spectrometry-based N-glycan profiling, gel electrophoresis, and immunoassays demonstrated that WT and hyper-glycosylated HBsAgS VLPs contain the same type and distribution of N-glycan structures, but the latter shows a higher glycan abundance per protein mass. The antigenic integrity of the modified VLPs was also shown to be retained. To assess whether hyper-glycosylated VLPs induce an enhanced immune response in the presence of the adjuvant aluminum hydroxide, the anti-HBV surface antigen (anti-HBsAgS) antibody response was monitored in BALB/c mice, subcutaneously injected with different VLP derivatives. In the absence and presence of adjuvant, hyper-glycosylated VLPs showed an enhanced immunogenicity compared to WT VLPs. The ability of hyper-glycosylated VLPs to promote potent anti-HBsAgS immune responses compared to VLPs with a native N-glycan level as well as non-glycosylated, yeast-derived HBsAgS VLPs opens exciting avenues for generating more efficacious VLP-based vaccines against hepatitis B and improved HBsAgS VLP carrier platforms using glycoengineering.

Hepatitis B virus-like particles expressing Plasmodium falciparum epitopes induce complement-fixing antibodies against the circumsporozoite protein.

The repetitive structure of compact virus-like particles (VLPs) provides high density displays of antigenic sequences, which trigger key parts of the immune system. The hepatitis B virus (HBV) and human papilloma virus (HPV) vaccines exploit the assembly competence of structural proteins, which are the effective immunogenic components of the prophylactic HBV and HPV vaccines, respectively. To optimize vaccine designs and to promote immune responses against protective epitopes, the "Asp-Ala-Asp-Pro" (NANP)-repeat from the Plasmodium falciparum circumsporozoite protein (CSP) was expressed within the exposed, main antigenic site of the small HBV envelope protein (HBsAgS); this differs from the RTS,S vaccine, in which CSP epitopes are fused to the N-terminus of HBsAgS. The chimeric HBsAgS proteins are assembly competent, produce VLPs, and provide a high antigenic density of the NANP repeat sequence. Chimeric VLPs with four or nine NANP-repeats (NANP4 and NANP9, respectively) were expressed in mammalian cells, the HBsAgS- and CSP-specific antigenicity of the VLPs was determined, and the immunogenicity of the VLPs assessed in relation to the induction of anti-HBsAgS and anti-CSP antibody responses. The chimeric VLPs induced high anti-CSP titres in BALB/c mice independent of the number of the NANP repeats. However, the number of NANP repeats influenced the activity of vaccine-induced antibodies measured by complement fixation to CSP, one of the proposed effector mechanisms for Plasmodium neutralization in vivo. Sera from mice immunized with VLPs containing nine NANP repeats performed better in the complement fixation assay than the group with four NANP repeats. The effect of the epitope-specific density on the antibody quality may instruct VLP platform designs to optimize immunological outcomes and vaccine efficacy.

A Divergent Hepatitis D-Like Agent in Birds.

Hepatitis delta virus (HDV) is currently only found in humans and is a satellite virus that depends on hepatitis B virus (HBV) envelope proteins for assembly, release, and entry. Using meta-transcriptomics, we identified the genome of a novel HDV-like agent in ducks. Sequence analysis revealed secondary structures that were shared with HDV, including self-complementarity and ribozyme features. The predicted viral protein shares 32% amino acid similarity to the small delta antigen of HDV and comprises a divergent phylogenetic lineage. The discovery of an avian HDV-like agent has important implications for the understanding of the origins of HDV and sub-viral agents.

Viral regulation of host cell biology by hijacking of the nucleolar DNA-damage response.

Recent studies indicate that nucleoli play critical roles in the DNA-damage response (DDR) via interaction of DDR machinery including NBS1 with nucleolar Treacle protein, a key mediator of ribosomal RNA (rRNA) transcription and processing. Here, using proteomics, confocal and single molecule super-resolution imaging, and infection under biosafety level-4 containment, we show that this nucleolar DDR pathway is targeted by infectious pathogens. We find that the matrix proteins of Hendra virus and Nipah virus, highly pathogenic viruses of the Henipavirus genus in the order Mononegavirales, interact with Treacle and inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1-Treacle interaction during a DDR. Furthermore, inhibition of Treacle expression/function enhances henipavirus production. These data identify a mechanism for viral modulation of host cells by appropriating the nucleolar DDR and represent, to our knowledge, the first direct intranucleolar function for proteins of any mononegavirus.

Signalome-wide assessment of host cell response to hepatitis C virus.

Host cell signalling during infection with intracellular pathogens remains poorly understood. Here we report on the use of antibody microarray technology to detect variations in the expression levels and phosphorylation status of host cell signalling proteins during hepatitis C virus (HCV) replication. Following transfection with HCV RNA, the JNK and NF-κB pathways are suppressed, while the JAK/STAT5 pathway is activated; furthermore, components of the apoptosis and cell cycle control machineries are affected in the expression and/or phosphorylation status. RNAi-based hit validation identifies components of the JAK/STAT, NF-κB, MAPK and calcium-induced pathways as modulators of HCV replication. Selective chemical inhibition of one of the identified targets, the JNK activator kinase MAP4K2, does impair HCV replication. Thus this study provides a comprehensive picture of host cell pathway mobilization by HCV and uncovers potential therapeutic targets. The strategy of identifying targets for anti-infective intervention within the host cell signalome can be applied to any intracellular pathogen.

Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens.

Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases.

Nuclear localization and secretion competence are conserved among henipavirus matrix proteins.

Viruses of the genus Henipavirus of the family Paramyxoviridae are zoonotic pathogens, which have emerged in Southeast Asia, Australia and Africa. Nipah virus (NiV) and Hendra virus are highly virulent pathogens transmitted from bats to animals and humans, while the henipavirus Cedar virus seems to be non-pathogenic in infection studies. The full replication cycle of the Paramyxoviridae occurs in the host cell's cytoplasm, where viral assembly is orchestrated by the matrix (M) protein. Unexpectedly, the NiV-M protein traffics through the nucleus as an essential step to engage the plasma membrane in preparation for viral budding/release. Comparative studies were performed to assess whether M protein nuclear localization is a common feature of the henipaviruses, including the recently sequenced (although not yet isolated) Ghanaian bat henipavirus (Kumasi virus, GH-M74a virus) and Mojiang virus. Live-cell confocal microscopy revealed that nuclear translocation of GFP-fused M protein is conserved between henipaviruses in both human- and bat-derived cell lines. However, the efficiency of M protein nuclear localization and virus-like particle budding competency varied. Additionally, Cedar virus-, Kumasi virus- and Mojiang virus-M proteins were mutated in a bipartite nuclear localization signal, indicating that a key lysine residue is essential for nuclear import, export and induction of budding events, as previously reported for NiV-M. The results of this study suggest that the M proteins of henipaviruses may utilize a similar nucleocytoplasmic trafficking pathway as an essential step during viral replication in both humans and bats.

The non-pathogenic Henipavirus Cedar paramyxovirus phosphoprotein has a compromised ability to target STAT1 and STAT2.

Immune evasion by the lethal henipaviruses, Hendra (HeV) and Nipah virus, is mediated by its interferon (IFN) antagonist P gene products, phosphoprotein (P), and the related V and W proteins, which can target the signal transducer and activator of transcription 1 (STAT1) and STAT2 proteins to inhibit IFN/STAT signaling. However, it is not clear if the recently identified non-pathogenic Henipavirus, Cedar paramyxovirus (CedPV), is also able to antagonize the STAT proteins. We performed comparative studies between the HeV P gene products (P/V/W) and CedPV-P (CedPV does not encode V or W) and demonstrate that differences exist in their ability to engage the STAT proteins using immunoprecipitation and quantitative confocal microscopic analysis. In contrast to HeV-P gene encoded proteins, the ability of CedPV-P to interact with and relocalize STAT1 or STAT2 is compromised, correlating with a reduced capacity to inhibit the mRNA synthesis of IFN-inducible gene MxA. Furthermore, infection studies with HeV and CedPV demonstrate that HeV is more potent than CedPV in inhibiting the IFN-α-mediated nuclear accumulation of STAT1. These results strongly suggest that the ability of CedPV to counteract the IFN/STAT response is compromised compared to HeV.

Modification of Asparagine-Linked Glycan Density for the Design of Hepatitis B Virus Virus-Like Particles with Enhanced Immunogenicity.

UNLABELLED: The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified virus-like particles (VLPs) exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wild-type (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced T116N and G130N sites were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs, with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamines, and sialylation. Antigenic fingerprints for the WT and hypo- and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their ability to bind to the different VLP glyco-analogues, suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titers. The T116N VLPs induced earlier and longer-lasting antibody responses than did the hypoglycosylated and WT VLPs. The ability of nonnative VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with cells of the innate immune system illustrates pathways for the design of immunogens for superior preventive applications. IMPORTANCE: The use of biochemically modified, nonnative immunogens represents an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized, and the results demonstrated that they carry the same types of glycans as wild-type VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer-lasting antibody immune responses than do wild-type or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.

Anti-hepatitis B activity of isoquinoline alkaloids of plant origin.

Hepatitis B virus (HBV) is the causative agent of B-type hepatitis in humans, a vaccine-preventable disease. Despite the availability of effective vaccines, globally, 2 billion people show evidence of past or current HBV infection, of which 350 million people are persistently infected, with an estimated annual increase of 1 million. There is no cure for chronic HBV infections, which are associated with cirrhotic liver failure and with an increased risk of developing hepatocellular carcinoma. Hepatitis antiviral research has focused primarily on the development of inhibitors of viral polymerase through the use of nucleoside analogues. Therefore, there is an urgent need for the development of non-nucleoside compounds to be used as an alternative or to complement the current therapy. To address this need, 18 isoquinoline alkaloids were evaluated for their potential antiviral activity against HBV in vitro.

Production, characterization, and immunogenicity of a secreted form of Plasmodium falciparum merozoite surface protein 4 produced in Bacillus subtilis.

Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.

Hepatitis B virus-like particles access major histocompatibility class I and II antigen presentation pathways in primary dendritic cells.

Virus-like particles (VLPs) represent high density displays of viral proteins that efficiently trigger immunity. VLPs composed of the small hepatitis B virus envelope protein (HBsAgS) are useful vaccine platforms that induce humoral and cellular immune responses. Notably, however, some studies suggest HBsAgS VLPs impair dendritic cell (DC) function. Here we investigated HBsAgS VLP interaction with DC subsets and antigen access to major histocompatibility complex (MHC) class I and II antigen presentation pathways in primary DCs. HBsAgS VLPs impaired plasmacytoid DC (pDC) interferon alpha (IFNα) production in response to CpG in vitro, but did not alter conventional DC (cDC) or pDC phenotype when administered in vivo. To assess cellular immune responses, HBsAgS VLPs were generated containing the ovalbumin (OVA) model epitopes OVA(257-264) and OVA(323-339) to access MHCI and MHCII antigen presentation pathways, respectively; both in vitro and following immunisation in vivo. HBsAgS VLP-OVA(257-264) elicited CTL responses in vivo that were not enhanced by inclusion of an additional MHCII helper epitope. HBsAgS VLP-OVA(257-264) administered in vivo was cross-presented by CD8(+) DCs, but not CD8(-) DCs. Therefore, HBsAgS VLPs can deliver antigen to both MHCI and MHCII antigen presentation pathways in primary DCs and promote cytotoxic and helper T cell priming despite their suppressive effect on pDCs.

Hepatitis C virus nonstructural protein 5B is involved in virus morphogenesis.

The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.

Modulation of the immunogenicity of virus-like particles composed of mutant hepatitis B virus envelope subunits.

Virus-like particles (VLPs) are non-infectious subviral protein complexes, which possess structural features identical or closely related to infectious virions. They are utilized as delivery tools for immunologically relevant antigenic sequences. In order to investigate whether mutant subunits can modulate the VLP immunogenicity, comparative immunization studies with wild-type and non-native VLPs were performed. To determine whether disulfide bonding impacts on the immunogenicity of hepatitis B virus envelope proteins (HBsAg), mutant HBsAg subunits with single, double and triple cysteine residue substitutions were generated. The mutant proteins were expressed in cell culture, secretion competent non-native VLPs generated, followed by immunization studies in mice to measure the cellular immune response. The reduced ability of mutant HBsAg proteins to form disulfide bonds does not interfere with their ability to assemble into secretion competent VLPs. Depending on specific cysteine to alanine changes, VLPs could be generated with or without an increased ratio of monomeric versus dimeric/oligomeric subunits compared to wild-type VLPs. The utilization of non-native VLPs resulted in enhanced cellular immune responses and does not seem to depend on the ratio between monomeric or dimeric/oligomeric subunits. Comparative immunization studies strongly indicate that changes in the disulfide bonding modulate the VLP immunogenicity most likely due to structural changes. We hypothesize that structural features have evolved with reduced immunogenicity to evade the constraints imposed by the immune system. Altering VLP conformation may represent an attractive strategy to modulate antigen processing resulting in an enhanced immune response and/or a changed hierarchy of epitope presentation.