Type I interferon rapidly restricts infectious hepatitis C virus particle genesis

Interferon-alpha (IFNα) has been used to treat chronic hepatitis C virus (HCV) infection for over 20 years with varying efficacy, depending on the infecting viral genotype. The mechanism of action of IFNα is not fully understood, but is thought to target multiple stages of the HCV lifecycle, inhibiting viral transcription and translation leading to a degradation of viral RNA and protein expression in the infected cell. IFNα induces the expression of an array of interferon-stimulated genes within minutes of receptor engagement; however, the impact of these early responses on the viral lifecycle are unknown. We demonstrate that IFNα inhibits the genesis of infectious extracellular HCV particles within 2 hours of treating infected cells, with minimal effect on the intracellular viral burden. Importantly, this short duration of IFNα treatment of infected cells significantly reduced cell-free and cell-to-cell dissemination. The secreted viral particles showed no apparent change in protein content or density, demonstrating that IFNα inhibits particle infectivity but not secretion rates. To investigate whether particles released from IFNα-treated cells have a reduced capacity to establish infection we used HCV lentiviral pseudotypes (HCVpp) and demonstrated a defect in cell entry. Using a panel of monoclonal antibodies targeting the E2 glycoprotein, we demonstrate that IFNα alters glycoprotein conformation and receptor utilization. Conclusion: These observations show a previously unreported and rapid effect of IFNα on HCV particle infectivity that inhibits de novo infection events. Evasion of this response may be a contributing factor in whether a patient achieves early or rapid virological response, a key indicator of progression to sustained virological response or clearance of viral infection. (Hepatology 2014;60:1890–1900

How have retrovirus pseudotypes contributed to our understanding of viral entry?

Study of virus entry into host cells is important for understanding viral tropism and pathogenesis. Studying the entry of in vitro cultured viruses is not always practicable. Study of highly pathogenic viruses, viruses that do not grow in culture, and viruses that rapidly change phenotype in vitro can all benefit from alternative models of entry. Retrovirus particles can be engineered to display the envelope proteins of heterologous enveloped viruses. This approach, broadly termed ‘pseudotyping’, is an important technique for interrogating virus entry. In this perspective we consider how retrovirus pseudotypes have addressed these challenges and improved our understanding of the entry pathways of diverse virus species, including Ebolavirus, human immunodeficiency virus and hepatitis C virus

How have retrovirus pseudotypes contributed to our understanding of viral entry?

Study of virus entry into host cells is important for understanding viral tropism and pathogenesis. Studying the entry of in vitro cultured viruses is not always practicable. Study of highly pathogenic viruses, viruses that do not grow in culture, and viruses that rapidly change phenotype in vitro can all benefit from alternative models of entry. Retrovirus particles can be engineered to display the envelope proteins of heterologous enveloped viruses. This approach, broadly termed ‘pseudotyping’, is an important technique for interrogating virus entry. In this perspective we consider how retrovirus pseudotypes have addressed these challenges and improved our understanding of the entry pathways of diverse virus species, including Ebolavirus, human immunodeficiency virus and hepatitis C virus

Hepatitis C virus vaccine: Challenges and prospects

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. The hepatitis C virus (HCV) causes both acute and chronic infection and continues to be a global problem despite advances in antiviral therapeutics. Current treatments fail to prevent reinfection and remain expensive, limiting their use to developed countries, and the asymptomatic nature of acute infection can result in individuals not receiving treatment and unknowingly spreading HCV. A prophylactic vaccine is therefore needed to control this virus. Thirty years since the discovery of HCV, there have been major gains in understanding the molecular biology and elucidating the immunological mechanisms that underpin spontaneous viral clearance, aiding rational vaccine design. This review discusses the challenges facing HCV vaccine design and the most recent and promising candidates being investigated

Troubleshooting methods for the generation of novel pseudotyped viruses

A pseudotyped virus (PV) is a virus particle with an envelope protein originating from a different virus. The ability to dictate which envelope proteins are expressed on the surface has made pseudotyping an important tool for basic virological studies such as determining the cellular targets of the envelope protein of the virus as well as identification of potential antiviral compounds and measuring specific antibody responses. In this review, we describe the common methodologies employed to generate PVs, with a focus on approaches to improve the efficacy of PV generation

The past, present and future of neutralizing antibodies for hepatitis C virus

Hepatitis C virus (HCV) is a major cause of liver disease and hepatocellular carcinoma worldwide. HCV establishes a chronic infection in the majority of cases. However, some individuals clear the virus, demonstrating a protective role for the host immune response. Although new all-oral drug combinations may soon replace traditional ribavirin–interferon therapy, the emerging drug cocktails will be expensive and associated with side-effects and resistance, making a global vaccine an urgent priority. T cells are widely accepted to play an essential role in clearing acute HCV infection, whereas the role antibodies play in resolution and disease pathogenesis is less well understood. Recent studies have provided an insight into viral neutralizing determinants and the protective role of antibodies during infection. This review provides a historical perspective of the role neutralizing antibodies play in HCV infection and discusses the therapeutic benefits of antibody-based therapies. This article forms part of a symposium in Antiviral Research on “Hepatitis C: next steps toward global eradication.

Troubleshooting methods for the generation of novel pseudotyped viruses

A pseudotyped virus (PV) is a virus particle with an envelope protein originating from a different virus. The ability to dictate which envelope proteins are expressed on the surface has made pseudotyping an important tool for basic virological studies such as determining the cellular targets of the envelope protein of the virus as well as identification of potential antiviral compounds and measuring specific antibody responses. In this review, we describe the common methodologies employed to generate PVs, with a focus on approaches to improve the efficacy of PV generation

Novel functional hepatitis C virus glycoprotein isolates identified using an optimised viral pseudotype entry assay

Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, pre-clinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from individual patient quasispecies were discovered to behave very differently in this entry model. Empirical optimisation of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterised as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) were also sensitive to the amount, and ratio, of plasmids used, and that protocols for optimal production of these pseudoviruses is dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilising pseudoviruses to conduct empirical optimisation of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping

Flexible and rapid construction of viral chimeras applied to Hepatitis C Virus

A novel and broadly applicable strategy combining site directed mutagenesis and DNA assembly for constructing seamless viral chimeras is described using Hepatitis C Virus as an exemplar. Full-length HCV genomic cloning cassettes, which contained flexibly situated restriction endonuclease sites, were prepared via a single site-directed mutagenesis reaction and digested to receive PCR amplified virus envelope genes by In-Fusion cloning. Using this method we were able to construct gene-shuttle cassettes for generation of cell culture-infectious JFH-1-based chimeras containing genotype 1-3 E1E2 genes. Importantly, using this method we also show that E1E2 clones that were not able to support cell entry in the HCV pseudoparticle assay did confer entry when shuttled into the chimeric cell culture chimera system. This method can be easily applied to other genes of study and other viruses and, as such, will greatly simplify reverse genetics studies of variable viruses

Immunization with a synthetic consensus hepatitis C virus E2 glycoprotein ectodomain elicits virus-neutralizing antibodies

Global eradication of hepatitis C virus (HCV) infection will require an efficacious vaccine capable of eliciting protective immunity against genetically diverse HCV strains. Natural spontaneous resolution of HCV infection is associated with production of broadly neutralizing antibodies targeting the HCV glycoproteins E1 and E2. As such, production of cross-neutralizing antibodies is an important endpoint for experimental vaccine trials. Varying success generating cross-neutralizing antibodies has been achieved with immunogens derived from naturally-occurring HCV strains. In this study the challenge of minimising the genetic diversity between the vaccine strain and circulating HCV isolates was addressed. Two novel synthetic E2 glycoprotein immunogens (NotC1 and NotC2) were derived from consensus nucleotide sequences deduced from samples of circulating genotype 1 HCV strains. These two synthetic sequences differed in their relative positions in the overall genotype 1a/1b phylogeny. Expression of these constructs in Drosophila melanogaster S2 cells resulted in high yields of correctly-folded, monomeric E2 protein, which were recognised by broadly neutralizing monoclonal antibodies. Immunization of guinea pigs with either of these consensus immunogens, or a comparable protein representing a circulating genotype 1a strain resulted in high titres of cross-reactive anti-E2 antibodies. All immunogens generated antibodies capable of neutralizing the H77 strain, but NotC1 elicited antibodies that more potently neutralized virus entry. These vaccine-induced antibodies neutralized some viruses representing genotype 1, but not strains representing genotype 2 or genotype 3. Thus, while this approach to vaccine design resulted in correctly folded, immunogenic protein, cross-neutralizing epitopes were not preferentially targeted by the host immune response generated by this immunogen. Greater immunofocussing by vaccines to common epitopes is necessary to successfully elicit broadly neutralizing antibodies