Mapping glycoprotein structure reveals Flaviviridae evolutionary history.

Viral glycoproteins drive membrane fusion in enveloped viruses and determine host range, tissue tropism and pathogenesis1. Despite their importance, there is a fragmentary understanding of glycoproteins within the Flaviviridae2, a large virus family that include pathogens such as hepatitis C, dengue and Zika viruses, and numerous other human, animal and emergent viruses. For many flaviviruses the glycoproteins have not yet been identified, for others, such as the hepaciviruses, the molecular mechanisms of membrane fusion remain uncharacterized3. Here we combine phylogenetic analyses with protein structure prediction to survey glycoproteins across the entire Flaviviridae. We find class II fusion systems, homologous to the Orthoflavivirus E glycoprotein in most species, including highly divergent jingmenviruses and large genome flaviviruses. However, the E1E2 glycoproteins of the hepaciviruses, pegiviruses and pestiviruses are structurally distinct, may represent a novel class of fusion mechanism, and are strictly associated with infection of vertebrate hosts. By mapping glycoprotein distribution onto the underlying phylogeny, we reveal a complex evolutionary history marked by the capture of bacterial genes and potentially inter-genus recombination. These insights, made possible through protein structure prediction, refine our understanding of viral fusion mechanisms and reveal the events that have shaped the diverse virology and ecology of the Flaviviridae.

Structures of the Hepaci-, Pegi-, and Pestiviruses envelope proteins suggest a novel membrane fusion mechanism.

Enveloped viruses encode specialised glycoproteins that mediate fusion of viral and host membranes. Discovery and understanding of the molecular mechanisms of fusion have been achieved through structural analyses of glycoproteins from many different viruses, and yet the fusion mechanisms of some viral genera remain unknown. We have employed systematic genome annotation and AlphaFold modelling to predict the structures of the E1E2 glycoproteins from 60 viral species in the Hepacivirus, Pegivirus, and Pestivirus genera. While the predicted structure of E2 varied widely, E1 exhibited a very consistent fold across genera, despite little or no similarity at the sequence level. Critically, the structure of E1 is unlike any other known viral glycoprotein. This suggests that the Hepaci-, Pegi-, and Pestiviruses may possess a common and novel membrane fusion mechanism. Comparison of E1E2 models from various species reveals recurrent features that are likely to be mechanistically important and sheds light on the evolution of membrane fusion in these viral genera. These findings provide new fundamental understanding of viral membrane fusion and are relevant to structure-guided vaccinology.

GB Virus B and Hepatitis C Virus, Distantly Related Hepaciviruses, Share an Entry Factor, Claudin-1.

Due to increased and broadened screening efforts, the last decade has seen a rapid expansion in the number of viral species classified into the Hepacivirus genus. Conserved genetic features of hepaciviruses suggest that they have undergone specific adaptation and have evolved to hijack similar host proteins for efficient propagation in the liver. Here, we developed pseudotyped viruses to elucidate the entry factors of GB virus B (GBV-B), the first hepacivirus described in an animal after hepatitis C virus (HCV). GBV-B-pseudotyped viral particles (GBVBpp) were shown to be uniquely sensitive to the sera of tamarins infected with GBV-B, validating their usefulness as a surrogate for GBV-B entry studies. We screened GBVBpp infection of human hepatoma cell lines that were CRISPR/Cas9 engineered to ablate the expression of individual HCV receptors/entry factors and found that claudin-1 is essential for GBV-B infection, indicating the GBV-B and HCV share an entry factor. Our data suggest that claudin-1 facilitates HCV and GBV-B entry through distinct mechanisms since the former requires the first extracellular loop and the latter is reliant on a C-terminal region containing the second extracellular loop. The observation that claudin-1 is an entry factor shared between these two hepaciviruses suggests that the tight junction protein is of fundamental mechanistic importance during cell entry. IMPORTANCE Hepatitis C virus (HCV) is a major public health burden; approximately 58 million individuals have chronic HCV infection and are at risk of developing cirrhosis and liver cancer. To achieve the World Health Organization's target of eliminating hepatitis by 2030, new therapeutics and vaccines are needed. Understanding how HCV enters cells can inform the design of new vaccines and treatments targeting the first stage of infection. However, the HCV cell entry mechanism is complex and has been sparsely described. Studying the entry of related hepaciviruses will increase the knowledge of the molecular mechanisms of the first stages of HCV infection, such as membrane fusion, and inform structure-guided HCV vaccine design; in this work, we have identified a protein, claudin-1, that facilitates the entry of an HCV-related hepacivirus but with a mechanism not described for HCV. Similar work on other hepaciviruses may unveil a commonality of entry factors and, possibly, new mechanisms.

More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation.

Serological assays detecting neutralising antibodies are important for determining the immune responses following infection or vaccination and are also often considered a correlate of protection. The target of neutralising antibodies is usually located in the Envelope protein on the viral surface, which mediates cell entry. As such, presentation of the Envelope protein on a lentiviral particle represents a convenient alternative to handling of a potentially high containment virus or for those viruses with no established cell culture system. The flexibility, relative safety and, in most cases, ease of production of lentiviral pseudotypes, have led to their use in serological assays for many applications such as the evaluation of candidate vaccines, screening and characterization of anti-viral therapeutics, and sero-surveillance. Above all, the speed of production of the lentiviral pseudotypes, once the envelope sequence is published, makes them important tools in the response to viral outbreaks, as shown during the COVID-19 pandemic in 2020. In this review, we provide an overview of the landscape of the serological applications of pseudotyped lentiviral vectors, with a brief discussion on their production and batch quality analysis. Finally, we evaluate their role as surrogates for the real virus and possible alternatives.