Entry and replication of positive-sense, RNA viruses

National Institute Of Allergy And Infectious Diseases

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Abstract

HCV represents a major global public health problem, infecting approximately 70 million people worldwide. There is currently no approved vaccine to counter HCV infection. The Center for Disease Control estimates more than 40,000 new infections annually in the US alone with an additional 1.5 million new infections in the rest of world. Chronic HCV infection is curable by an effective, albeit expensive, antiviral therapy. Drug treatment is not, however, a feasible route to worldwide eradication of HCV infection, as the cost of doing so would be prohibitive. Moreover, successful treatment of a patient infected with one viral genotype does not preclude re-infection with another. The drug treatment approach is also complicated by the fact that most affected individuals are unaware that they are infected, and many engage in risky behaviors, such as intravenous drug use. Simply put, the best long term solution is to invest considerable intellectual and financial resources in discovery and development of a polyvalent vaccine effective against most, if not all, HCV viral genotypes. Early appearance of neutralizing antibodies during acute HCV infection is associated with spontaneous viral clearance. Longitudinal changes in E2 glycoprotein-specific memory B cells from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection were characterized by single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (46 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV. HCV enters hepatocytes through a multistep process requiring a series of host cellular factors and the viral envelope glycoproteins E1 and E2. The HCV glycoproteins mediate cell targeting, endocytosis, and membrane fusion ultimately stimulated by endosomal acidification. At least four cellular factors are critical for HCV attachment and entry: CD81, scavenger receptor class B type I (SRBI), claudin-1 (CLDN), and occludin (OCLN). CD81 is ubiquitously expressed on a variety of cell lines, indicating a role secondary to hepatocyte-specific receptor binding. CD81 translocates with the virion to tight junctions and engages with late entry factors, CLDN and OCLN, in the endosome for acidification and entry. The majority of broadly neutralizing antibodies preclude interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81. We observed that low pH enhances CD81-LEL binding to E2 and determined the crystal structure of E2/CD81-LEL. Upon binding CD81, E2 extends an internal loop along the surface of CD81. Docking of the E2/CD81-LEL complex onto a membrane embedded, full length CD81 places the loop proximal to the membrane. Liposome flotation assays demonstrate that low pH and CD81-LEL increase E2 interaction with membranes, while structure-based mutants of highly conserved, hydrophobic amino acids abolish membrane binding. These data support a model that acidification and receptor binding result in a conformation change in E2 in preparation for membrane fusion.The molecular mechanisms thereafter, for mediating cell entry and membrane fusion for HCV, remain undefined. These comprehensive structural, biochemical, and biophysical results have established a foundation to better define the functional roles of the envelope glycoproteins in HCV infection for vaccine design.