A Novel Approach To Display Structural Proteins of Hepatitis C Virus Quasispecies in Patients Reveals a Key Role of E2 HVR1 in Viral Evolution

ABSTRACT Hepatitis C virus (HCV) infection remains a major worldwide health problem despite development of highly effective direct-acting antivirals. HCV rapidly evolves upon acute infection and generates multiple viral variants (quasispecies), leading to immune evasion and persistent viral infection. Identification of epitopes of broadly neutralizing anti-HCV antibodies (nAbs) is critical to guide HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins. The HCV genome (JFH1 strain) lacking the structural protein-coding sequence can be efficiently rescued by ectopic expression of core-E1-E2-p7-NS2 (core-NS2) or core-E1-E2-p7 (core-p7) in trans, leading to production of single-round infectious virions designated HCVΔS. JFH1-based HCVΔS can be also rescued by expressing core-NS2 of other HCV genotypes, rendering it an efficient tool to display the structural proteins of HCV strains of interests. Furthermore, we successfully rescued HCVΔS with structural proteins from clinical isolates. Multiple viral structural proteins with different sensitivities to nAbs were identified from a same patient serum, demonstrating the genetic diversity of HCV quasispecies in vivo. Interestingly, the structural protein-coding sequences of highly divergent viral quasispecies from the same patient can be clustered based on their hypervariable region 1 (HVR1) in viral envelope protein E2, which critically dictates the sensitivity to neutralizing antibodies. In summary, we developed a novel reverse genetics system that efficiently displays viral structural proteins from HCV clinical isolates, and analysis of quasispecies from the same patient using this system demonstrated that E2 HVR1 is the major determinant of viral evolution in vivo. IMPORTANCE A cell culture model that can recapitulate the diversity of HCV quasispecies in patients is important for analysis of neutralizing epitopes and HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins (HCVΔS). This system can be used to display structural proteins of HCV strains of multiple genotypes as well as clinical isolates. By using this system, we showed that multiple different HCV structural proteins from a same patient were displayed on HCVΔS. Interestingly, these variant structural proteins within the same patient can be classified according to the sequence of HVR1in E2, which dictates viral sensitivity to nAbs and viral evolution in vivo. Our work provided a new tool to study highly divergent HCV quasispecies and shed light on underlying mechanisms driving HCV evolution.

Control of Alphavirus Replication in Neurons

Sindbis virus causes age-dependent encephalomyelitis in mice. Young mice and immature neurons replicate the virus to high titers and die from infection while older mice and mature neurons restrict replication and survive infection. Studies to identify factors that affect maturation-dependent virus replication in neurons have used AP-7 rat olfactory neuronal cells that can be differentiated in vitro, and have identified roles for host transcription factors interferon regulatory protein (IRF) 7 and NF-B and viral proteins E2 and nsP3. IRF7 is required for neuronal survival and deficiency leads to paralysis and death of 4–6 weeks old C57BL/6 mice. Activation of NF-κB works in conjunction with PKR to facilitate replication in mature neurons by promoting shut-off of host protein synthesis and increasing translation of the viral structural proteins from subgenomic RNA. The macro domain of nsP3 binds and hydrolyses ADP-ribose from ADP-ribosylated proteins and is important for initiation of neuronal infection and for synthesis of viral structural proteins. Thus, neurons regulate translation of the structural proteins from subgenomic RNA required for the production of the infectious virus.

Hijacking of Host Calreticulin is Required for the White Spot Syndrome Virus Replication Cycle.

We have previously shown that multifunctional calreticulin (CRT), which resides in the endoplasmic reticulum (ER) and is involved in ER-associated protein processing, responds to infection with white spot syndrome virus (WSSV) by increasing mRNA and protein expression and by forming a complex with gC1qR and thereby delaying apoptosis. Here, we show that CRT can directly interact with WSSV structural proteins, including VP15 and VP28, during an early stage of virus infection. The binding of VP28 with CRT does not promote WSSV entry, and CRT-VP15 interaction was detected in the viral genome in virally infected host cells, and thus may have an effect on WSSV replication. Moreover, CRT was detected in the viral envelope of purified WSSV virions. CRT was also found to be of high importance for proper oligomerization of the viral structural proteins VP26 and VP28, and when CRT glycosylation was blocked with tunicamycin, a significant decrease in both viral replication and assembly was detected. Together, these findings suggest that CRT confers several advantages to WSSV from the initial steps of WSSV infection, to the assembly of virions. Therefore, CRT is required as a “vital factor” and is hijacked by WSSV for its replication cycle.ImportanceWhite spot syndrome virus (WSSV) is a double-stranded DNA virus and the cause of a serious disease in a wide range of crustaceans that often leads to high mortality rates. We have previously shown that the protein calreticulin (CRT), which resides in the endoplasmic reticulum (ER) of the cell, is important in the host response to the virus. In this report, we show that the virus uses this host protein to enter the cell and to make the host produce new viral structural proteins. Through its interaction with two viral proteins, the virus “hijacks” host calreticulin and uses it for its own needs. These findings provide new insight into the interaction between a large DNA virus and the host protein CRT, and may help in understanding the viral infection process in general.

Efficient Axonal Localization of Alphaherpesvirus Structural Proteins in Cultured Sympathetic Neurons Requires Viral Glycoprotein E

ABSTRACT Pseudorabies virus (PRV) glycoprotein E (gE) is a type I viral membrane protein that facilitates the anterograde spread of viral infection from the peripheral nervous system to the brain. In animal models, a gE-null mutant infection spreads inefficiently from presynaptic neurons to postsynaptic neurons (anterograde spread of infection). However, the retrograde spread of infection from post- to presynaptic neurons remains unaffected. Here we show that gE is required for wild-type localization of viral structural proteins in axons of infected neurons. During a gE-null PRV infection, a subset of viral glycoproteins, capsids, and tegument proteins enter and localize to the axon inefficiently. This defect is most obvious in the distal axon and growth cones. However, axonal entry and localization of other viral membrane proteins and endogenous cellular proteins remains unaffected. Neurons infected with gE-null mutants produce wild-type levels of viral structural proteins and infectious virions in the cell body. Our results indicate that reduced axonal targeting of viral structural proteins is a compelling explanation for the lack of anterograde spread in neural circuits following infection by a gE-null mutant.

Overexpression of 7a, a Protein Specifically Encoded by the Severe Acute Respiratory Syndrome Coronavirus, Induces Apoptosis via a Caspase-Dependent Pathway

ABSTRACT Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these “accessory” viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.