Expression Of Virus Glycoprotein Research Articles

Green tea extract reduces viral proliferation and ROS production during Feline Herpesvirus type-1 (FHV-1) infection

BackgroundFeline Herpesvirus type-1 (FHV-1) is a worldwide spread pathogen responsible for viral rhinotracheitis and conjunctivitis in cats that, in the most severe cases, can lead to death. Despite the availability of a variety of antiviral medications to treat this illness, mainly characterized by virostatic drugs that alter DNA replication, their use is often debated. Phytotherapeutic treatments are a little-explored field for FHV-1 infections and reactivations. In this scenario, natural compounds could provide several advantages, such as reduced side effects, less resistance and low toxicity. The purpose of this study was to explore the potential inhibitory effects of the green tea extract (GTE), consisting of 50% of polyphenols, on FHV-1 infection and reactive oxygen species (ROS) production.ResultsCrandell-Reese feline kidney (CRFK) cells were treated with different doses of GTE (10–400 µg/mL) during the viral adsorption and throughout the following 24 h. The MTT and TCID50 assays were performed to determine the cytotoxicity and the EC50 of the extract, determining the amounts of GTE used for the subsequent investigations. The western blot assay showed a drastic reduction in the expression of viral glycoproteins (i.e., gB and gI) after GTE treatment. GTE induced not only a suppression in viral proliferation but also in the phosphorylation of Akt protein, generally involved in viral entry. Moreover, the increase in cell proliferation observed in infected cells upon GTE addition was supported by enhanced expression of Bcl-2 and Bcl-xL anti-apoptotic proteins. Finally, GTE antioxidant activity was evaluated by dichloro-dihydro-fluorescein diacetate (DCFH-DA) and total antioxidant capacity (TAC) assays. The ROS burst observed during FHV-1 infection was mitigated after GTE treatment, leading to a reduction in the oxidative imbalance.ConclusionsAlthough further clinical trials are necessary, this study demonstrated that the GTE could potentially serve as natural inhibitor of FHV-1 proliferation, by reducing viral entry. Moreover, it is plausible that the extract could inhibit apoptosis by modulating the intrinsic pathway, thus affecting ROS production.

Abstract 1483: Sugar analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response

Abstract Background: Kaposi´s Sarcoma (KS) herpesvirus (KSHV) is one of the seven known human oncogenic viruses. KSHV infection precedes the development of KS, an AIDS-associated malignancy. While the incidence of AIDS-KS globally has seen a decline, this endothelial cancer is refractory to treatment. The standard of care for AIDS-KS has gone virtually unchanged for over 20 years and effective treatments against the oncovirus KSHV have yet to be developed. 2-DG (2-deoxy-D-glucose), 2-FDG (2-fluoro-deoxy-D-glucose), and2-DFM (2-deoxy-2-fluoro-D-mannose) are sugar analogs. 2-DG and 2-FDG are D-glucose analogs and strong glycolysis inhibitors. 2-DG is also a D-mannose analog so that can inhibit N-glycosylation. In this work we aimed to further characterize and compare the inhibition of the three drug analogs on KSHV replication and pathogenesis. Design/Methods: We determined the ability of each drug to inhibit KSHV reactivation and infection by flow cytometry using the KSHV producer cell line iSLK.219 Virus titers were measured by de novo infection of naïve AdHEK293 cells and quantifying the number of AdHEK293 infected cells. We further characterized sugar analog inhibition assessing viral glycoprotein expression by western blot and quantifying virion production by qPCR. To show that N-glycosylation impairment is the main contributor of virus replication inhibition, we carried out D-mannose rescue experiments and analyzed activation of the unfolded protein response (UPR). When UPR cannot be resolved, it leads to either apoptosis or autophagy. For this reason, we analyzed the cellular fate of reactivated sugar analog-treated cells by western blot using apoptotic and autophagic markers. Additionally, we calculated the cell death index using the IncuCyte® machine. Results: All three sugar analogs negatively affected KSHV replication and virion infectivity, but 2-DFM displayed the strongest inhibition. Viral glycoproteins K8.1 and gB were down-regulated and virion production was diminished especially with 2-FDG and 2-DFM sugar analogs. D-mannose could rescue viral glycoprotein expression and virion infectivity, but could not reverse the number of reactivated cells, indicating that N-glycosylation is the main target of the drugs. In line with this, all analogs triggered UPR and even overcame KSHV-induced suppression of the PERK pathway. Finally, 2-DG, 2-FDG, and 2-DFM played a protective role in reactivated cells, displaying the autophagic marker instead of the apoptotic signature observed in the untreated cells. Conclusion: Collectively, this work identifies the non-toxic inhibition of N-glycosylation by sugar analogs as an important viral target and reinforces the potential of 2-DG as an antiviral for KSHV and other enveloped viruses. We also found that 2-DFM is a more potent and target-oriented compound and a promising therapeutic antiviral agent against an oncogenic virus. Citation Format: Mariana Schlesinger, Christian McDonald, Anuj Ahuja, Carolina Alvez-Canete, Zelmira Nunez Del Prado, Julian Naipauer, Theodore Lampidis, Enrique Mesri, Noula Shembade. Sugar analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1483.

Apoptosis is mediated by FeHV-1 through the intrinsic pathway and interacts with the autophagic process

BackgroundAlthough FeHV-1 is a primary feline pathogen, little is known about its interactions with host cells. Its relationship with several cellular pathways has recently been described, whereas its interplay with the apoptotic process, unlike other herpesviruses, has not yet been clarified. The aim of this work was to evaluate whether FeHV-1 induces apoptosis in its permissive cells, as well as the pathway involved and the effects of induction and inhibition of apoptosis on viral replication.MethodsMonolayers of CRFK cells were infected at different times with different viral doses. A cytofluorimetric approach allowed the quantification of cells in early and late apoptosis. All infections and related controls were also subjected to Western blot analysis to assess the expression of apoptotic markers (caspase 3-8-9, Bcl-2, Bcl-xL, NF-κB). An inhibitor (Z-VAD-FMK) and an inducer (ionomycin) were used to evaluate the role of apoptosis in viral replication. Finally, the expression of autophagy markers during the apoptosis inhibition/induction and the expression of apoptosis markers during autophagy inhibition/induction were evaluated to highlight any crosstalk between the two pathways.ResultsFeHV-1 triggered apoptosis in a time- and dose-dependent manner. Caspase 3 cleavage was evident 48 h after infection, indicating the completeness of the process at this stage. While caspase 8 was not involved, caspase 9 cleavage started 24 h post-infection. The expression of other mitochondrial damage markers also changed, suggesting that apoptosis was induced via the intrinsic pathway. NF- κB was up-regulated at 12 h, followed by a gradual decrease in levels up to 72 h. The effects of apoptosis inhibitors and inducers on viral replication and autophagy were also investigated. Inhibition of caspases resulted in an increase in viral glycoprotein expression, higher titers, and enhanced autophagy, whereas induction of apoptosis resulted in a decrease in viral protein expression, lower viral titer, and attenuated autophagy. On the other hand, the induction of autophagy reduced the cleavage of caspase 3.ConclusionsIn this study, we established how FeHV-1 induces the apoptotic process, contributing to the understanding of the relationship between FeHV-1 and this pathway.

Autophagy up-regulation upon FeHV-1 infection on permissive cells.

FeHV-1 is a member of the Herpesviridae family that is distributed worldwide and causes feline viral rhinotracheitis (FVR). Since its relationship with the autophagic process has not yet been elucidated, the aim of this work was to evaluate the autophagy mediated by FeHV-1 and to determine its proviral or antiviral role. Our data showed that autophagy is induced by FeHV-1 in a viral dose and time-dependent manner. Phenotypic changes in LC3/p62 axis (increase of LC3-II and degradation of p62) were detected from 12 h post infection using western blot and immuno-fluorescence assays. In a second step, by using late autophagy inhibitors and inducers, the possible proviral role of autophagy during FeHV-1 infection was investigating by assessing the effects of each chemical in terms of viral yield, cytotoxic effects, and expression of viral glycoproteins. Our findings suggest that late-stage autophagy inhibitors (bafilomycin and chloroquine) have a negative impact on viral replication. Interestingly, we observed an accumulation of gB, a viral protein, when cells were pretreated with bafilomycin, whereas the opposite effect was observed when an autophagy inducer was used. The importance of autophagy during FeHV-1 infection was further supported by the results obtained with ATG5 siRNA. In summary, this study demonstrates FeHV-1-mediated autophagy induction, its proviral role, and the negative impact of late autophagy inhibitors on viral replication.

Modifications of the PI3K/Akt/mTOR axis during FeHV-1 infection in permissive cells.

FeHV-1 is the causative agent of infectious rhinotracheitis in cats. The relationship between viral infection and the PI3K/Akt/mTOR pathway, as well as its function in crucial physiological processes like as autophagy, apoptosis or the IFN induction cascade is known for other varicelloviruses. However, there is no information on whether autophagy is activated during FeHV-1 infection nor on how this infection modifies PI3K/Akt/mTOR pathway. In this work, we aim to elucidate the involvement of this pathway during cytolytic infection by FeHV-1 in permissive cell lines. Using a phenotypic approach, the expression of proteins involved in the PI3K/Akt/mTOR pathway was examined by Western blot analysis. The findings demonstrated the lack of modifications in relation to viral dose (except for phospho-mTOR), whereas there were changes in the expression of several markers in relation to time as well as a mismatch in the time of activation of this axis. These results suggest that FeHV-1 may interact independently with different autophagic signaling pathways. In addition, we found an early phosphorylation of Akt, approximately 3 h after infection, without a concomitant decrease in constitutive Akt. This result suggests a possible role for this axis in viral entry. In a second phase, the use of early autophagy inhibitors was examined for viral yield, cytotoxic effects, viral glycoprotein expression, and autophagy markers and resulted in inefficient inhibition of viral replication (12 h post-infection for LY294002 and 48 h post-infection for 3-methyladenine). The same markers were examined during Akt knockdown, and we observed no differences in viral replication. This result could be explained by the presence of a protein kinase in the FeHV-1 genome (encoded by the Us3 gene) that can phosphorylate various Akt substrates as an Akt surrogate, as has already been demonstrated in genetically related viruses (HSV-1, PRV, etc.). For the same reasons, the use of LY294002 at the beginning of infection did not affect FeHV-1-mediated Akt phosphorylation. Our findings highlight changes in the PI3K/Akt/mTOR pathway during FeHV-1 infection, although further research is needed to understand the importance of these changes and how they affect cellular processes and viral propagation.

HSV1716 Infects Multiple Myeloma Cells and Leads to CD138 Downregulation

Multiple myeloma (MM) is a plasma cell malignancy that accounts for approximately 10% of all hematological cancers. MM is classified as incurable due to a high relapse rate after the initial therapy and failure to achieve long-term minimal residual disease (MRD). Therefore, new therapies and combinations are needed. Oncolytic viruses (OVs) are genetically modified viruses that preferentially infect and lyse malignantly transformed cells. HSV1716 is a replication-restricted herpes-simplex virus type I (HSV-1)-based OV (HSV-OV) which has been evaluated in several clinical trials. HSV-OVs have been shown to infect and kill MM cell lines and primary malignant plasma cells. Furthermore, HSV-OV mediated target cell killing was demonstrated to be enhanced by natural killer (NK) cells, an immune cell subset that naturally recognizes malignantly transformed or virally infected cells. The underlying mechanisms behind HSV-OV-enhanced NK-tumor cell killing, however, have not yet been elucidated. Here, we aim to monitor phenotypic changes of MM cells upon HSV1716 infection and thus explore the basis for NK cell recognition of HSV-OV infected target MM cells. The MM cell lines RPMI8226 and MM1S were infected with HSV1716 at various multiplicities of infection (MOIs) over a three-day timecourse. Target cell death and expression of viral glycoproteins were measured at different timepoints by flow cytometry. HSV1716 caused oncolysis of 60% of RPMI8226, and 70% of MM1S cells 48h post-infection (p.i.) at an MOI of 0.5. Under these conditions, the expression of viral glycoprotein E on live cells reached 74% on RPMI8226 cells and 37% on MM1S cells. Primary NK (pNK) cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and co-cultured with infected and uninfected MM cells for 4 hours. CD107a as a marker for NK cell degranulation, as well as the release of IFNγ and TNF were measured on CD3-CD56+ pNK cells by flow cytometry. CD107a expression increased from 15.6% (+/- 0.9%) to 56.2% (+/- 2.4%) following infection of RPMI8226 cells and from 15.6% (+/- 3.4%) to 59.2% (+/- 4.5%) following infection of MM1S cells. Similarly, the expression of IFNγ and TNF increased by four- and three-fold, respectively, as compared to uninfected target cells. To monitor changes in the expression of NK cell ligands on infected MM cells, a 16-color flow cytometry panel was designed. RPMI8226 and MM1S cells were infected at different MOIs, and ligand expression was analyzed at 24h, 48h, and 72h p.i.. Most significantly, an early and progressive downregulation of CD138 expression from 96% on uninfected cells to 79 % (24h MOI 0.5), 70% (48h MOI 0.5), and 22% (72h MOI 0.5) on live RPMI8226 cells and from 99% on uninfected cells to 82% (24h MOI 0.5), 65% (48h MOI 0.5) and 23% (72h MOI 0.5) on live MM1S cells was observed. Of note, the expression of CD38 remained stable at each of these timepoints. While downregulation of HLA class I molecules was observed, the expression of other NK cells ligands on RPMI8226 or MM1S cells did not notably differ between infected and uninfected cells. Together, our data confirm that HSV-OV enhances NK cell-mediated killing of MM cells. While expression of ligands for activating NK cell receptors was not altered on infected versus uninfected RPMI8226 and MM1S cells, a strong downregulation of CD138 with maintained CD38 expression was observed upon HSV1716 infection. The implementation of oncolytic virotherapy as an immunotherapeutic platform for cancer treatment adds a new and promising therapeutic tool for combination therapies to the treatment options of MM.

Secretion of functional α1-antitrypsin is cell type dependent: Implications for intramuscular delivery for gene therapy

Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.

Synthesized Magnolol Derivatives Improve Anti-Micropterus salmoides Rhabdovirus (MSRV) Activity In Vivo.

Micropterus salmoides rhabdovirus (MSRV) is a primary viral pathogen in largemouth bass aquaculture, which leads to tremendous economic losses yearly. Currently, there are no approved drugs for the treatment and control of this virus. Our previous studies screened the herb Magnolia officinalis from many traditional Chinese medicines, and we isolated and identified magnolol as its main active compound against multiple rhabdoviruses, including MSRV. On the basis of the structure–activity relationship and pharmacophore model of magnolol, two new magnolol derivatives, namely, hydrogenated magnolol and 2,2′-dimethoxy-magnolol, were designed and synthesized. Their anti-MSRV activities were systematically investigated both in vitro and in vivo. By comparing the half-maximal inhibitory concentration (IC50), it was found that hydrogenated magnolol possessed a higher anti-MSRV activity than magnolol and 2,2′-dimethoxy-magnolol, with an IC50 of 13.37 μM. Furthermore, hydrogenated magnolol exhibited a protective effect on the grass carp ovary (GCO) cell line by reducing the cytopathic effect induced by MSRV. Further studies revealed that hydrogenated magnolol did not directly impact virions or interfere with MSRV adsorption. It worked within the 6–8 h of the phase of virus replication. In vivo treatment of MSRV infection with magnolol and hydrogenated magnolol showed that they significantly improved the survival rate by 44.6% and 62.7%, respectively, compared to MSRV-infected groups. The viral load measured by the expression of viral glycoprotein in the organs including the liver, spleen, and kidney also significantly decreased when fish were intraperitoneally injected at a dose of 20 mg/kg. Altogether, the structural optimization of magnolol via hydrogenation of the propylene groups increased its anti-MSRV activity both in vitro and in vivo. These results may provide a valuable reference for anti-MSRV drug discovery and development in aquaculture.

MARCH8 Restricts Influenza A Virus Infectivity but Does Not Downregulate Viral Glycoprotein Expression at the Surface of Infected Cells.

ABSTRACTMembrane-associated RING-CH8 (MARCH8) impairs the cell surface expression of envelope glycoproteins from different viruses, reducing their incorporation into virions. Using stable cell lines with inducible MARCH8 expression, we show that MARCH8 did not alter susceptibility to influenza A virus (IAV) infection, but virions released from infected cells were markedly less infectious. Knockdown of endogenous MARCH8 confirmed its effect on IAV infectivity. The expression of MARCH8 impaired the infectivity of both H3N2 and H1N1 strains and was dependent on its E3 ligase activity. Although virions released in the presence of MARCH8 expressed smaller amounts of viral hemagglutinin (HA) and neuraminidase (NA) proteins, there was no impact on levels of the viral HA, NA, or matrix 2 (M2) proteins detected on the surface of infected cells. Moreover, mutation of lysine residues in the cytoplasmic tails of HA, NA, and/or M2, or in the viral M1 protein, did not abrogate MARCH8-mediated restriction. While MARCH1 and -8 target similar immunological ligands and both restrict HIV-1, only MARCH8 inhibited IAV infectivity. Deletion of the N-terminal cytoplasmic (N-CT) domain of MARCH8 confirmed it to be a critical determinant of IAV inhibition. Of interest, deletion of the MARCH1 N-CT or its replacement with the MARCH8 N-CT resulted in acquisition of IAV restriction. Together, these data demonstrate that MARCH8 restricts a late stage in IAV replication by a mechanism distinct to its reported activity against other viruses. Moreover, we show that the N-CT of MARCH8 is essential for anti-IAV activity, whereas the MARCH1 N-CT inhibits its ability to restrict IAV.

Antiviral effect of Chinese herbal prescription JieZe-1 on adhesion and penetration of VK2/E6E7 with herpes simplex viruses type 2.

Ethnopharmacological relevanceThe Chinese Herbal Prescription JieZe-1(JZ-1), added and subtracted from Yihuang Decoction, a famous formula in the 12th year of Kangxi in Qing Dynasty, has a clear effect on Genital Herpes (GH) and no obvious adverse reactions occur clinically. JZ-1 also has preventive and therapeutic effects on Trichomonas vaginitis, Candida albicans vaginitis and GH in vitro and in vivo experiments.Aim of studyThe effect and mechanism of JZ-1 on anti-herpes simplex virus type 2(HSV-2) in vitro focusing on adhesion and penetration stages were investigated.Materials and methodsA model of HSV-2 infection of VK2/E6E7 was developed. In order to explore JZ-1's anti-HSV-2 effect in vitro, cell morphology, ultrastructural pathology, cell viability and expression of viral glycoprotein D (gD) were assessed at 6 h, 12 h, 18 h, and 24 h of JZ-1 treatment. Then we measured the exact time required for adhesion and penetration of HSV-2 into VK2/E6E7 among a series of times at room temperature and under temperature control techniques. We treated VK2/E6E7 with JZ-1, penciclovir, or berberine and explored the mechanism of JZ-1 in blocking HSV-2 adhesion and penetration of host cells by assessing the cell ultrastructural pathology, viability, viral proteins gB, gD, VP16, ICP5, and ICP4 and host cell proteins HVEM, Nectin-1, and Nectin-2.ResultsHSV-2 can fully adhere and penetrate into VK/E6E7 within 5 mins at room temperature while it takes 60mins under temperature control techniques. JZ-1 and penciclovir showed significant anti-HSV-2 effects, with improved host cell morphologies and increased host cell viabilities observed after treatment for 24 h. The anti-HSV-2 effect of JZ-1 can be detected after treatment for 6 h while that of penciclovir was not obvious until treatment for 12 h. JZ-1 showed distinct effect on HSV-2 adhesion and penetration stages by significantly reducing the expression of viral proteins gB, gD, VP16, ICP5, and ICP4, improving cell morphology and increasing cell viability. However, these effects were not exerted via downregulated expression of membrane fusion-related proteins such as HVEM, Nectin-1, or Nectin-2. The specific anti-HSV-2 mechanism of JZ-1 need to be further explored.ConclusionThe anti-HSV-2 effect of JZ-1 was superior to that of penciclovir and berberine in vitro, and was mainly mediated by enhancing host cell defense and blocking adhesion and penetration of HSV-2.

Anti-herpes activity of polysaccharide fractions from Stevia rebaudiana leaves

The antiviral potential of natural polysaccharide compounds has been demonstrated, especially against enveloped viruses and members of the Herpesviridae family. Two polysaccharide fractions obtained from Stevia rebaudiana (Bertoni) leaves, that were active against Herpes simplex virus type 1 (HSV-1) were studied to investigate their mode of action. Both polysaccharides - SFW (crude faction) and SSFK (homogeneous alkaline fraction) - exerted antiviral effects on the initial stages of HSV-1 infection by inhibiting viral adsorption and penetration. When added after virus internalization, both fractions decreased plaque size. The effect of the fractions was confirmed by investigating viral glycoprotein expression. Based on the mode of action of the polysaccharides demonstrated in the present work and on their selectivity index, the polysaccharides obtained from S. rebaudiana could be an alternative treatment of infections caused by HSV-1.

IC-Tagging methodology applied to the expression of viral glycoproteins and the difficult-to-express membrane-bound IGRP autoantigen

We have previously developed a methodology to produce protein microspheres (MS) that can be loaded with proteins of interest in living cells through their C or N-terminal tagging with the so-called IC-Tag. The IC-Tagging method has many applications ranging from the production of immobilized enzymes for industrial use to the production of subunit vaccines due to its intrinsic adjuvancy. Here we show the adaptation of the IC-Tagging to work inside the endoplasmic reticulum and bacteria, allowing us to produce properly modified viral glycoproteins. Additionally, we were able to express the Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), whose expression remained elusive to date possibly due to its toxicity when over-expressed. IGRP is an antigen of enormous pharmaceutical interest as it is specifically targeted during the autoimmune response taking place in both the Non-Obese Diabetic (NOD) mice and type 1 diabetes (T1D) patients leading to the destruction of insulin-producing beta cells.

A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

Responses to fibroblast-adapted human cytomegalovirus (HCMV) vaccines conform to CD8 T cell recognition paradigms

Abstract Recently, a rhesus CMV-based SIV vaccine that clears SIV from half of vaccinated monkeys was shown to elicit unconventional CD8 T cell responses to both SIV and RhCMV (IE1) antigens in all immunized monkeys. These responses were characterized by i) recognition of an unusually large number of peptide epitopes within each antigen, and ii) MHCII restriction by ~2/3 of responding CD8 T cells (Hansen et al, Science, 2013). Unconventional responses were only elicited by fibroblast-adapted rhCMV strain 68-1 that lacks ability to infect cells other than fibroblasts due to loss of pentameric viral glycoprotein expression. To determine whether fibroblast-adapted human CMV (HCMV) vaccines also elicit unconventional CD8 T cell responses, we exploited an ongoing phase I clinical trial of live HCMV vaccines. These vaccines have similar fibroblast-restricted tropism to rhCMV68-1, although the precise genetic lesions differ. We analyzed CD8 T cell responses in 12 seroconverters by stimulating PBMC with overlapping peptide pools covering 20 HCMV antigens. Seven subjects in this low dose trial (100 or 1000 pfu) made consistent, measurable CD8 T cell responses. All 7 responded to IE1; 3 also responded to 1–2 additional antigens. We have mapped responses to individual 15mers in 3 subjects, and all revealed highly focused responses to 1–3 epitopes within IE1. In one HLA-B08 positive subject, the two responses mapped to known immunodominant B08-restricted IE1 epitopes. We have mapped 4 other epitopes to date; of these, 3 are 9mers and are likely conventional, but one may be a 13mer. Thus far, in contrast to RhCMV in monkeys, the CD8 T cell response to fibroblast-adapted HCMV vaccines in humans appears to be predominantly conventional. Supported by NIH R21 AI116107

Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.

SummaryIn this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV‐G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV‐G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV‐G gene was not tremendously increased as compared with the non‐optimized one. In addition, similar levels of RABV‐G were obtained when α‐factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high‐level expression of RABV‐G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV‐G expression cassette indicating that folding was the limiting step for RABV‐G secretion. To circumvent this limitation, co‐overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1,ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV‐G reached 1230 ng ml−1. Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.

Controlled viral glycoprotein expression as a safety feature in a bivalent rabies-ebola vaccine

Using a recombinant rabies (RABV) vaccine platform, we have developed several safe and effective vaccines. Most recently, we have developed a RABV-based ebolavirus (EBOV) vaccine that is efficacious in nonhuman primates. One safety feature of this vaccine is the utilization of a live but replication-deficient RABV construct. In this construct, the RABV glycoprotein (G) has been deleted from the genome, requiring G trans complementation in order for new infectious viruses to be released from the initial infected cell. Here we analyze this safety feature of the bivalent RABV-based EBOV vaccine comprised of the G-deleted RABV backbone expressing EBOV glycoprotein (GP). We found that, while the level of RABV genome in infected cells is equivalent regardless of G supplementation, the production of infectious virus is indeed restricted by the lack of G, and most importantly, that the presence of EBOV GP does not substitute for G. These findings further support the safety profile of this replication-deficient RABV–EBOV bivalent vaccine.

Anterograde glycoprotein-dependent transport of newly generated rabies virus in dorsal root ganglion neurons.

Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

Influence of recombinant S2 cell population enrichment on rabies virus glycoprotein expression and especific RNA and DNA quantities

Background Different strategies have been evaluated for increasing the productivity of Drosophila melonogaster (S2) cells expressing recombinant rabies virus glycoprotein (RVGP). The maintenance of selective pressure during all the cultivation time and new methods for population enrichment with high level expressing cells are two methods which can increase the productivity through increased expression of RVGP gene. To understand how these methods can improve glycoprotein expression, analysis of the quantities of heterologous DNA and RNA were performed [1,2].

Attachment protein G of an African bat henipavirus is differentially restricted in chiropteran and nonchiropteran cells.

Henipaviruses are associated with pteropodid reservoir hosts. The glycoproteins G and F of an African henipavirus (strain M74) have been reported to induce syncytium formation in kidney cells derived from a Hypsignathus monstrosus bat (HypNi/1.1) but not in nonchiropteran BHK-21 and Vero76 cells. Here, we show that syncytia are also induced in two other pteropodid cell lines from Hypsignathus monstrosus and Eidolon helvum bats upon coexpression of the M74 glycoproteins. The G protein was transported to the surface of transfected chiropteran cells, whereas surface expression in the nonchiropteran cells was detectable only in a fraction of cells. In contrast, the G protein of Nipah virus is transported efficiently to the surface of both chiropteran and nonchiropteran cells. Even in chiropteran cells, M74-G was predominantly expressed in the endoplasmic reticulum (ER), as indicated by colocalization with marker proteins. This result is consistent with the finding that all N-glycans of the M74-G proteins are of the mannose-rich type, as indicated by sensitivity to endo H treatment. These data indicate that the surface transport of M74-G is impaired in available cell culture systems, with larger amounts of viral glycoprotein present on chiropteran cells than on nonchiropteran cells. The restricted surface expression of M74-G explains the reduced fusion activity of the glycoproteins of the African henipavirus. Our results suggest strategies for the isolation of infectious viruses, which is necessary to assess the risk of zoonotic virus transmission. Importance: Henipaviruses are highly pathogenic zoonotic viruses associated with pteropodid bat hosts. Whether the recently described African bat henipaviruses have a zoonotic potential as high as that of their Asian and Australian relatives is unknown. We show that surface expression of the attachment protein G of an African henipavirus, M74, is restricted in comparison to the G protein expression of the highly pathogenic Nipah virus. Transport to the cell surface is more restricted in nonchiropteran cells than it is in chiropteran cells, explaining the differential fusion activity of the M74 surface proteins in these cells. Our results imply that surface expression of viral glycoproteins may serve as a major marker to assess the zoonotic risk of emerging henipaviruses.

Varicella-zoster virus glycoprotein expression differentially induces the unfolded protein response in infected cells.

Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.