Virus-host coevolution in a persistently coxsackievirus B3-infected cardiomyocyte cell line

Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1CVB3. CVB3 persistence in HL-1CVB3 cells represented a typical carrier-state infection with high levels (106 to 108 PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1CVB3 cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1CVB3 cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence

Anti-adenoviral artificial microRNAs Expressed from AAV9 vectors inhibit human adenovirus infection in immunosuppressed Syrian hamsters

Infections of immunocompromised patients with human adenoviruses (hAd) can develop into life-threatening conditions, whereas drugs with anti-adenoviral efficiency are not clinically approved and have limited efficacy. Small double-stranded RNAs that induce RNAi represent a new class of promising anti-adenoviral therapeutics. However, as yet, their efficiency to treat hAd5 infections has only been investigated in vitro. In this study, we analyzed artificial microRNAs (amiRs) delivered by self-complementary adeno-associated virus (scAAV) vectors for treatment of hAd5 infections in immunosuppressed Syrian hamsters. In vitro evaluation of amiRs targeting the E1A, pTP, IVa2, and hexon genes of hAd5 revealed that two scAAV vectors containing three copies of amiR-pTP and three copies of amiR-E1A, or six copies of amiR-pTP, efficiently inhibited hAd5 replication and improved the viability of hAd5-infected cells. Prophylactic application of amiR-pTP/amiR-E1A- and amiR-pTP-expressing scAAV9 vectors, respectively, to immunosuppressed Syrian hamsters resulted in the reduction of hAd5 levels in the liver of up to two orders of magnitude and in reduction of liver damage. Concomitant application of the vectors also resulted in a decrease of hepatic hAd5 infection. No side effects were observed. These data demonstrate anti-adenoviral RNAi as a promising new approach to combat hAd5 infection

Prevention of cardiac dysfunction in acute coxsackievirus B3 cardiomyopathy by inducible expression of a soluble coxsackievirus-adenovirus receptor

Background— Group B coxsackieviruses (CVBs) are the prototypical agents of acute myocarditis and chronic dilated cardiomyopathy, but an effective targeted therapy is still not available. Here, we analyze the therapeutic potential of a soluble (s) virus receptor molecule against CVB3 myocarditis using a gene therapy approach. Methods and Results— We generated an inducible adenoviral vector (AdG12) for strict drug-dependent delivery of sCAR-Fc, a fusion protein composed of the coxsackievirus-adenovirus receptor (CAR) extracellular domains and the carboxyl terminus of human IgG1-Fc. Decoy receptor expression was strictly doxycycline dependent, with no expression in the absence of an inducer. CVB3 infection of HeLa cells was efficiently blocked by supernatant from AdG12-transduced cells, but only in the presence of doxycycline. After liver-specific transfer, AdG12 (plus doxycycline) significantly improved cardiac contractility and diastolic relaxation compared with a control vector in CVB3-infected mice if sCAR-Fc was induced before infection (left ventricular pressure 59±3.8 versus 45.4±2.7 mm Hg, median 59 versus 45.8 mm Hg, P<0.01; dP/dtmax 3645.1±443.6 versus 2057.9±490.2 mm Hg/s, median 3526.6 versus 2072 mm Hg/s, P<0.01; and dP/dtmin −2125.5±330.5 versus −1310.2±330.3 mm Hg/s, median −2083.7 versus −1295.9 mm Hg/s, P<0.01) and improved contractility if induced concomitantly with infection (left ventricular pressure 76.4±19.2 versus 56.8±10.3 mm Hg, median 74.8 versus 54.4 mm Hg, P<0.05; dP/dtmax 5214.2±1786.2 versus 3011.6±918.3 mm Hg/s, median 5182.1 versus 3106.6 mm Hg/s, P<0.05), respectively. Importantly, hemodynamics of animals treated with AdG12 (plus doxycycline) were similar to uninfected controls. Preinfection induction of sCAR-Fc completely blocked and concomitant induction strongly reduced cardiac CVB3 infection, myocardial injury, and inflammation. Conclusion— AdG12-mediated sCAR-Fc delivery prevents cardiac dysfunction in CVB3 myocarditis under prophylactic and therapeutic conditions

Gene therapy as antiviral treatment by Coxsackievirus infection in the myokardium

Entzündungen des Myokards können zu schwerwiegenden akuten und chronischen Beeinträchtigungen der Herzfunktion bis hin zur Herzinsuffizienz führen. Coxsackie-B-Viren gelten dabei zu den häufigsten Viren, die eine virale Myokarditis verursachen können. Molekulare und gentherapeutische Verfahren können gezielt gegen definierte Strukturen vorgehen und besitzen somit das Potential, die Spezifität und Effizienz einer antiviralen Therapie bedeutend zu erhöhen. In der vorliegenden Arbeit wurden drei verschiedene gentherapeutische Strategien hinsichtlich ihrer Effizienz und Sicherheit in vitro und in vivo untersucht. Dabei ging es in erster Linie darum, die Infektion kardialer Zellen durch CVB3, dessen Replikation und Schädigung der Wirtszelle zu verhindern. Die ersten beiden Strategien basierten auf dem Mechanismus der RNA-Interferenz, wobei der ersten Ansatz das Ziel hatte, die Anheftung des Virus an die Wirtszelle durch Silencing seines zellulären Rezeptors zu inhibieren. Dabei gelang es, die Expression des Coxsackie- Adenovirus-Rezeptors (CAR) mittels Adenovektor-expremierter short hairpin (sh)RNAs über 90 % zu inhibieren. Daraus resultierend, konnte eine Verringerung der viralen Replikation um 97 % in der kardialen Mauszelllinie HL-1 und 90 % in Rattenkardiomyozyten (NRCM) erzielt werden. In der zweiten Strategie wurden zwei shRNAs, spezifisch gegen die CVB3-RNA-abhängige RNA- Polymerase (RdRP) von einem Adeno-assozierten Virus (AAV)-Vektor (scAAV2/6-shRdRP2.4) in kardiale Zielzellen eingebracht und somit die virale Replikation in NRCM bis zu 3log10-Stufen inhibiert. Durch AAV2/9-Vektoren konnte anschließend mittels kardiospezifischen Gentransfers, die virale Replikation in einem CVB3-Myokarditis-Mausmodell um 70 % verringert werden. Zusätzlich wurde erstmals mit RNAi-Technologie ein therapeutischer Effekt auf die Virus-induzierte kardiale Pumpfunktionsstörung erzielt. Der dritte Teil der Arbeit beschäftigte sich mit der Blockade der Virus-Rezeptor-Interaktion durch lösliche Rezeptorproteine. Dabei wurde mittels Adenovektoren ein lösliches, rekombinantes Rezeptorprotein (sCAR-Fc) expremiert und dessen inhibitorische Wirkung auf die CVB3-Infektion untersucht. Um die Sicherheit bei in vivo-Anwendung zu erhöhen, wurde die Expression des sCAR-Fc mittels Tet-On-Genexpressionssystems pharmakologisch regulierbar gemacht. Dabei war es möglich die Basalaktivität des Tet-On-Systems auf ein undetektierbares Level zu minimieren, ohne die Transaktivierbarkeit negativ zu beeinflussen. Durch die Expression von sCAR-Fc konnte in vitro eine Inhibierung von CVB3 bis zu 11log10-Stufen erreicht werden. Anschließend wurde in einem CVB3-Myokarditis- Mausmodel durch prophylaktische Anwendung des Vektor-expremierten sCAR-Fc eine komplette Inhibierung viral bedingten Myokarditis erreicht.. Wurde die sCAR-Fc Expression zeitgleich mit einer CVB3-Infektion bzw. 24 Stunden später induziert, konnte eine Inhibierung der kardialen Virusinfektion und Inflammation beobachtet werden, sowie eine signifikante Abmilderung der Virus- bedingte Verschlechterung der kardialen Pumpfunktion.Inflammation of the myocard can lead to severe acute and chronical impairment of heart function and even to cardiac insufficiency. Enteroviruses and especially coxsackie-B-viruses are one of the most common causes of viral myocarditis. Molecular and gene therapeutic trials can be directed against definite structures. They offer the potential to considerably increase specificity and thus efficiency of therapy. In this thesis different gene therapeutic methods were tested for their in vitro and in vivo efficiency in preventing infection of cardiac cells with coxsackievirus B3 (CVB3). The first and second strategy was based on RNA interference with the first strategy being directed against virus attachment to the host cell through down- regulation of the cellular coxsackievirus-adenovirs receptor (CAR). CAR was down-regulated by over 90% by adenoviral vectors encoding short hairpin (sh)RNAs directed against CAR. This leads to reduction of viral replication by 97% in the cardiac cell line HL-1 and by 90% in neonatal rat cardiomyocytes (NRCM). The second RNAi based strategy employed two shRNAs directed against a viral target, namely the RNA-dependent RNA-polymerase (RdRP) of CVB3. The corresponding shRNAs were encoded by an adenovirus-associated virus (AAV) vector (scAAV2/6-shRdRP2.4). NRCM transduced by this vector before CVB3 infection reduced viral replication up to 3log10. Furthermore AAV2/9 vectors were used for cardiac-specific gene transfer and scAAV2/9-shRdRP2.4 reduced viral replication in a CVB3-myocarditis mouse model by 70 %. For the first time it was possible to achieve a therapeutic effect on virus-induced deficiency of the cardiac function by an RNAi-based antiviral therapy by improvement of systolic and diastolic parameters. The third part of this thesis was based on a block of virus-receptor interaction by soluble receptor proteins. Therefore a recombinant soluble receptor protein (sCAR-Fc) was expressed by adenoviral vectors to evaluate the inhibitory effect on CVB3-infection. To improve the safety of this approach by in vivo application, the sCAR-Fc was expressed with a regulatable tet-on gene expression system. The tet-on system in this thesis shows no leakiness by strong trans-activation of sCAR-Fc expression. Expression of sCAR-Fc inhibited an in vitro infection of CVB3 up to 11log10. In a myocarditis mouse model cardiac virus infection and virus-induced inflammation was completely inhibited through application of vector-expressed sCAR-Fc before CVB3 infection. Additionally, sCAR-Fc was able to protected mice for CVB3-induced cardiac dysfunction. Concomitant induction of sCAR-Fc led to strong reduction of cardiac CVB3 infection, myocardial injury and inflammation. Furthermore, sCAR-Fc expression concomitant with CVB3 infection shows significantly improvement of cardiac contractility and diastolic relaxation, compared to CVB3 infected control mice. This work shows that antiviral RNAi-based therapy with shRNAs directed against CAR as a cellular target and RdRP as a viral target and the soluble receptor protein, sCAR-Fc are able to prevent or reduce CVB3-induced myokarditis

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence

A Conserved Cysteine Residue in Coxsackievirus B3 Protein 3A with Implication for Elevated Virulence

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence

A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca²⁺ Uptake into the Sarcoplasmic Reticulum

<div><p>In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca²⁺ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca²⁺ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca²⁺ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.</p></div

Vector dose dependence of amiR-155-PLBr, shPLBr and PLB mRNA expression in cardiomyocytes (CM) at post transduction days 4 and 14.

<p>CM were transduced with indicated doses of scAAV6-shPLBr and scAAV6-amiR155-PLBr and analyzed for expression of processed shPLBr and amiR155-PLBr normalized to 18 S rRNA (A) as well as PLB mRNA normalized to GAPDH mRNA using qRT-PCR (B). All values in A and B were normalized to that obtained for the cells transduced with the lowest scAAV6-shPLBr vector dose. The column labeled no add contains nontransduced cells. *p<0.05 vs. cells transduced with the same dose of the scAAV6-shPLBr vector. ^#p<0.05 vs. non-transduced cells. a.u., arbitrary units.</p

Alterations in the global protein pattern in scAAV6-amiR155-PLBr and scAAV6-shPLB transduced cardiomyocytes assessed by shot gun proteomics and validation by Western Blot.

<p>(A) Scatter plot of protein intensities of cardiomyocytes (CM) 14 days after transduction with 25×10³ vg/c of scAAV6-shPLBr (shPLBr), scAAV6-amiR155-PLBr (amiR155-PLBr) or the control vectors scAAV6-shCon (shCon) and scAAV6-amiR155-Con (amiR155-Con). Each dot represents one protein. Red dots represent proteins displaying significantly different levels in comparison to control experiment (fold change >1.5, p<0.01). PLB is phospholamban protein. (B and C) Western Blot analysis of STAT1 and STAT3. CM were transduced and analyzed at 14 days after transduction as in A. (B) Quantitative analysis of three bioreplicates per assay. STAT-specific signal intensities were normalized to GAPDH as loading control. (C) Western Blot images of total and phosphorylated STAT1 and STAT3 were analyzed on 3 separate membranes. GAPDH was detected as a house keeping protein on each membrane. *p<0.05 significant intergroup difference as indicated; n.s., not significantly different.</p