Inhibition of Hepatitis E Virus Replication by Peptide-Conjugated Morpholino Oligomers

Inhibition of Hepatitis E Virus Replication by Peptide-Conjugated Morpholino Oligomers

Inhibition of Hepatitis E Virus Replication by Peptide-Conjugated Morpholino Oligomers Nan, Y., Ma, Z., Kannan, H., Stein, D. A., Iversen, P. I., Meng, X. J., & Zhang, Y. J. (2015). Inhibition of hepatitis E virus replication by peptide-conjugated morpholino oligomers. Antiviral Research, 120, 134-139. doi:10.1016/j.antiviral.2015.06.006 10.1016/j.antiviral.2015.06.006 Elsevier Accepted Manuscript http://cdss.library.oregonstate.edu/sa-termsofuse *Manuscript Click here to view linked References 1 1 2 3 4 Inhibition of Hepatitis E Virus Infection by Peptide-Conjugated Morpholino Oligomers 5 6 7 8 a a a‡ c d 9 Yuchen Nan , Zexu Ma , Harilakshmi Kannan , David A. Stein , Patrick I. Iversen , Xiang-Jin 10 Menge, and Yan-Jin Zhanga,b* 11 12 13 14 15 16 17 aVA-MD College of Veterinary Medicine, and bMaryland Pathogen Research Institute, 18 19 20 University of Maryland, College Park, MD; 21 22 c d 23 Department of Biomedical Science, and Department of Environmental and Molecular 24 25 Toxicology, Oregon State University, Corvallis, OR; 26 27 e 28 Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, 29 30 Virginia Polytechnic Institute and State University, Blacksburg, VA 31 32 33 34 35 36 37 38 39 ‡Present address: Merck & Co., Inc. West Point, PA. 40 41 42 43 * Address correspondence to: [email protected] 44 45 46 47 48 49 50 51 52 Total text words: 2952 53 54 55 56 57 58 59 60 61 62 63 64 65 1 2 2 3 4 ABSTRACT 5 6 7 Hepatitis E virus (HEV) infection is a cause of hepatitis in humans worldwide. Recently, 8 9 persistent and chronic HEV infections have been recognized as a serious clinical problem, 10 11 12 especially in immunocompromised individuals. To date, there are no FDA-approved HEV- 13 14 specific antiviral drugs. In this study, we designed and evaluated antisense peptide-conjugated 15 16 morpholino oligomers (PPMO) as potential HEV-specific antiviral compounds. Two genetically- 17 18 19 distinct strains of human HEV, genotype 1 Sar55 and genotype 3 Kernow C1, which cause acute 20 21 and chronic hepatitis, respectively, were used to evaluate PPMO inhibition of viral replication in 22 23 24 liver cells. Four anti-HEV PPMOs tested led to a significant reduction in the levels of viral RNA 25 26 and HEV capsid protein as well as luciferase yield from Sar55 replicons in S10-3 liver cells, 27 28 29 indicating an effective inhibition of HEV replication. PPMO HP1, which targets the ORF1 30 31 translation initiation region, was also effective against the genotype 3 Kernow C1 strain in 32 33 34 stably-infected HepG2/C3A liver cells. The antiviral activity observed was specific, dose- 35 36 responsive, potent and effective, suggesting that further exploration of HP1 as a potential HEV- 37 38 specific antiviral agent is warranted. 39 40 41 42 43 Keywords: hepatitis E virus, morpholino oligomers, antisense, PPMO, antiviral. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 3 2 3 4 1. INTRODUCTION 5 6 7 Hepatitis E virus (HEV) is a single-stranded positive-sense RNA virus in the family 8 9 Hepeviridae (Emerson et al., 2004). HEV is an etiologic agent of acute hepatitis in humans, and 10 11 12 is endemic to various tropical and subtropical regions of the world, where it causes both sporadic 13 14 cases and epidemic outbreaks. Sporadic HEV infections with disease consequences also occur in 15 16 non-endemic regions (Kamar et al., 2014; Khuroo, 2011). In pregnant women, HEV infection 17 18 19 can lead to fulminant hepatitis that has a mortality rate of up to 30% (Jameel, 1999; Kumar et al., 20 21 2013). The World Health Organization (WHO) estimates that there are over 3 million acute cases 22 23 24 of hepatitis E and over 56,600 deaths annually (WHO, 2014). Hepatitis E is now a recognized 25 26 zoonotic disease, and strains of HEV from pig, chicken, mongoose, rabbit, rat, ferret, bat, fish 27 28 29 and deer have been genetically characterized (Haqshenas et al., 2001; Li et al., 2005; Meng, 2011; 30 31 Meng et al., 1997). More recently, chronic and persistent HEV infections have been reported in 32 33 34 increasing numbers of immunocompromised individuals in industrialized countries, including 35 36 organ transplant recipients and leukemia, lymphoma and HIV/AIDS patients (Kamar et al., 37 38 2014). Chronic hepatitis E has now become a significant clinical problem, warranting an 39 40 41 effective antiviral drug, especially for management of HEV infection in immunosuppressed 42 43 individuals (Kamar et al., 2014). 44 45 46 47 The HEV genome is approximately 7.2 kb in length and consists of three open reading 48 49 frames (ORFs) (Tam et al., 1991). ORF1 encodes a polyprotein which is supposed to be cleaved 50 51 to produce all the putative nonstructural proteins involved in HEV replication. ORF2 encodes the 52 53 54 capsid protein, the major structural protein in the HEV virion. ORF3 encodes a multi-functional 55 56 phosphoprotein that is essential for establishing HEV infection in macaques and pigs (Graff et al., 57 58 59 2005; Huang et al., 2007). A single bicistronic RNA was found to encode both ORF2 and ORF3, 60 61 62 63 64 65 1 4 2 3 4 which start from two closely spaced initiation codons in different reading frames (Graff et al., 5 6 7 2006). 8 9 10 HEV strains are highly diverse in sequence and those strains infecting humans are 11 12 classified into four major genotypes (genotypes 1-4) (Lu et al., 2006). All four genotypes of 13 14 15 human HEV belong to the genus Orthohepevirus. HEV genotypes 1 and 2 are restricted to 16 17 humans with no known animal reservoir, whereas genotypes 3 and 4 are known to be zoonotic, 18 19 20 and infect several animal species in addition to humans (Ahmad et al., 2011; Meng, 2010). The 21 22 identification of new HEV strains has prompted a recent proposal from the HEV Study Group of 23 24 25 ICTV to reorganize the family Hepeviridae in order to accommodate a more elaborate taxonomy 26 27 (Smith et al., 2014). 28 29 30 There is no specific anti-HEV drugs though it has been over two decades since the 31 32 33 sequence of the first full-length HEV genome was published (Tam et al., 1991). Off-label use of 34 35 ribavirin and pegylated interferon for treatment of acute and chronic hepatitis E patients has been 36 37 38 reported (Gerolami et al., 2011; Kamar et al., 2010; Mallet et al., 2010; Wedemeyer et al., 2012), 39 40 but there are safety and efficacy concerns with respect to those options. Ribavirin belongs to the 41 42 43 FDA Pregnancy Risk Category X and is not recommended for use by pregnant women. Thus, 44 45 there is a pressing need for the development of a specific anti-HEV therapeutic, especially for 46 47 treating immunocompromised patients and for chronic infections. 48 49 50 Phosphorodiamidate morpholino oligomers (PMO) are nuclease-resistant single-stranded 51 52 53 DNA analogs containing a backbone of morpholine rings and phosphorodiamidate linkages 54 55 (Summerton, 1999). PMO bind to mRNA by Watson–Crick base pairing and can interfere with 56 57 58 translation through steric blockade of the AUG-translation start site region. Conjugation of PMO 59 60 to an arginine-rich cell penetrating peptide, yielding peptide-conjugated PMO (PPMO), facilitate 61 62 63 64 65 1 5 2 3 4 delivery into cells (Abes et al., 2006; Summerton, 1999). PPMOs are water soluble and enter 5 6 7 cells readily. 8 9 10 In this study, PPMOs were tested for their ability to inhibit HEV replication in liver cells. 11 12 Several PPMOs demonstrated potent inhibition of HEV genotype 1 strain replication. Notably, 13 14 15 PPMO HP1 also effectively inhibited infection of genotype 3 Kernow C1 strain in liver cells. 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 6 2 3 4 2. MATERIALS AND METHODS 5 6 7 8 2.1. PPMO design and synthesis. Based on previous studies targeting viral RNAs with PPMOs 9 10 (Stein, 2008), the PPMOs for this study were designed to target genomic sequence of HEV Sar55 11 12 strain. PPMO HP1 and HP2 are complementary to the 5’end of genomic and subgenomic RNA, 13 14 15 respectively (Fig. 1 and Table 1). HP3U is complementary to sequence in the terminal region of 16 17 the 3’ UTR. HPN3 is reverse complement to HP1 and was intended to interfere with the 18 19 20 synthesis of genomic RNA. A nonsense-sequence PPMO CP1 (Zhang et al., 2007), having little 21 22 agreement with HEV or human mRNA sequences, was used as a negative control PPMO. CP1 23 24 25 with fluorescein conjugated at its 3’end (CP1-Fl) was used in the PPMO uptake assay. PPMOs 26 27 were synthesized with an arginine-rich cell-penetrating peptide (P7) conjugated at the 5’end at 28 29 30 AVI BioPharma Inc (Corvallis, OR) as previously described (Abes et al., 2006). 31 32 33 2.2. Cell-free translation. PPMO target sequences were cloned upstream of the luciferase gene 34 35 in reporter vector pCiNeoLucr as previously described (Zhang et al., 2007).

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