Hepatitis C virus genetics affects miR-122 requirements and response to miR-122 inhibitors

Israelow, Benjamin; Mullokandov, Gavriel; Agudo, Judith; Sourisseau, Marion; Bashir, Ali Kashif; Maldonado, Andres Y.; Dar, Arvin C.; Brown, Brian D.; Evans, Matthew J.

doi:10.1038/ncomms6408

Cited by 69 publications

(75 citation statements)

References 40 publications

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“…For example, miR-122 has two seed-binding sites in the 5=UTR of the HCV RNA genome that are upstream of the internal ribosomal entry site and are necessary for viral replication (34). Reduced miR-122 binding decreases, while increased binding elevates HCV replication (3,22). Alternatively, we have postulated a role for apoptosis in HCV propagation, and miR-34a is known to regulate apoptosis in hepatocytes (10).…”

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confidence: 99%

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde

Ganesan

Natarajan

Zhang

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV ϩ AB expressed more IL-1␤, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV Ϫ AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression. HCV RNA; acetaldehyde; apoptosis; hepatocytes; macrophages ALCOHOL EXPOSURE EXACERBATES hepatitis C virus (HCV) infection severity, increases liver inflammation, and potentiates liver injury progression. However, the pathogenesis of HCV-alcohol interactions is not clear yet. The major difficulty in HCValcohol studies is related to the lack of adequate models that can recapitulate both viral replication and ethanol metabolism-two important features that potentiate liver injury progression in alcohol-abusing HCV patients. Human liver cell lines permissive for HCV (Huh7.5 or Huh 7.5.1) that are currently used for HCV-alcohol-related in vitro studies do not metabolize ethanol, while ethanol-metabolizing rodent liver cells cannot support HCV replication. Studies conducted with isolated human hepatocytes also have failed, since these cells require at least 4 -5 days to become infected with HCV; however, by this time, hepatocytes lose the expression and functional activity of CYP2E1 and alcohol dehydrogenase (ADH), the main ethanol-metabolizing enzymes (47). In vivo models of HCV infection are also questionable, since small rodents cannot be infected with human HCV, and they only transgenically express HCV proteins, in the absence of replicating virus. The best option i...

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confidence: 99%

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde

Ganesan

Natarajan

Zhang

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…2b, c), suggesting that these viruses are still dependent on miR-122. A recent study showed that HCV recombinant Jc1 with a G1A change in the S1 site (G28A in the genotype 2a genome) was selected from miR-122-antagonized Huh7.5 cells and was responsible for virus resistance to miR-122 inhibition (Israelow et al, 2014). We here tested G fi A, G fi U and G fi C changes at position 1 of the S1 and found that these mutations did not appear to affect the replication of the virus, with a high percentage of infected cells on day 1 post-transfection (Table 2 and Li et al, 2011a).…”

Section: Discussionmentioning

confidence: 99%

“…We demonstrated previously that introduction of selected mutations in the miR-122 binding site S1 conferred virus resistance to miravirsen treatment in cell culture (Li et al, 2011a); subsequent studies identified virus resistant to miravirsen treatment (Israelow et al, 2014;Ottosen et al, 2015). These observations indicate that HCV therapy using miR-122 antagomirs would also encounter the issue of virus escape.…”

Section: Introductionmentioning

confidence: 89%

Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir

Pham

Uzcátegui

et al. 2016

Journal of General Virology

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MicroRNA 122 (miR-122) stimulates the replication and translation of hepatitis C virus (HCV) RNA by binding to two adjacent sites, S1 and S2, within the HCV 5¢UTR. We demonstrated previously that the miR-122 antagomir miravirsen (SPC3649) suppresses the infection of HCV strain JFH1-based recombinants with HCV genotypes 1-6 5¢UTR-NS2 in human hepatoma Huh7.5 cells. However, specific S1 mutations were permitted and conferred virus resistance to miravirsen treatment. Here, using the J6 (genotype 2a) 5¢UTR-NS2 JFH1-based recombinant, we performed reverse-genetics analysis of S1 (ACACUCCG, corresponding to miR-122 seed nucleotide positions 8-1), S2 (CACUCC, positions 7-2), and ACCC (positions 1-4) at the 5¢ end of the HCV genome (5¢E); the CC at positions 2-3 of 5¢E is involved in miR-122 binding. We demonstrated that the 5¢E required four nucleotides for optimal function, and that G or A at position 3 or combined GA at positions 2-3 of 5¢E was permitted. In S1 and S2, several single mutations were allowed at specific positions. A UCC fi CGA change at positions 4-3-2 of S1, S2, or both S1 and S2 (S1/S2), as well as a C fi G change at position 2 of S1/S2 were permitted. We found that 5¢E mutations did not confer virus resistance to miravirsen treatment. However, mutations in S1 and S2 induced virus resistance, and combined S1 and/or S2 mutations conferred higher resistance than single mutations. Identification of miR-122 antagomir resistance-associated mutations will facilitate the study of additional functions of miR-122 in the HCV life cycle and the mechanism of virus escape to host-targeting antiviral approaches.

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“…We expressed ApoE or CAMP in 293T cells expressing CLDN1 (Fig. 8D) and infected them with an HCV mutant possessing a G28A substitution in the 5= untranslated region (UTR) of the genotype 2a JFH-1 strain (HCV122KO) that facilitates viral RNA replication in miR-122-deficient Huh7.5.1 cells (60). The results showed that the expression of CAMP has no effect on the replication of HCV122KO (Fig.…”

Section: Expression Screening and Identification Of Campmentioning

confidence: 99%

Human Cathelicidin Compensates for the Role of Apolipoproteins in Hepatitis C Virus Infectious Particle Formation

Puig-Basagoiti

Fukuhara

Tamura

et al. 2016

J Virol

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Exchangeable apolipoproteins (ApoA, -C, and -E) have been shown to redundantly participate in the formation of infectious hepatitis C virus (HCV) particles during the assembly process, although their precise role in the viral life cycle is not well understood. Recently, it was shown that the exogenous expression of only short sequences containing amphipathic ␣-helices from various apolipoproteins is sufficient to restore the formation of infectious HCV particles in ApoB and ApoE double-gene-knockout Huh7 (BE-KO) cells. In this study, through the expression of a small library of human secretory proteins containing amphipathic ␣-helix structures, we identified the human cathelicidin antimicrobial peptide (CAMP), the only known member of the cathelicidin family of antimicrobial peptides (AMPs) in humans and expressed mainly in bone marrow and leukocytes. We showed that CAMP is able to rescue HCV infectious particle formation in BE-KO cells. In addition, we revealed that the LL-37 domain in CAMP containing amphipathic ␣-helices is crucial for the compensation of infectivity in BE-KO cells, and the expression of CAMP in nonhepatic 293T cells expressing claudin 1 and microRNA miR-122 confers complete propagation of HCV. These results suggest the possibility of extrahepatic propagation of HCV in cells with low-level or no expression of apolipoproteins but expressing secretory proteins containing amphipathic ␣-helices such as CAMP. IMPORTANCEVarious exchangeable apolipoproteins play a pivotal role in the formation of infectious HCV during the assembly of viral particles, and amphipathic ␣-helix motifs in the apolipoproteins have been shown to be a key factor. To the best of our knowledge, we have identified for the first time the human cathelicidin CAMP as a cellular protein that can compensate for the role of apolipoproteins in the life cycle of HCV. We have also identified the domain in CAMP that contains amphipathic ␣-helices crucial for compensation and show that the expression of CAMP in nonhepatic cells expressing claudin 1 and miR-122 confers complete propagation of HCV. We speculate that low levels of HCV propagation might be possible in extrahepatic tissues expressing secretory proteins containing amphipathic ␣-helices without the expression of apolipoproteins. According to recent estimates, more than 160 million people worldwide are chronically infected with hepatitis C virus (HCV) (1), which causes liver-related pathologies that can result in life-threatening diseases such as cirrhosis and hepatocellular carcinoma (2). Chronic HCV infection is also associated with several extrahepatic manifestations, including immunological disorders such as cryoglobulinemia and B-cell non-Hodgkin lymphoma (3). A vaccine is not yet available, but recent advances in antiviral treatment, improving upon what was for many years the standard therapy of pegylated interferon alpha plus ribavirin, now include several direct-acting antivirals (DAAs) that have increased the rate of sustained virological response (SVR) up to 80 ...

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Hepatitis C virus genetics affects miR-122 requirements and response to miR-122 inhibitors

Cited by 69 publications

References 40 publications

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde

Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir

Human Cathelicidin Compensates for the Role of Apolipoproteins in Hepatitis C Virus Infectious Particle Formation

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