Longer Poly(U) Stretches in the 3′UTR Are Essential for Replication of the Hepatitis C Virus Genotype 4a Clone in in vitro and in vivo

The 3′ untranslated region (UTR) of the hepatitis C virus (HCV) genome plays a significant role in replication including the poly(U) tract (You and Rice, 2008). Here we established an HCV clone that is infectious in vitro and in vivo, from an Egyptian patient with chronic HCV infection and hepatocellular carcinoma (HCC). First, we inoculated the patient plasma into a humanized chimeric mouse and passaged. We observed HCV genotype 4a propagation in the chimeric mouse sera at 1.7 × 107 copies/mL after 6 weeks. Next, we cloned the entire HCV sequence from the HCV-infected chimeric mouse sera using RT-PCR, and 5′ and 3′ RACE methodologies. We obtained first a shorter clone (HCV-G4 KM short, GenBank: AB795432.1), which contained 9,545 nucleotides with 341 nucleotides of the 5′UTR and 177 nucleotides of the 3′UTR, and this was frequently obtained for unknown reasons. We also obtained a longer clone by dividing the HCV genome into three fragments and the poly (U) sequences. We obtained a longer 3′UTR sequence than that of the HCV-G4 KM short clone, which contained 9,617 nucleotides. This longer clone possessed a 3′-UTR of 249 nucleotides (HCV-G4 KM long, GenBank: AB795432.2), because of a 71-nucleotide longer poly (U) stretch. The HCV-G4-KM long clone, but not the HCV-G4-KM short clone, could establish infection in human hepatoma HuH-7 cells. HCV RNAs carrying a nanoluciferase (NL) reporter were also constructed and higher replication activity was observed with G4-KM long-NL in vitro. Next, both short and long RNAs were intra-hepatically injected into humanized chimeric mice. Viral propagation was only observed for the chimeric mouse injected with the HCV-G4 KM long RNA in the sera after 21 days (1.64 × 106 copies/mL) and continued until 10 weeks post inoculation (wpi; 1.45–4.74 × 107 copies/mL). Moreover, sequencing of the HCV genome in mouse sera at 6 wpi revealed the sequence of the HCV-G4-KM long clone. Thus, the in vitro and in vivo results of this study indicate that the sequence of the HCV-G4-KM long RNA is that of an infectious clone.


INTRODUCTION
Hepatitis C virus (HCV) belongs to the Flaviviridae family and genus Hepacivirus and possesses a single-stranded RNA with positive polarity (Choo et al., 1989). Approximately, 58 million people are estimated to be currently infected with HCV (WHO, 2021). Based on the diversity of the HCV genome, it has been classified into seven genetically distinct genotypes (HCV 1-7) (Smith et al., 2014;Tsukiyama-Kohara and Kohara, 2017). A recent report suggested the existence of eight HCV genotypes (Borgia et al., 2018). HCV genotype 4 appears to be mainly found in Africa and the Middle East; in particular, HCV isolated in Egypt is mostly classified as genotype 4 (Iles et al., 2014). The characteristics of HCV genotype 4 have not been fully clarified, mostly because of the lack of an available infection system. An infectious clone of HCV genotype 4a has been previously developed in vivo (Strain ED43) (Gottwein et al., 2010), and was cloned from the plasma of chimpanzees inoculated with plasma from patients with chronic hepatitis C.
After the discovery of HCV (Choo et al., 1989;Kolykhalov et al., 1997), an efficient infection system was first established using the HCV genotype 2a clone, JFH-1 (Lindenbach et al., 2005;Wakita et al., 2005). The HCV JFH-1 clones can replicate both in vitro and in vivo. This HCV infection system has contributed to the development of direct-acting antivirals (DAAs), which target HCV protease and polymerase and efficiently suppress viral replication (Spengler, 2018).
To better understand the characteristics of HCV genotype 4a in patient plasma, we aimed to construct an infectious clone for HCV genotype 4a from chimeric mice with humanized livers (Mercer et al., 2001) inoculated with Egyptian chronic hepatitis C patient plasma. We first cloned the full-length sequence of the HCV genome from the sera of these chimeric mice. We obtained clones with a shorter 3 UTR (G4 KM short) and longer 3 UTR (G4 KM long). It has been previously reported that 3 UTR plays a significant role in HCV replication, and this is composed of a stem-loop structure, poly(U) stretch and 3 X region (Tanaka et al., 1995;You and Rice, 2008). The infectivity of the HCV genotype 4a clones was examined by transfecting of RNA into cell culture and chimeric mice with humanized livers.

Infection of Hepatitis C Virus Genotype 4a in Chimeric Mice With Humanized Liver
Chimeric mice with humanized livers were purchased from PhoenixBio Co., Ltd. They were intravenously infected with plasma from Egyptian patients (HCV-positive, multiple hepatic B G4-KM long / 9,617 ntds. FIGURE 1 | Strategy for HCV-G4 cloning. (A) Strategy for HCV-G4 KM short cloning. Fragment A, B, C, D and 3 X were amplified from cDNA which was reverse transcribed with RNA isolated from HCV-G4-infected chimeric mouse sera. HCV genes were amplified using primer sets of each fragment as described in Table 1. (B) Fragment E was generated from Fragment A, B, and C using restriction enzymes. We obtained Fragment G with a 71-nucleotide longer poly U sequence, which was used for the construction of HCV-G4 KM long. focal lesions, splenomegaly) containing 10 4 copies of viral RNA (Katsume et al., 2013). After a few weeks, the mice were sacrificed, and sera were obtained for further experimentation. Isolation and quantitation of HCV-RNA were performed, as described previously (Takeuchi et al., 1999).

Cloning of Hepatitis C Virus Genotype 4a cDNA and Construction of an Infectious Clone
Construction of the infectious HCV genotype 4a clone was performed as follows. First, RNA was extracted from the serum of a chimeric mouse using the acid guanidinium thiocyanatephenol-chloroform extraction method, as described previously (Katsume et al., 2013) or SepaGene RV-R (Sanko Junyaku Co.). Then, cDNA was synthesized using Superscript III reverse transcriptase and amplified using PrimeSTAR GXL DNA polymerase (Takara Bio Co.) in fragment A-G and 3 X (Figure 1) with the primer sets described in Table 1. PCR conditions were as follows: 98 • C for 5 min, 30 cycles of 98 • C for 10 s, 60 • C for 15 s, and 68 • C for 1 min/kb, and finally 68 • C for 7 min. Amplified DNA fragments were subcloned into a low-copy number vector, pSMART (Lucigen Co.). Construction of the infectious HCV genotype 4a clone was performed using restriction enzymes or an In-Fusion HD cloning kit (Clontech Co.).
To clone the 5 UTR, total RNA was extracted from chimeric mouse sera (10 mL) with ISOGEN-LS and treated with DNase, followed by cDNA synthesis using Superscript III reverse transcriptase (Thermo Fisher Scientific Co.) and a 5 RACE antisense primer (5 RACE System for amplification of cDNA ends, ver. 2, Invitrogen Co.). The target region was amplified after dCTP tailing, according to the manufacturer's protocol. Cloning of the 3 UTR was performed using RNA extracted from chimeric mice; cDNA was synthesized using Superscript III reverse transcriptase and amplified, as reported previously (Tanaka et al., 1996;Tsuchihara et al., 1997).

Immunohistochemistry
The sectioned liver tissue of HCV-infected chimeric mice in OCT blocks was fixed with cold acetone at −20 • C for 5 min, washed once with PBS, treated with 0.03% H 2 O 2 for 10 min at room temperature, blocked with Odyssey blocking buffer (LI-COR Co.), and reacted with anti-HCV core RR8 (Ishida et al., 2001) biotinylated antibody and streptavidin-Alexa488. Stained tissues were observed using fluorescent microscope (BZ-X700, Keyence Co.).

Construction of Hepatitis C Virus -G4 Expressing NL Reporter and Replication Assay
To monitor replication, the HCV-G4 NL reporter clone was constructed by digesting HCV-G4KM long or G4 KM short clones with NsiI and inserted in the NL gene fused at its C-terminus to the foot and mouth disease virus (FMDV) 2A autoprotease after amplification with primers (IF-G4p7-nLuc-F21 5 -gcccgaaagagcttatgcaATGAACTCCTTCTCCACAAGC-3 , IF-G4NS2-2A-R20 5 -cacctcctgatcataGGGCCCTGGGTTGGACT CGA-3 ) using PCR targeting a region between p7 and NS2, as described (Shimakami et al., 2011;Yamane et al., 2014), with the In Fusion-HD cloning kit. Transfection of HCV-G4 NL reporter RNA was performed by electroporating 10 µg of in vitro transcribed viral RNA into 5 × 10 6 Huh-7.5 cells stably expressing SEC14L2 (Saeed et al., 2015) with a Gene Pulser Xcell Total System (250V, 950 µF and 50 ) (Yamane et al., 2014). Secreted NL activity was measured in 20 µL aliquots of the supernatant fluids using the Nano-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol. The luminescent signal was measured using a Mithras LB940 Multimode Microplate Reader (Berthold).

Ethical Statement
All experimental protocols in this study, including animal experiments, were approved by the regional ethics committee of Kagoshima University (K28002), Tokyo Metropolitan Institute of Medical Science, and Phoenix Bio Co. Patient samples were obtained according to the Declaration of Helsinki and approved by the Suez Canal University (Egypt).

Construction of a Full Genome Hepatitis C Virus -Genotype 4a Clone
After obtaining sufficient quantities of mouse sera containing the infectious HCV genotype 4a, we started the cloning process. To obtain the entire 5 UTR region, we performed 5 RACE and obtained 30 clones ( Figure 3A). Three clones contained the fulllength sequence of the 5 UTR, which was 341 bases. Next, we obtained the 3 UTR using the 3 RACE method and obtained a 177-nucleotide sequence ( Figure 3B).
We amplified the HCV genome with high-fidelity DNA polymerase; the amplification region was divided into four sections (fragments A, B, C, D and 3 X; Figure 1A) and subcloned into the pSMART vector. As a result of cloning of the fragment D, we obtained a 71-nucleotide longer stretch of poly(U) sequence in the 3 UTR in Fragment G than that of the HCV-G4 KM short clone ( Figure 1B). We determined the entire sequence of this clone and named it HCV-G4 KM short (9,545 nucleotides, GenBank: AB795432.1, Figure 1A and Supplementary Figure 1). We also obtained a 71-nucleotide longer 3 -UTR sequence than that of the HCV-G4 KM short clone, and the 3 UTR of this clone was 249 nucleotides (HCV-G4 KM long; 9,617 nucleotides; GenBank: AB795432.2, Figure 1B and Supplementary Figure 2).

Construction of the Infectious HCV-Genotype 4a Clone
Next we synthesized the RNA from HCV-G4 KM short and HCV-G4 KM long clones using T7 RNA polymerase and examined their lengths via formaldehyde agarose gel electrophoresis ( Figure 4A). The expression of viral proteins from the HCV-G4 KM short and HCV-G4 KM long clones was determined by T7 vaccinia virus infection and examined by immunoblotting using specific antibodies. As a result, all HCV proteins were expressed with an appropriate size ( Figure 4B).
To further define the replication activity of HCV-G4 KM clones, we constructed NL-inserted chimeric clones ( Figure 5A). After 5-8 days of transfection into Huh-7.5 cells expressing SEC14L2 (Yamane et al., 2014), we observed replication of the G4 KM long clone, which could be significantly suppressed by the treatment with DAA. In contrast, the G4 KM short clone failed to replicate to detectable levels ( Figure 5B).
HCV-G4 KM short or HCV-G4 KM long RNA was synthesized in vitro, and purified RNA was intra-hepatically FIGURE 5 | Construction of NL-containing HCV-G4 expression vectors, HCV-G4 KM long-NL and HCV-G4 KM short-NL (A). Poly (U) stretch in 3 UTR of HCV-G4 KM long is indicated as a black box. (B) These clones were used to infect Huh-7.5 cells expressing SEC14L2 and measured after day 5, 6, 7 and 8 (n = 4). Their replication activity was measured by NL assays. Treatment with DAA (30 µM sofosbuvir) decreased their replication. Significant differences based on the control were evaluated by statistical analysis (t-test, 2-sided), and a p-value less than 0.05 was considered significant.
injected into chimeric mice (30 µg/mouse; Figure 6A). Mice were bled every week for 10 weeks and then sacrificed. HCV RNA could not be detected in any of the sera obtained from chimeric mice injected with HCV-G4 KM short RNA. After injection of the HCV-G4 KM long RNA, the level of HCV RNA in chimeric mouse sera showed a significant increase after 3 weeks post-infection (wpi; t-test, p < 0.05, 1.59 × 10 6 copies/mL), reaching 4.84 × 10 7 copies/mL at 6 wpi and it was continuously detected until 10 wpi (3.57 × 10 7 copies/mL, Figure 6B). HCV RNA was isolated from chimeric mouse sera and quantitated by RT-PCR at 6 wpi. We also confirmed the same sequence as that of HCV-G4 KM long by sequencing the isolated RNA. HCV core protein was only detected in HCV-G4 KM long infected chimeric mouse liver tissue ( Figure 6C). These results indicated infectivity in naïve chimeric mice. Together, the results of this study show the replication activity of the HCV-G4 KM long sequence in vivo.

DISCUSSION
The results of this study show the significance of the 3 UTR region in HCV-G4 infection. The HCV-G4 KM long clone has a 3 UTR that is 71-nucleotides longer than that of the HCV-G4 KM short clone. The HCV-G4 KM long clone can replicate in vitro and in vivo, but the HCV-G4 KM short clone could not. Thus, this study highlighted the importance of the 3 UTR for HCV genotype 4a viral infectivity. The reason why HCV-G4 KM short was abundantly cloned or its characteristics as a viral clone are still unclear at present. It might be due to the higher cloning efficiency of HCV-G4 KM short than HCV-G4 KM long. A previous study reported that deletion of the poly(U/UC) region in the 3 UTR decreases the suppressive effect of NS5A on translation of the full-length HCV genomic RNA (Hoffman et al., 2015). This suggests that translation of HCV-G4 KM short could be more pronounced than HCV-G4 KM long. When compared nucleotide sequence of G4-KM short and long, there were two silent mutations (No. 8939 G to A, No. 9080 G to A (Supplementary Figure 3A) in the coding region, which did not cause amino acid changes. In addition, the 3 UTR ply(U/C) stretch is 71 nucleotides shorter in HCV-G4 KM short than the long clone (Supplementary Figure 3B). The entire nucleotide homology of HCV-G4 KM long with that of an in vitro and in vivo infectious HCV JFH-1 clone (9,678 nucleotide, GenBank AB047639.1) was 62% (Wakita et al., 2005). The 5 UTR of the JFH-1 clone was 341 nucleotides, and the 3 UTR was 236 nucleotides ( Table 2). The 5 UTR of HCV-G4 KM long was 341 nucleotides, and the length of the 3 UTR was 249 nucleotides.

CONCLUSION
The HCV-G4 KM long clone is an infectious clone in vitro and in vivo. The infectious HCV genotype 4a clone and chimeric clones with NL established in this study should help to characterize the biological features and clarify the molecular basis of the pathogenesis of HCV genotype 4a.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Egypt-Japan University for Science and Technology. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Kagoshima University and Tokyo Metropolitan Institute.

AUTHOR CONTRIBUTIONS
KT-K and MK designed the experiments, reviewed the data, planned the experimental strategy, and wrote the manuscript. AT, DY, BK, YT, and YA performed the experiments. AE-G obtained patient samples and helped in the experimental design. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by grants from the Ministry of Health, Labor, and Welfare of Japan and Japan Agency for Medical Research and Development.