Repressors of viral infection

Abstract

The present invention pertains to a non-human genetically modified animal with increased susceptibility to infection with a human virus. The invention suggests to genetically impair the expression of newly identified viral infection repression factors CD302, Cr11, Ndufc2, AW112010, Scarb2 and Zc3hav1, which markedly improves infection with human viruses in none-human hosts. Furthermore provided are methods for the generation of the animal of the invention, methods for increasing or reducing the susceptibility of a cell to viral infection, methods for screening novel modulators of viral infection as well as new therapy options for the treatment of viral diseases, in particular hepatitis C.

Claims

1. A genetically modified non-human animal with increased susceptibility to an infection with a hepatitis C virus, wherein the genetically modified non-human animal comprises a genome with at least one genetic modification compared to a wild-type genome of said non-human animal, characterized in that the at least one genetic modification reduces the expression, function or stability of CD302, and/or Cr11, and the genome comprising at least one transgene for ectopic expression of human Occludin (OCLN), human SCARB1, human CLDN1 and/or human CD81.

2. The genetically modified non-human animal according to claim 1, wherein the at least one genetic modification reduces the expression, function or stability of CD302 and Cr11.

3. The genetically modified non-human animal according to claim 1, which is a mouse, rat, rabbit or guinea pig.

4. The genetically modified non-human animal according to claim 1, wherein the at least one genetic modification is a mutation of at least one nucleic acid residue in the genes of CD302 and CR11.

5. The genetically modified non-human animal according to claim 1, wherein the at least one genetic modification is a dominant negative expression construct, or a transgenic RNA interference construct comprising a sequence targeting the expression of one or more genes selected from the group consisting of CD302 and Cr11.

6. The genetically modified non-human animal according to claim 1, wherein the genome comprises at least one transgene for ectopic expression of OCLN, and/or human CD81.

7. The genetically modified non-human animal according to claim 1, further having a reduced expression of at least one endogenous anti-viral immune factor.

8. A method for enhancing susceptibility of a non-human cell to an infection with a hepatitis C virus, comprising genetically modifying the non-human cell to reduce in the non-human cell the expression, function and/or stability of CD302, and/or Cr11, wherein the genetically modified non-human cell comprises a genome comprising at least one transgene for ectopic expression of human Occludin (OCLN), human SCARB1, human CLDN1 and/or human CD81.

9. The method according to claim 8, wherein said non-human cell is a mammalian cell.

10. The method, according to claim 9, wherein the mammalian cell is a mouse cell.

11. The method, according to claim 8, which is conducted in vitro.

Description

(1) The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:

(2) FIG. 1: cDNA library generation

(3) FIG. 2: Library screening protocol for HCV species-specific restriction factors

(4) FIG. 3: Significant enrichment of x17 murine genes—potential restriction factors

(5) FIG. 4: Murine restriction factors do not inhibit viral replication (A) but viral entry (B).

(6) FIG. 5: Murine repression factors inhibit viral entry for many different viral genotypes

(7) FIG. 6: Murine Cd302 & Cr11 are expressed on the cell-surface of Huh7.5 over-expressing cells

(8) FIG. 7: siRNA knock-down of repression factors

(9) FIG. 8: SiRNA knockdown rescues infectivity in Huh7.5 over-expressing cells

(10) FIG. 9: human CD302 has antiviral activity (A) but is poorly expressed (not shown); combined expression of human CD302 and human Cr11 enhances the phenotype significantly.

(11) FIG. 10: Cd302 & Cr11 are expressed on the surface primary mouse hepatocytes

(12) FIG. 11: Infection by primary HCV from patient sera (sera 1a, 1b and 3a) is inhibited by mouse CD302 and mouse Cr11

EXAMPLES

Example 1

cDNA Library Generation (FIG. 1)

(13) To generate a Mouse IFNα-Induced Liver Library (MIILL), a single mouse was injected with IFNα and sacrificed at 4 hrs post IFN-induction. The liver (in an antiviral state) was immediately homogenized in Trizol and total RNA extracted. The PolyA+ component of the total RNA was enriched and reverse-transcribed into cDNA. The cDNA was amplified for x15 cycles using SMART oligos containing SfiI sites (extremely rare sequence in mammalian coding DNAs). Amplified cDNA was SfiI digested and size fractionated prior to ligation into pV1 (lentiviral vector).

Example 2

Library Screening Protocol for HCV Species-specific Restriction Factors (FIG. 2)

(14) Ligated fractions were electrorporated into ElectroMAX cells and resulting colonies sequenced to ascertain the library quality. Ligated fractions which contained high percentages of diverse full-length mouse ORFs were pooled and electroporated prior to solid phase amplication in approximately 8L medium. Prior to solid phase amplification the MIILL contained 4×10.sup.6 independent clones (good coverage considering there are only 2.2×10.sup.4 described mouse genes and an average tissue expresses 8×10.sup.3 genes—multiple copies of each gene are contained in the library before amplification). After solid phase amplification, the pV1 MIILL was extracted from transformed bacteria using a maxiprep protocol, aliquoted and stored at −20° C. The library was packaged into VSV-G enveloped pseudoparticles (pps) using via 3-plasmid transfection into 293T cells. VSV-G, HIV-1gag/pol and pV1 MIILL plasmids were transfected in equimolar amounts using Lipofectamine 2000 and supernatents harvested at 24 h and 48 h post sodium butyrate induction. Titres of MIILLpps were calculated via TCID50 limiting dilution onto Hela-TZMbl cells. Cells were fixed and stained 72 hrs post-infection and MIILLpps aliquoted and stored at −80° C. Permissiveness of different cell-lines for MIILLpp transduction was determined via FACS.

(15) Entry factor transgenic mice with a STAT1 knockout (4EF STAT1-/-) support only weak viral replication, indicating either a missing host factor or potent murine restriction factors. The hypothesis underlying the experimental design was that the human/murine species barrier to HCV infection is, in part, determined by murine specific restriction factors which are highly expressed in mouse hepatocytes. The inventors hypothesized that these factors may be IFN-inducible. To test this hypothesis, the inventors delivered the MIILL (1 or 2 particles per cell) to highly permissive n4mBid hepatoma cells (Kindly supplied by Charles Rice, Rockefeller). These cells are modified Huh7.5 cells which undergo programmed cell death upon HCV replication. The apoptosis cascade is started by cleavage of the Bid protein, however in n4mBid cells the cleavage site has been replaced by NS3/4A junction which is the recognition site for the HCV NS3 protease. Thus, when HCV replicates in n4mBid cells, the NS3 protease cleaves the modified Bid protein and apoptosis occurs. This was the platform on which the screen was based. First the MIILL was transduced into the n4mBid cells (4×10.sup.6 cells) so that every cell contained at least 1 integrated provirus encoding a murine ORF. Then iterative rounds of selection were performed with WT HCV (Jc1: MIILL2 MOI 100). Permissiveness to HCV/CoV infection was assessed using luciferase reporter viruses. After the selection experiment, a 200-fold reduction in permissiveness for HCV was observed in the cell population, which was attributable to enrichment of library delivered mouse genes which restrict HCV replication.

Example 3

Significant Enrichment of x17 Murine Genes—Potential Restriction Factors (FIG. 3)

(16) To identify the determinants of the reduction HCV permissiveness, RNAseq was performed on RNA extracted from transduced cells at the start of the experiment (S0) and compared with the cell population at the end of the experiment which was highly refractive for HCV infection (S2). RNAseq data was first mapped to the human transcriptome, to remove the cellular background and also to check the reduction in permissiveness was not due to depletion of an essential human co-factor over the course of the experiment (eg CD81). The remaining unmapped reads (1% of the total) were then mapped to the mouse transcriptome and represent the delivered library. The S0 mouse transcriptome was then compared to the S2 mouse transcriptome. Murine genes which were significantly enriched over the course of both experiments (FC>5; final RPKM >100) were further investigated (17 candidate genes). All transcriptomic data was analysed using CLC Genomics Workbench and individual points represent averages from 2 RNAseq technical replicates.

Example 4

Murine Restriction Factors do not Inhibit Viral Replication (FIG. 4A) but Viral Entry (FIG. 4B).

(17) Lentiviral overexpression of individual restriction candidates in Huh7.5 cells (Kindly supplied by Charles Rice, Rockefeller) identified 3 murine genes which are potent restrictors of HCV infection (>50% reduction in permissiveness). Further characterization of Cd302 and Cr11 then commenced. (Panel A) The effect on JFH-1 subgenome replication was assessed by comparison with 2 well characterized human ISGs (NOS2 and IRF1). Subgenome replication was largely unaffected by over-expression of murine Cd302 and Cr11. Over-expressing cells (Control, Cd302, Cr11 and Cd302/Cr11) were infected with Jc1 R2a reporter virus (panel B). Time course infections were conducted, again using NOS2 and IRF1 as controls. Cd302 and Cr11 potently restrict infection by HCV. Over-expression of both factors simultaneously results in 2.5 log reduction in the ability to infect hepatoma cells

Example 5

Murine Repression Factors Inhibit Viral Entry for Many Different Viral Genotypes (FIG. 5)

(18) Over-expressing cells (Cd302, Cr11, Ndufc2, AW112010, Scarb2 and Zc3hav1) were infected with renilla reporter viruses representing HCV genotypes 1-7 and luciferase counts measured at 72 hrs. Cells transduced with the empty vector (pWPI-empty) served as control and infection efficiency in these control cells was normalized to 100% (black bars). B: combining the disruption of multiple repression factors enhances the phenotype.

Example 6

Murine Cd302 & Cr11 are Expressed on the Cell-surface of Huh7.5 Over-expressing Cells (FIG. 6)

(19) Over-expressing cells (Control, Cd302 and Cr11) were trypsinized. Non-permeabilized cells were stained with anti-Cd302 or anti-Cr11 antibodies, with secondary only and isotype controls also included in addition to staining of a control cell-line. Fluorescence Activated Cell Sorting (FACS) was performed and these data indicate that murine Cd302 & Cr11 are expressed on the cell-surface of Huh7.5 over-expressing cells.

Example 7

siRNA Knock-down of Repression Factors (FIG. 7)

(20) Characterized siRNAs were ordered from Ambion targeting either Cd302 or Cr11. Three different siRNAs were tested for each gene in both over-expressing cells and in plated primary mouse hepatocytes. SiRNAs were transfected using Lipofectamine and incubated for 48hrs prior to RNA extraction. Knockdown of specific mRNA was then assessed by qPCR. These data revealed that Cd302_siRNA_1 and Cr11_siRNA_2 were most effective at silencing their respective mRNAs.

Example 8

SiRNA Knockdown Rescues Infectivity in Huh7.5 Over-expressing Cells (FIG. 8)

(21) Over-expressing cells (Control, Cd302, Cr11 and Cd302/Cr11) were transfected with siRNAs targeting CD81, Cd302 and Cr11 or a scrambled control using Lipofectamine and incubated for 24 hrs. Transfected cells were then infected with Jc1 R2a virus and incubated for 48 hrs prior to lysis and luciferase measurement. Transfection of control cells with siRNAs targeting the viral receptor CD81 resulted in a 50% reduction in the ability to infect. Conversely, transfection of over-expressing cells with cognate siRNAs targeting Cd302, Cr11 and Cd302/Cr11 resulted in a 50% 100% and 300% increase in the ability to infect respectively.

Example 9

Human CD302 has Antiviral Activity Alone, which is Increased in Combination with Human Cr11

(22) The human homologs of CD302 and Cr11 were tested for antiviral activity using the lentiviral over-expression system (panel A). Time course infections demonstrate the human CD302 is a potent restrictor of HCV entry while human Cr11 is not. However, a combined expression of human CD302 and Cr11 significantly increased antiviral activity (panel B). Total transcriptomic analyses of human primary hepatocytes from 3 donors indicates that human CD302 is expressed to modest levels in human hepatocytes (not shown). However, human Cr11 was expressed at a level below 1 RPKM.

Example 10

Cd302 & Cr11 are Expressed on the Surface Primary Mouse Hepatocytes (FIG. 10)

(23) Plated primary mouse hepatocytes from an FVBN mouse were trypsinized and non-permeabilized cells were stained with anti-Cd302 or anti-Cr11 antibodies and secondary controls (panel A). Fluorescence Activated Cell Sorting (FACS) was performed and these data indicate that murine Cd302 & Cr11 are expressed on the cell-surface of mouse hepatocytes. Control Huh7.5 cells were also included with no cell surface shift observed (panel B).

Example 11

HCV from Infected Human Patient Sera is Inhibited by Murine CD302 and Cr11

(24) Sec14L2 over-expressing Huh-7.5 cells (compare Saeed et al. 2015 Aug. 27;524(7566):471-5) were transduced with a control lentiviral, with mCD302 or with mCr11. Subsequently, these cells were inoculated with sera from patients infected with genotype 1a (black), 1b (blue) or 3a (red) viruses. Infection efficiency was quantified by RT-PCR. Virus load is decreased in mCD302 and mCR1L expressing cells indicating that these factors restrict primary, patient serum derived HCV.