HCV genome-wide analysis for development of efficient culture systems and unravelling of antiviral resistance in genotype 4.

OBJECTIVE: HCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at developing infectious genotype 4 cell culture systems to understand the evolutionary genetic landscapes of antiviral resistance, which can help preserve the future efficacy of DAA-based therapy. DESIGN: HCV recombinants were tested in liver-derived cells. Long-term coculture with DAAs served to induce antiviral-resistance phenotypes. Next-generation sequencing (NGS) of the entire HCV-coding sequence identified mutation networks. Resistance-associated substitutions (RAS) were studied using reverse-genetics. RESULT: The in-vivo infectious ED43(4a) clone was adapted in Huh7.5 cells, using substitutions identified in ED43(Core-NS5A)/JFH1-chimeric viruses combined with selected NS5B-changes. NGS, and linkage analysis, permitted identification of multiple genetic branches emerging during culture adaptation, one of which had 31 substitutions leading to robust replication/propagation. Treatment of culture-adapted ED43 with nine clinically relevant protease-DAA, NS5A-DAA and NS5B-DAA led to complex dynamics of drug-target-specific RAS with coselection of genome-wide substitutions. Approved DAA combinations were efficient against the original virus, but not against variants with RAS in corresponding drug targets. However, retreatment with glecaprevir/pibrentasvir remained efficient against NS5A inhibitor and sofosbuvir resistant variants. Recombinants with specific RAS at NS3-156, NS5A-28, 30, 31 and 93 and NS5B-282 were viable, but NS3-A156M and NS5A-L30Δ (deletion) led to attenuated phenotypes. CONCLUSION: Rapidly emerging complex evolutionary landscapes of mutations define the persistence of HCV-RASs conferring resistance levels leading to treatment failure in genotype 4. The high barrier to resistance of glecaprevir/pibrentasvir could prevent persistence and propagation of antiviral resistance.

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants.

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance-associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1-4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156-RASs were not maintained. For genotypes 1 and 2, persistence of 156-RASs depended on genome-wide substitution networks, co-selected under continued PI treatment and identified by next-generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156-RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre-existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156-RASs, we observed genome-wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1-6 revealed 156-RASs as key determinants of high-level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1-3 156-variants, which might pose a threat to clinically relevant combination treatments.

HCV genotype 1-6 NS3 residue 80 substitutions impact protease inhibitor activity and promote viral escape.

BACKGROUND & AIMS: Protease inhibitors (PIs) are of central importance in the treatment of patients with chronic hepatitis C virus (HCV) infection. HCV NS3 protease (NS3P) position 80 displays polymorphisms associated with resistance to the PI simeprevir for HCV genotype 1a. We investigated the effects of position-80-substitutions on fitness and PI-resistance for HCV genotypes 1-6, and analyzed evolutionary mechanisms underlying viral escape mediated by pre-existing Q80K. METHODS: The fitness of infectious NS3P recombinants of HCV genotypes 1-6, with engineered position-80-substitutions, was studied by comparison of viral spread kinetics in Huh-7.5 cells in culture. Median effective concentration (EC50) and fold resistance for PIs simeprevir, asunaprevir, paritaprevir, grazoprevir, glecaprevir and voxilaprevir were determined in short-term treatment assays. Viral escape was studied by long-term treatment of genotype 1a recombinants with simeprevir, grazoprevir, glecaprevir and voxilaprevir and of genotype 3a recombinants with glecaprevir and voxilaprevir, next generation sequencing, NS3P substitution linkage and haplotype analysis. RESULTS: Among tested PIs, only glecaprevir and voxilaprevir showed pan-genotypic activity against the original genotype 1-6 culture viruses. Variants with position-80-substitutions were all viable, but fitness depended on the specific substitution and the HCV isolate. Q80K conferred resistance to simeprevir across genotypes but had only minor effects on the activity of the remaining PIs. For genotype 1a, pre-existing Q80K mediated accelerated escape from simeprevir, grazoprevir and to a lesser extent glecaprevir, but not voxilaprevir. For genotype 3a, Q80K mediated accelerated escape from glecaprevir and voxilaprevir. Escape was mediated by rapid and genotype-, PI- and PI-concentration-dependent co-selection of clinically relevant resistance associated substitutions. CONCLUSIONS: Position-80-substitutions had relatively low fitness cost and the potential to promote HCV escape from clinically relevant PIs in vitro, despite having a minor impact on results in classical short-term resistance assays. LAY SUMMARY: Among all clinically relevant hepatitis C virus protease inhibitors, voxilaprevir and glecaprevir showed the highest and most uniform activity against cell culture infectious hepatitis C virus with genotype 1-6 proteases. Naturally occurring amino acid changes at protease position 80 had low fitness cost and influenced sensitivity to simeprevir, but not to other protease inhibitors in short-term treatment assays. Nevertheless, the pre-existing change Q80K had the potential to promote viral escape from protease inhibitors during long-term treatment by rapid co-selection of additional resistance changes, detected by next generation sequencing.

Adapting SHIVs In Vivo Selects for Envelope-Mediated Interferon-α Resistance.

Lentiviruses are able to establish persistent infection in their respective hosts despite a potent type-I interferon (IFN-I) response following transmission. A number of IFN-I-induced host factors that are able to inhibit lentiviral replication in vitro have been identified, and these studies suggest a role for IFN-induced factors as barriers to cross-species transmission. However, the ability of these factors to inhibit viral replication in vivo has not been well characterized, nor have the viral determinants that contribute to evasion or antagonism of the host IFN-I response. In this study, we hypothesized that the host IFN-I response serves as a strong selective pressure in the context of SIV/HIV chimeric virus (SHIV) infection of macaques and sought to identify the viral determinants that contribute to IFN-I resistance. We assessed the ability of SHIVs encoding HIV-1 sequences adapted by serial passage in macaques versus SHIVs encoding HIV sequences isolated directly from infected individuals to replicate in the presence of IFNα in macaque lymphocytes. We demonstrate that passage in macaques selects for IFNα resistant viruses that have higher replication kinetics and increased envelope content. SHIVs that encode HIV-1 sequences derived directly from infected humans were sensitive to IFNα -induced inhibition whereas SHIVs obtained after passage in macaques were not. This evolutionary process was directly observed in viruses that were serially passaged during the first few months of infection-a time when the IFNα response is high. Differences in IFNα sensitivity mapped to HIV-1 envelope and were associated with increased envelope levels despite similar mRNA expression, suggesting a post-transcriptional mechanism. These studies highlight critical differences in IFNα sensitivity between HIV-1 sequences in infected people and those used in SHIV models.

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle.

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research.

Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance.

Various protease inhibitors (PIs) currently are becoming available for treatment of hepatitis C virus (HCV). For genotype 1, substitutions at NS3 protease positions 155, 156, and 168 are the main determinants of PI resistance. For other genotypes, similar substitutions were selected during PI treatment but were not characterized systematically. To elucidate the impact of key PI resistance substitutions on genotypes 2 to 6, we engineered the substitutions R155A/E/G/H/K/Q/T, A156G/S/T/V, and D/Q168A/E/G/H/N/V into HCV recombinants expressing genotype 2 to 6 proteases. We evaluated viral fitness and sensitivity to nine PIs (telaprevir, boceprevir, simeprevir, asunaprevir, vaniprevir, faldaprevir, paritaprevir, deldeprevir, and grazoprevir) in Huh7.5 cells. We found that most variants showed decreased fitness compared to that of the original viruses. Overall, R155K, A156G/S, and D/Q168A/E/H/N/V variants showed the highest fitness; however, genotype 4 position 168 variants showed strong fitness impairment. Most variants tested were resistant to several PIs. Resistance levels varied significantly depending on the specific substitution, genotype, and PI. For telaprevir and boceprevir, specific 155 and 156, but not 168, variants proved resistant. For the remaining PIs, most genotype 2, 4, 5, and 6, but not genotype 3, variants showed various resistance levels. Overall, grazoprevir (MK-5172) had the highest efficacy against original viruses and variants. This is the first comprehensive study revealing the impact of described key PI resistance substitutions on fitness and PI resistance of HCV genotypes 2 to 6. In conclusion, the studied substitutions induced resistance to a panel of clinically relevant PIs, including the newer PIs paritaprevir, deldeprevir, and grazoprevir. We discovered complex patterns of resistance, with the impact of substitutions varying from increased sensitivity to high resistance.

Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors.

BACKGROUND & AIMS: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A) from genotypes 1-6 and 2a(JFH1) NS5B-3' untranslated region, and tested the effects of NS3 protease and NS5A inhibitors on these recombinants. METHODS: The HCV 5-5A recombinants with previously identified mutations in the NS3-helicase (F1464L), NS4A (A1672S), and NS5B (D2979G) were adapted and improved, by incorporating additional recovered mutations that increased their propagation in Huh7.5 cells. Concentration-response profiles were determined for each DAA agent in replicate infected Huh7.5 cells. RESULTS: Developed efficient 1a(H77), 1a(TN), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants did not require mutations after viral passage in the NS3 protease or NS5A domain-I regions targeted by the drugs. They were inhibited in a concentration-dependent manner by the NS3 protease inhibitors telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172 and by the NS5A inhibitor daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency observed, but with genotype-dependent activity. CONCLUSIONS: The mutations F1464L, A1672S, and D2979G permitted the development of efficient HCV recombinants comprising genotype-specific 5' untranslated region-NS5A (5-5A), which include the natural NS3 protease and NS5A domain-I drug targets. The robust replication of adapted 5-5A recombinants allowed for direct comparison of NS3 protease and NS5A inhibitors against HCV strains of genotypes 1-6.

A species-specific amino acid difference in the macaque CD4 receptor restricts replication by global circulating HIV-1 variants representing viruses from recent infection.

HIV-1 replicates poorly in macaque cells, and this had hindered the advancement of relevant nonhuman primate model systems for HIV-1 infection and pathogenesis. Several host restriction factors have been identified that contribute to this species-specific restriction to HIV-1 replication, but these do not fully explain the poor replication of most strains of HIV-1 in macaque cells. Only select HIV-1 envelope variants, typically those derived from viruses that have been adapted in cell culture, result in infectious chimeric SIVs encoding HIV-1 envelope (SHIVs). Here we demonstrate that most circulating HIV-1 variants obtained directly from infected individuals soon after virus acquisition do not efficiently mediate entry using the macaque CD4 receptor. The infectivity of these viruses is ca. 20- to 50-fold lower with the rhesus and pig-tailed macaque versus the human CD4 receptor. In contrast, culture-derived HIV-1 envelope variants that facilitate efficient replication in macaques showed similar infectivity with macaque and human CD4 receptors (within ∼2-fold). The ability of an envelope to mediate entry using macaque CD4 correlated with its ability to mediate entry of cells expressing low levels of the human CD4 receptor and with soluble CD4 sensitivity. Species-specific differences in the functional capacity of the CD4 receptor to mediate entry mapped to a single amino acid difference at position 39 that is under strong positive selection, suggesting that the evolution of CD4 may have been influenced by its function as a viral receptor. These results also suggest that N39 in human CD4 may be a critical residue for interaction of transmitted HIV-1 variants. These studies provide important insights into virus-host cell interactions that have hindered the development of relevant nonhuman primate models for HIV-1 infection and provide possible markers, such as sCD4 sensitivity, to identify potential HIV-1 variants that could be exploited for development of better SHIV/macaque model systems.

Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4 + T cells.

Type I interferon (IFN) upregulates proteins that inhibit HIV within infected cells. Prior studies have identified IFN-stimulated genes (ISGs) that impede lab-adapted HIV in cell lines, yet the ISG(s) that mediate IFN restriction in HIV target cells, primary CD4 + T cells, are unknown. Here, we interrogate ISG restriction of primary HIV in CD4 + T cells. We performed CRISPR-knockout screens using a custom library that specifically targets ISGs expressed in CD4 + T cells and validated top hits. Our investigation identified new HIV-restricting ISGs (HM13, IGFBP2, LAP3) and found that two previously studied factors (IFI16, UBE2L6) are IFN effectors in T cells. Inactivation of these five ISGs in combination further diminished IFN’s protective effect against six diverse HIV strains. This work demonstrates that IFN restriction of HIV is multifaceted, resulting from several effectors functioning collectively, and establishes a primary cell ISG screening model to identify both single and combinations of HIV-restricting ISGs.

Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4+ T cells.

Type I interferon (IFN) upregulates proteins that inhibit HIV within infected cells. Prior studies have identified IFN-stimulated genes (ISGs) that impede lab-adapted HIV in cell lines, yet the ISG(s) that mediate IFN restriction in HIV target cells, primary CD4+ T cells, are unknown. Here, we interrogate ISG restriction of primary HIV in CD4+ T cells by performing CRISPR-knockout screens with a custom library that specifically targets ISGs expressed in CD4+ T cells. Our investigation identifies previously undescribed HIV-restricting ISGs (HM13, IGFBP2, LAP3) and finds that two factors characterized in other HIV infection models (IFI16 and UBE2L6) mediate IFN restriction in T cells. Inactivation of these five ISGs in combination further diminishes IFN's protective effect against diverse HIV strains. This work demonstrates that IFN restriction of HIV is multifaceted, resulting from several effectors functioning collectively, and establishes a primary cell ISG screening model to identify both single and combinations of HIV-restricting ISGs.

Host cell glycosylation selects for infection with CCR5- versus CXCR4-tropic HIV-1.

HIV-1 infection involves a selection bottleneck that leads to transmission of one or a few HIV variants, which nearly always use CCR5 as the coreceptor (R5 viruses) for viral entry as opposed to CXCR4 (X4 viruses). The host properties that drive this selection are not well understood and may hold keys to factors that govern HIV susceptibility. In this report, we identified SLC35A2, a transporter of UDP-galactose, as a candidate X4-specific restriction factor in CRISPR-knockout screens in primary target CD4+ T cells. SLC35A2 inactivation in CD4+ T cells, which resulted in truncation of glycans due to the absence of galactose, not only increased X4 infection levels, but also consistently decreased infection levels of R5 HIV strains. Single cycle infections demonstrated that the effect is host cell dependent. SLC35A2 is expressed in CD4+ T cells at different tissue sites, with high levels in the genital tract - the site of most HIV infections. These data support a role for a host cell protein that regulates glycan structure on HIV infection, with enhanced R5 infection but reduced X4 infection associated with SLC35A2-mediated glycosylation. Host cell glycosylation may therefore contribute to R5 selection and host susceptibility during HIV transmission.

Adaptation of subtype a human immunodeficiency virus type 1 envelope to pig-tailed macaque cells.

The relevance of simian/human immunodeficiency virus (SHIV) infection of macaques to HIV-1 infection in humans depends on how closely SHIVs mimic HIV-1 transmission, pathogenesis, and diversity. Circulating HIV-1 strains are predominantly subtypes C and A and overwhelmingly require CCR5 for entry, yet most SHIVs incorporate CXCR4-using subtype B envelopes (Envs). While pathogenic subtype C-based SHIVs have been constructed, the subtype A-based SHIVs (SHIV-As) constructed to date have been unable to replicate in macaque cells. To understand the barriers to SHIV-A replication in macaque cells, HIVA(Q23)/SIV(vif) was constructed by engineering a CCR5-tropic subtype A provirus to express SIV vif, which counters the macaque APOBEC3G restriction. HIVA(Q23)/SIV(vif) replicated poorly in pig-tailed macaque (Ptm) lymphocytes, but viruses were adapted to Ptm lymphocytes. Two independent mutations in gp120, G312V (V3 loop) and A204E (C2 region), were identified that increased peak virus levels by >100-fold. Introduction of G312V and A204E to multiple subtype A Envs and substitution of G312 and A204 with other residues increased entry into Ptm cells by 10- to 100-fold. G312V and A204E Env variants continued to require CCR5 for entry but were up to 50- and 200-fold more sensitive to neutralization by IgG1b12 and soluble CD4 and had a 5- to 50-fold increase in their ability to utilize Ptm CD4 compared to their wild-type counterparts. These findings identify the inefficient use of Ptm CD4 as an unappreciated restriction to subtype A HIV-1 replication in Ptm cells and reveal amino acid changes to gp120 that can overcome this barrier.

The TOP vector: a new high-titer lentiviral construct for delivery of sgRNAs and transgenes to primary T cells.

Efficient delivery of nucleic acids for the engineering of primary T cells is central to the study of the basic biology of these key immune effector cells and has clinical implications. To date, lentiviral vectors delivering guide RNAs for CRISPR-Cas9 editing are not optimal for use in primary cells. Herein, we describe the T cell optimized for packaging (TOP) vector for delivering guide RNAs and transgenes into primary T cells. The TOP vector produces high-titer virus compared to a routinely used guide RNA vector, resulting in a ~10-fold increase in transduction in T cells. Moreover, a TOP vector expressing a chimeric antigen receptor and a guide RNA targeting the T cell receptor showed an ~5- to 9-fold increased transduction efficiency with ~2- to 3-fold higher expression compared to the commonly used epHIV7 vector and was simultaneously able to mediate efficient knockout of the endogenous T cell receptor in >71% of transduced cells upon Cas9 electroporation. The increased packaging of the TOP vector genome into viral particles appears to contribute to its higher transduction efficiency. The TOP vector represents an optimal tool for tandem delivery of transgenes and guide RNAs to primary T cells for use in functional screens and immunotherapy applications.

Analysis of the percentage of human immunodeficiency virus type 1 sequences that are hypermutated and markers of disease progression in a longitudinal cohort, including one individual with a partially defective Vif.

Hypermutation, the introduction of excessive G-to-A substitutions by host proteins in the APOBEC family, can impair replication of the human immunodeficiency virus (HIV). Because hypermutation represents a potential antiviral strategy, it is important to determine whether greater hypermutation is associated with slower disease progression in natural infection. We examined the level of HIV-1 hypermutation among 28 antiretroviral-naive Kenyan women at two times during infection. By examining single-copy gag sequences from proviral DNA, hypermutation was detected in 16 of 28 individuals. Among individuals with any hypermutation, a median of 15% of gag sequences were hypermutated (range, 5 to 43%). However, there was no association between the level of gag hypermutation and the viral load or CD4 count. Thus, we observed no overall relationship between hypermutation and markers of disease progression among individuals with low to moderate levels of hypermutation. In addition, one individual sustained a typical viral load despite having a high level of hypermutation. This individual had 43% of gag sequences hypermutated and harbored a partially defective Vif, which was found to permit hypermutation in a peripheral blood mononuclear cell culture. Overall, our results suggest that a potential antiviral therapy based on hypermutation may need to achieve a substantially higher level of hypermutation than is naturally seen in most individuals to impair virus replication and subsequent disease progression.

The Robust Restriction of Zika Virus by Type-I Interferon in A549 Cells Varies by Viral Lineage and Is Not Determined by IFITM3.

Type-I interferon (IFN-I) is a major antiviral host response but its impact on Zika virus (ZIKV) replication is not well defined, particularly as it relates to different circulating strains. Interferon stimulated genes (ISGs) that inhibit ZIKV, such as IFITM3, have been identified largely using overexpression studies. Here, we tested whether diverse ZIKV strains differed in their susceptibility to IFN-I-mediated restriction and the contribution of IFITM3 to this restriction. We identified a robust IFN-I-mediated antiviral effect on ZIKV replication (>100-fold reduction) in A549 cells, a commonly used cell line to study ZIKV replication. The extent of inhibition depended on the IFN-I type and the virus strain tested. Viruses from the American pathogenic outbreak were more sensitive to IFNα (p = 0.049) and IFNß (p = 0.09) than African-lineage strains, which have not been linked to severe pathogenesis. Knocking out IFITM3 expression did not dampen the IFN-I antiviral effect and only high overexpression of IFITM3 led to ZIKV inhibition. Moreover, IFITM3 expression levels in different cells were not associated with IFN-mediated ZIKV inhibition. Taken together, our findings indicate that there is a robust IFN-I-mediated antiviral effect on ZIKV infection, particularly for American viruses, that is not due to IFITM3. A549 cells, which are a commonly used cell line to study ZIKV replication, present an opportunity for the discovery of novel antiviral ISGs against ZIKV.

A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV.

Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Recombinant hepatitis C virus genotype 5a infectious cell culture systems expressing minimal JFH1 NS5B sequences permit polymerase inhibitor studies.

The six major epidemiologically important hepatitis C virus (HCV) genotypes differ in global distribution and antiviral responses. Full-length infectious cell-culture adapted clones, the gold standard for HCV studies in vitro, are missing for genotypes 4 and 5. To address this challenge for genotype 5, we constructed a consensus full-length clone of strain SA13 (SA13fl), which was found non-viable in Huh7.5 cells. Step-wise adaptation of SA13fl-based recombinants, beginning with a virus encoding the NS5B-thumb domain and 3´UTR of JFH1 (SA13/JF372-X), resulted in a high-titer SA13 virus with only 41 JFH1-encoded NS5B-thumb residues (SA13/JF470-510cc); this required sixteen cell-culture adaptive substitutions within the SA13fl polyprotein and two 3´UTR-changes. SA13/JF372-X and SA13/JF470-510cc were equally sensitive to nucleoside polymerase inhibitors, including sofosbuvir, but showed differential sensitivity to inhibitors targeting the NS5B palm or thumb. SA13/JF470-510cc represents a model to elucidate the influence of HCV RNA elements on viral replication and map determinants of sensitivity to polymerase inhibitors.

X chromosome transmission ratio distortion in Cftr +/- intercross-derived mice.

BACKGROUND: Cystic fibrosis (CF) mice, created with a genetically engineered mutation in the Cystic fibrosis transmembrane conductance regulator (Cftr) gene, may develop intestinal plugs which limit their survival past weaning. In a studied population of genetically mixed CF mice differences in allelic ratios at particular loci, between surviving CF mice and mice with the lethal intestinal defect, were used to map cystic fibrosis modifier gene one, Cfm1. Using this approach, we previously identified an X chromosome locus which may influence the survival to weaning of C57BL/6J x BALB/cJ F2 CF mice. We also detected two regions of transmission ratio distortion, independent of Cftr genotype, in a limited dataset. To investigate these findings, in this study we have genotyped 1208 three-week old F2 mice, and 186 day E15.5 embryos, derived from a congenic (C57BL/6J x BALB/cJ) F1 Cftr +/- intercross, for the putative distortion regions. RESULTS: An excess of homozygous BALB genotypes, compared to Mendelian expectations, was detected on chromosomes 5 (p = 5.7 x 10-15) and X (p = 3.0 x 10-35) in three-week old female mice but transmission ratio distortion was not evident in the tested region of chromosome 3 (p = 0.39). Significant pre-weaning lethality of CF mice occurred as 11.3% (137/1208) of the three-week old offspring were identified as CF mice. X chromosome genotypes were not, however, distorted in the female CF mice (p = 0.62), thus the significant non-Mendelian inheritance of this locus was dependent on CF status. The survival of CF embryos to day E15.5 was consistent with Mendelian expectations (42/186 = 23%), demonstrating the loss of CF mice to have occurred between E15.5 and three weeks of age. The excess of X chromosome homozygous BALB genotypes was recorded in female embryos (p = 0.0048), including CF embryos, indicating the distortion to be evident at this age. CONCLUSION: Two of three previously suggested loci of transmission ratio distortion were replicated as distorted in this mouse cross. The non-Mendelian inheritance of X chromosome genotypes implicates this region in the survival to weaning of non-CF mice.

Glucocorticoid receptor disruption delays structural maturation in the lungs of newborn mice.

In order to better understand the regulation of lung maturation by glucocorticoid-glucocorticoid receptor signaling, we studied glucocorticoid receptor (GR) hypomorphic mice with a mixed C57Bl6/129 sv background, in which disruption of exon 2 of the GR gene produces an N-terminal truncated GR protein. Four groups of mice were compared: homozygous mice that die at birth (non-survivors), homozygous mice that survive the neonatal period (survivors), heterozygotes and wild-type mice. Newborn non-survivors had 50% thicker airspace walls and a 46% decrease in the formation of secondary crests (the beginning of alveolar secondary septation) compared to either survivor or wild-type littermates (n = 9 mice in each group). The lung tissue to airspace ratio in homozygous mice not expressing wild-type GR (non-survivor and survivor) was increased compared to heterozygotes and wild-type mice that do express wild-type GR (0.91 +/- 0.08 vs. 0.49 +/- 0.02, n = 4 in each of the four subgroups), suggesting that complete morphological maturation of the lung is dependent on effective glucocorticoid signaling through a fully functional GR. Moreover, the relatively mature lung morphology of survivor versus non-survivor newborns suggests that a partial reduction in mesenchymal thickness is compatible with capillary remodeling, alveolar septation, and viable respiratory function after birth. Our findings suggest that in mice homozygous for disrupted GR, the severity of newborn respiratory insufficiency correlates with the degree of lung structural immaturity.

Intestinal phenotype of variable-weight cystic fibrosis knockout mice.

Cystic fibrosis (CF) transmembrane conductance regulator (Cftr) knockout mice present the clinical features of low body weight and intestinal disease permitting an assessment of the interrelatedness of these phenotypes in a controlled environment. To identify intestinal alterations that are affected by body weight in CF mice, the histological phenotypes of crypt-villus axis height, goblet cell hyperplasia, mast cell infiltrate, crypt cell proliferation, and apoptosis were measured in a population of 12-wk-old (C57BL/6 x BALB/cJ) F2 Cftr(tm1UNC) and non-CF mice presenting a range of body weight. In addition, cardiac blood samples were assessed, and gene expression profiling of the ileum was completed. Crypt-villus axis height decreased with increasing body weight in CF but not control mice. Intestinal crypts from CF mice had fewer apoptotic cells, per unit length, than did non-CF mice, and normalized cell proliferation was similar to control levels. Goblet cell hyperplasia and mast cell infiltration were increased in the CF intestine and identified to be independent of body weight. Blood triglyceride levels were found to be significantly lower in CF mice than in control mice but were not dependent on CF mouse weight. By expression profiling, genes of DNA replication and lipid metabolism were among those altered in CF mice relative to non-CF controls, and no differences in gene expression were measured between samples from CF mice in the 25th and 75th percentile for weight. In this CF mouse model, crypt elongation, due to an expanded proliferative zone and decreased apoptosis, was identified to be dependent on body weight.