IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans.

The intracellular protozoan parasite Leishmania donovani causes debilitating human diseases that involve visceral and dermal manifestations. Type 3 interferons (IFNs), also referred to as lambda IFNs (IFNL, IFN-L, or IFN-λ), are known to play protective roles against intracellular pathogens at the epithelial surfaces. Herein, we show that L. donovani induces IFN-λ3 in human as well as mouse cell line-derived macrophages. Interestingly, IFN-λ3 treatment significantly decreased parasite load in infected cells, mainly by increasing reactive oxygen species production. Microscopic examination showed that IFN-λ3 inhibited uptake but not replication, while the phagocytic ability of the cells was not affected. This was confirmed by experiments that showed that IFN-λ3 could decrease parasite load only when added to the medium at earlier time points, either during or soon after parasite uptake, but had no effect on parasite load when added at 24 h post-infection, suggesting that an early event during parasite uptake was targeted. Furthermore, the parasites could overcome the inhibitory effect of IFN-λ3, which was added at earlier time points, within 2-3 days post-infection. BALB/c mice treated with IFN-λ3 before infection led to a significant increase in expression of IL-4 and ARG1 post-infection in the spleen and liver, respectively, and to different pathological changes, especially in the liver, but not to changes in parasite load. Treatment with IFN-λ3 during infection did not decrease the parasite load in the spleen either. However, IFN-λ3 was significantly increased in the sera of visceral leishmaniasis patients, and the IFNL genetic variant rs12979860 was significantly associated with susceptibility to leishmaniasis.

Gender-dependent multiple cross-phenotype association of interferon lambda genetic variants with peripheral blood profiles in healthy individuals.

BACKGROUND: Type III interferons (IFN), also called as lambda IFNs (IFN-λs), are antiviral and immunomodulatory cytokines that are evolutionarily important in humans. Given their central roles in innate immunity, they could be influencing other aspects of human biology. This study aimed to examine the association of genetic variants that control the expression and/or activity of IFN-λ3 and IFN-λ4 with multiple phenotypes in blood profiles of healthy individuals. METHODS: In a cohort of about 550 self-declared healthy individuals, after applying several exclusion criteria to determine their health status, we measured 30 blood parameters, including cellular, biochemical, and metabolic profiles. We genotyped them at rs12979860 and rs28416813 using competitive allele-specific PCR assays and tested their association with the blood profiles under dominant and recessive models for the minor allele. IFN-λ4 variants rs368234815 and rs117648444 were also genotyped or inferred. RESULTS: We saw no association in the combined cohort under either of the models for any of the phenotypes. When we stratified the cohort based on gender, we saw a significant association only in males with monocyte (p = 1 × 10-3 ) and SGOT (p = 7 × 10-3 ) levels under the dominant model and with uric acid levels (p = 0.01) under the recessive model. When we tested the IFN-λ4 activity modifying variant within groupings based on absence or presence of one or two copies of IFN-λ4 and on different activity levels of IFN-λ4, we found significant (p < 0.05) association with several phenotypes like monocyte, triglyceride, VLDL, ALP, and uric acid levels, only in males. All the above significant associations did not show any confounding when we tested for the same with up to ten different demographic and lifestyle variables. CONCLUSIONS: These results show that lambda interferons can have pleiotropic effects. However, gender seems to be an effect modifier, with males being more sensitive than females to the effect.

A Novel Inactive Isoform with a Restored Reading Frame Is Expressed from the Human Interferon Lambda 4 TT Allele at rs368234815.

The TT allele of the dinucleotide variant rs368234815 (TT/ΔG) abolishes the open reading frame (ORF) created by the ancestral ΔG allele of the human interferon lambda 4 (IFNL4) gene, thus preventing the expression of a functional IFN-λ4 protein. While probing the expression of IFN-λ4 in human peripheral blood mononuclear cells (PBMCs), using a monoclonal antibody that binds to the C-terminus of IFN-λ4, surprisingly, we observed that PBMCs obtained from TT/TT genotype individuals could also express proteins that reacted with the IFN-λ4-specific antibody. We confirmed that these products did not emanate from the IFNL4 paralog, IF1IC2 gene. Using cell lines and overexpressing human IFNL4 gene constructs, we obtained evidence from Western blots to show that the TT allele could express a protein that reacted with the IFN-λ4 C-terminal-specific antibody. It had a molecular weight similar if not identical to IFN-λ4 expressed from the ΔG allele. Furthermore, the same start and stop codons used by the ΔG allele were used to express the novel isoform from the TT allele suggesting that a restoration of the ORF had occurred in the body of the mRNA. However, this TT allele isoform did not induce any IFN-stimulated gene expression. Our data do not support a ribosomal frameshift that leads to the expression of this new isoform, implying that an alternate splicing event may be responsible. An N-terminal-specific monoclonal antibody did not react with the novel protein isoform suggesting that the alternate splicing event likely occurs beyond exon 2. The new isoform is glycosylated similar to the functional IFN-λ4 and is also secreted. Furthermore, we show that the ΔG allele can also potentially express a similarly frameshifted isoform. The splicing event that leads to the generation of these novel isoforms and their functional significance remains to be elucidated.

Distinct molecular phenotypes involving several human diseases are induced by IFN-λ3 and IFN-λ4 in monocyte-derived macrophages.

Human Interferon (IFN) lambda 3 (IFN-λ3) and IFN-λ4 are closely linked at the IFNL locus and show association with several diseases in genetic studies. Since they are only ~30% identical to each other, to better understand their roles in disease phenotypes, comparative studies are needed. Monocytes are precursors to macrophages (monocyte-derived macrophages; MDMs) that get differentiated under the influence of various immune factors, including IFNs. In a recent study, we characterized lipopolysaccharide-activated M1 and M2-MDMs that were differentiated in presence of IFN-λ3 or IFN-λ4. In this study, we performed transcriptomics on these M1 and M2-MDMs to further understand their molecular phenotypes. We identified over 760 genes that were reciprocally regulated by IFN-λ3 and IFN-λ4, additionally we identified over 240 genes that are significantly affected by IFN-λ4 but not IFN-λ3. We observed that IFN-λ3 was more active in M2-MDMs while IFN-λ4 showed superior response in M1-MDMs. Providing a structural explanation for these functional differences, molecular modeling showed differences in expected interactions of IFN-λ3 and IFN-λ4 with the extracellular domain of IFN-λR1. Further, pathway analysis showed several human infectious diseases and even cancer-related pathways being significantly affected by IFN-λ3 and/or IFN-λ4 in both M1 and M2-MDMs.

Functional genetic variants of the IFN-λ3 (IL28B) gene and transcription factor interactions on its promoter.

Interferon lambda 3 (IFN-λ3 or IFNL3, formerly IL28B), a type III interferon, modulates immune responses during infection/inflammation. Several human studies have reported an association of single nucleotide polymorphisms (SNP) in the IFNL3 locus with expression level of IFNL3. Previous genetic studies, in the context of hepatitis C virus infections, had predicted three regulatory SNPs: rs4803219, rs28416813 and rs4803217 that could have functional/causal roles. Subsequent studies confirmed this prediction for rs28416813 and rs4803217. A dinucleotide TA-repeat variant (rs72258881) has also been reported to be regulating the IFN-λ3 promoter. In this study, we tested all these genetic variants using a sensitive reporter assay. We show that the minor/ancestral alleles of both rs28416813 and rs4803217, together have a strong inhibitory effect on reporter gene expression. We also show an interaction between the two principal transcription factors regulating IFNL3 promoter: IRF7 and NF-kB RelA/p65. We show that IRF7 and p65 physically interact with each other. By using a transient ChIP assay, we show that presence of p65 increases the promoter occupancy of IRF7, thereby leading to synergistic activation of the IFNL3 promoter. We reason that, in contrast to p65, a unique nature of IRF7 binding to its specific DNA sequence makes it more sensitive to changes in DNA phasing. As a result, we see that IRF7, but not p65-mediated transcriptional activity is affected by the phase changes introduced by the TA-repeat polymorphism. Overall, we see that three genetic variants: rs28416813, rs4803217 and rs72258881 could have functional roles in controlling IFNL3 gene expression.

SARS-CoV-2 infection in India bucks the trend: Trained innate immunity?

SARS-CoV-2, the causative agent of COVID-19 pandemic caught the world unawares by its sudden onset in early 2020. Memories of the 1918 Spanish Flu were rekindled raising extreme fear for the virus, but in essence, it was the host and not the virus, which was deciding the outcome of the infection. Age, gender, and preexisting conditions played critical roles in shaping COVID-19 outcome. People of lower socioeconomic strata were disproportionately affected in industrialized countries such as the United States. India, a developing country with more than 1.3 billion population, a large proportion of it being underprivileged and with substandard public health provider infrastructure, feared for the worst outcome given the sheer size and density of its population. Six months into the pandemic, a comparison of COVID-19 morbidity and mortality data between India, the United States, and several European countries, reveal interesting trends. While most developed countries show curves expected for a fast-spreading respiratory virus, India seems to have a slower trajectory. As a consequence, India may have gained on two fronts: the spread of the infection is unusually prolonged, thus leading to a curve that is "naturally flattened"; concomitantly the mortality rate, which is a reflection of the severity of the disease has been relatively low. I hypothesize that trained innate immunity, a new concept in immunology, may be the phenomenon behind this. Biocultural, socioecological, and socioeconomic determinants seem to be influencing the outcome of COVID-19 in different regions/countries of the world.

Monocytes differentiated into macrophages and dendritic cells in the presence of human IFN-λ3 or IFN-λ4 show distinct phenotypes.

Human IFN-λ4 is expressed by only a subset of individuals who possess the ΔG variant allele at the dinucleotide polymorphism rs368234815. Recent genetic studies have shown an association between rs368234815 and different infectious and inflammatory disorders. It is not known if IFN-λ4 has immunomodulatory activity. The expression of another type III IFN, IFN-λ3, is also controlled by genetic polymorphisms that are strongly linked to rs368234815. Therefore, it is of interest to compare these two IFNs for their effects on immune cells. Herein, using THP-1 cells, it was confirmed that IFN-λ4 could affect the differentiation status of macrophage-like cells and dendritic cells (DCs). The global gene expression changes induced by IFN-λ4 were also characterized in in vitro generated primary macrophages. Next, human PBMC-derived CD14+ monocytes were used to obtain M1 and M2 macrophages and DCs in the presence of IFN-λ3 or IFN-λ4. These DCs were cocultured with CD4+ Th cells derived from allogenic donors and their in vitro cytokine responses were measured. The specific activity of recombinant IFN-λ4 was much lower than that of IFN-λ3, as shown by induction of IFN-stimulated genes. M1 macrophages differentiated in the presence of IFN-λ4 showed higher IL-10 secretion than those differentiated in IFN-λ3. Coculture experiments suggested that IFN-λ4 could confer a Th2-biased phenotype to allogenic Th cells, wherein IFN-λ3, under similar circumstances, did not induce a significant bias toward either a Th1 or Th2 phenotype. This study shows for the first time that IFN-λ4 may influence immune responses by immunomodulation.

Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility.

Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S2 domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in SD domain) and G1124V (in S2 subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird's eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India.

The Genetic Association of IFN-λs with Human Inflammatory Disorders Remains a Conundrum.

Type III Interferons (IFNs) or lambda IFNs (IFN-λs or IFNLs) although are primarily antiviral cytokines, may have roles to play in shaping immune responses, including those during inflammation. Genetic variants within the IFNL locus have been shown to be associated with various inflammatory conditions in humans ranging from metabolic to autoimmune and allergic diseases. The mechanism behind these genetic associations is not clear. Appropriate data analysis methods and functional evidence should be complimentarily used to identify the causal variants and mechanisms.

Identifying causal variants at the interferon lambda locus in case-control studies: Utilizing non-synonymous variant rs117648444 to probe the role of IFN-λ4.

Genetic variants at the interferon lambda (IFNL) locus have been associated with several human phenotypes in both disease and health. In chronic hepatitis C virus (HCV) infections, where the IFNL variants were first identified to be associated with response to interferon-α-ribavirin therapy, the available data clearly suggests that the causal variant could be the dinucleotide polymorphism rs368234815 that causes an open reading frame-shift in the IFNL4 gene resulting in expression of a functional IFN-λ4, a new type III IFN. In other human diseases/phenotypes where IFNL variants have been recently associated with, the causal mechanism remains unclear. In vitro evidence has shown that other IFNL variants (rs28416813, rs4803217) may regulate expression of another type III IFN, IFN-λ3. Therefore, expression of a functional IFN-λ4 and quantitative differences in IFN-λ3 expression are two potential causal mechanisms behind the observed phenotypes. Since these two potential causal mechanisms involve features of mutual exclusivity and overlapping functions, it is difficult to differentiate one from the other, in vivo, in absence of other implicating evidences. In addition, the strong linkage disequilibrium (LD) observed in many populations at the IFNL locus makes it difficult to tease out the actual functional/causal variants responsible for the phenotypes. The non-synonymous single nucleotide polymorphism rs117648444 that alters the activity of IFN-λ4 and the LD structure in the IFNL region which leads to a confounding effect of rs117648444 on other IFNL variants, provide us with additional tools in case-control studies to probe the role of IFN-λ4.

A functional IFN-λ4-generating DNA polymorphism could protect older asthmatic women from aeroallergen sensitization and associate with clinical features of asthma.

Lambda interferons (IFNLs) have immunomodulatory functions at epithelial barrier surfaces. IFN-λ4, a recent member of this family is expressed only in a subset of the population due to a frameshift-causing DNA polymorphism rs368234815. We examined the association of this polymorphism with atopy (aeroallergen sensitization) and asthma in a Polish hospital-based case-control cohort comprising of well-characterized adult asthmatics (n = 326) and healthy controls (n = 111). In the combined cohort, we saw no association of the polymorphism with asthma and/or atopy. However, the IFN-λ4-generating ΔG allele protected older asthmatic women (>50 yr of age) from atopic sensitization. Further, ΔG allele significantly associated with features of less-severe asthma including bronchodilator response and corticosteroid usage in older women in this Polish cohort. We tested the association of related IFNL locus polymorphisms (rs12979860 and rs8099917) with atopy, allergic rhinitis and presence/absence of asthma in three population-based cohorts from Europe, but saw no significant association of the polymorphisms with any of the phenotypes in older women. The polymorphisms associated marginally with lower occurrence of asthma in men/older men after meta-analysis of data from all cohorts. Functional and well-designed replication studies may reveal the true positive nature of these results.

Confounding by Single Nucleotide Polymorphism rs117648444 (P70S) Affects the Association of Interferon Lambda Locus Variants with Response to Interferon-α-Ribavirin Therapy in Patients with Chronic Genotype 3 Hepatitis C Virus Infection.

Genome-wide association studies discovered interferon lambda (IFNL or IFN-λ) locus on chromosome 19 to be involved in clearance of chronic hepatitis C virus (HCV) infection in patients following interferon-α-ribavirin (IFN-RBV) therapy. Subsequent studies established a dinucleotide polymorphism rs368234815, as the prime causal variant behind this association. The ΔG allele of this variant gives rise to a new IFNL gene, IFNL4, coding for IFN-λ4 whose activity paradoxically associates with lesser viral clearance rates. A low-frequency, nonsynonymous single nucleotide polymorphism (SNP) rs117648444 within the 2nd exon of IFNL4 changes the 70th amino acid from proline to serine resulting in lower activity of the functional IFN-λ4 protein, thereby increasing HCV clearance rates. In the present study, we used a cohort of genotype 3 HCV-infected patients, drawn from different geographical regions of India who underwent IFN-RBV therapy, to examine the association of several important IFNL locus SNPs/variants with sustained virological response (SVR). Intriguingly, the causal variant rs368234815 did not show the best strength and significance of association with SVR, while further analysis revealed that a negative confounding effect of rs117648444 was responsible for this phenomenon. Our results indicate that IFNL locus SNPs are subject to either a positive or a negative confounding effect by rs117648444; the nature of confounding depends on the linkage of the IFNL SNPs with the low-activity IFN-λ4-generating minor allele of rs117648444. Thus, our work demonstrates that the linkage disequilibrium structure of the IFNL region may confound the results of association studies. These results have implications for the design and understanding of future case-control studies involving IFNL locus SNPs/variants.

Roles for Transcription Factors Sp1, NF-κB, IRF3, and IRF7 in Expression of the Human IFNL4 Gene.

The expression of a biologically active human IFNλ4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFNλ4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFNλ4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.

Association of IL28B single nucleotide polymorphism rs8099917 with response to treatment in genotype 3 HCV-infected patients from India.

BACKGROUND AND AIM: IL28B gene polymorphisms have been associated with treatment-response (sustained virological response, SVR) in genotype 1 hepatitis C virus (HCV)-infected patients, but only with early phase of viral decline (rapid virological response, RVR) with genotype 3 HCV-infected patients. Association between IL28B variants and SVR in genotype 3 HCV- infected patients is unclear. Our study aimed to replicate the association of IL28Bsingle nucleotide polymorphism (SNP) rs8099917 with SVR and to validate its association with RVR in genotype 3 HCV-infected patients. METHODS: 72 patients receiving combination therapy (interferon-alpha and ribavirin) at different Indian centers were retrospectively recruited and their genotype atrs8099917 was determined. The association with RVR and SVR was tested taking in to account the variation in relevant covariates such as age, gender, baseline HCV RNA copy number and liver enzymes. RESULTS: The minor allele frequency (MAF) in the pooled samples was 0.17 at rs8099917 (G allele). 68% had TT, 29% had GT and 3% had the GG genotype. SVR was achieved in 71% of patients. A significant association ofrs8099917 with both RVR (p = 0.026) and SVR (p = 0.016) was observed with none of the covariates showing any significant association. The relapse rate was high (20%) but no association of rs8099917 was observed with relapse (p = 0.420). CONCLUSION: An IL28B SNP associates with both early phase of viral decline and sustained response in a cohort of genotype 3 HCV-infected patients from India.

Genetic variants at the IFNL3 locus and their association with hepatitis C virus infections reveal novel insights into host-virus interactions.

Human genetic variation plays a critical role in both spontaneous clearance of and response to interferon (IFN)-based therapies against hepatitis C virus (HCV) as shown by the success of recent genome-wide association studies (GWAS). Several GWAS and later validation studies have shown that single nucleotide polymorphisms (SNPs) at the IFNL3 (formerly IL28B) locus on chromosome 19 are involved in eliminating HCV in human patients. No doubt that this information is helping clinicians worldwide in making better clinical decisions in anti-HCV therapy, but the biological mechanisms involving the SNPs leading to differential responses to therapy and spontaneous clearance of HCV remain elusive. Recent reports including the discovery of a novel IFN (IFN-λ4) gene at the IFNL3 locus and in vitro functional studies implicating 2 SNPs as causal variants lead to novel conclusions and perhaps to new directions in research. An attempt is made in this review to summarize the major findings of the GWAS, the efforts involved in the discovery of causal SNPs; and to explain the biological basis for spontaneous clearance and response to treatment in HCV infections.

A single nucleotide polymorphism associated with hepatitis C virus infections located in the distal region of the IL28B promoter influences NF-κB-mediated gene transcription.

Persistence of hepatitis C virus (HCV) infection is observed only in a subset of infected individuals and among them only some respond to treatment. Genome-wide association studies (GWAS) carried out around the world identified single nucleotide polymorphisms (SNPs) in the IL28B locus that are strongly associated with both HCV clearance and treatment response. The functional significance of these associations however, is not clear. In this report we show that an SNP rs28416813 in the distal promoter region of IL28B that is in close proximity to a non-consensus NF-κB-binding site affects downstream reporter gene expression. The effect is likely due to differential binding of NF-κB at the non-consensus site. The non-protective allele showed a reduction in luciferase reporter gene expression compared to the protective allele in HEK293T cells under different experimental conditions including treatment with tumor necrosis factor alpha (TNF-α) and 5' triphosphorylated dsRNA. Furthermore, the HCV RNA polymerase was able to induce transcription from the IL28B promoter in a RIG-I-dependent manner. This induction was influenced by the alleles present at rs28416813. We also demonstrate strong linkage disequilibrium between rs28416813 and another important SNP rs12979860 in two ethnic populations. These results suggest possible mechanisms by which SNPs at the IL28B locus influence spontaneous clearance and treatment response in chronic HCV infections.

Mutations in the hepatitis C virus polymerase that increase RNA binding can confer resistance to cyclosporine A.

UNLABELLED: Hepatitis C virus (HCV) infection leads to acute and chronic liver diseases, and new classes of anti-HCV therapeutics are needed. Cyclosporine A (CsA) inhibits HCV replication and CsA derivatives that lack the immunosuppressive function are currently in clinical trials as candidate anti-HCV drugs. Here we characterize several independently derived HCV replicons with varying levels of CsA resistance due to mutations in nonstructural protein 5B (NS5B), the HCV-encoded polymerase. Mutant HCV replicons engineered with these mutations showed resistance to CsA. The mutations reside in two distinct patches in the polymerase: the template channel and one face of a concave surface behind the template channel. Mutant NS5B made by cells expressing the HCV replicon had increased ability to bind to RNA in the presence of CsA. Purified recombinant NS5B proteins containing the mutations were better at de novo initiated RNA synthesis than the wild-type control. Furthermore, the mutant proteins were able to bind RNA with approximately 8-fold higher affinity. Last, mutation near the template channel alleviated the lethal phenotype of a mutation in the concave patch, P540A. This intramolecular compensation for the HCV replicase function by amino acid changes in different domains was further confirmed in an infectious cell culture-derived virus system. CONCLUSION: An increased level of CsA resistance is associated with distinct mutations in the NS5B gene that increase RNA binding in the presence of CsA, and the intramolecular communications between residues of the thumb and the C-terminal domains are important for HCV replicase function.

Impaired replication of hepatitis C virus containing mutations in a conserved NS5B retinoblastoma protein-binding motif.

Hepatitis C virus (HCV) downregulates the retinoblastoma tumor suppressor protein (Rb), a central cell cycle regulator which is also targeted by oncoproteins expressed by DNA tumor viruses. HCV genome replication is also enhanced in proliferating cells. Thus, it is possible that HCV interactions with host cell cycle regulators, such as Rb, have evolved to modify the intracellular environment to promote viral replication. To test this hypothesis and to determine the impact of viral regulation of Rb on HCV replication, we constructed infectious viral genomes containing mutations in the Rb-binding motif of NS5B which ablate the ability of HCV to regulate Rb. These genomes underwent replication in transfected cells but produced variably reduced virus yields. One mutant, L314A, was severely compromised for replication and rapidly mutated to L314V, thereby restoring both Rb regulation and replication competence. Another mutant, C316A, also failed to downregulate Rb abundance and produced virus yields that were about one-third that of virus with the wild-type (wt) NS5B sequence. Despite this loss of replication competence, purified NS5B-C316A protein was two- to threefold more active than wt NS5B in cell-free polymerase and replicase assays. Although small interfering RNA knockdown of Rb did not rescue the replication fitness of these mutants, we conclude that the defect in replication fitness is not due to defective polymerase or replicase function and is more likely to result from the inability of the mutated NS5B to optimally regulate Rb abundance and thereby modulate host gene expression.

A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase.

Mutational analysis of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) template channel identified two residues, Trp(397) and His(428), which are required for de novo initiation but not for extension from a primer. These two residues interact with the Delta1 loop on the surface of the RdRp. A deletion within the Delta1 loop also resulted in comparable activities. The mutant proteins exhibit increased double-stranded RNA binding compared with the wild type, suggesting that the Delta1 loop serves as a flexible locking mechanism to regulate the conformations needed for de novo initiation and for elongative RNA synthesis. A similar locking motif can be found in other viral RdRps. Products associated with the open conformation of the HCV RdRp were inhibited by interaction with the retinoblastoma protein but not cyclophilin A. Different conformations of the HCV RdRp can thus affect RNA synthesis and interaction with cellular proteins.