Antibody-independent protection against heterologous SARS-CoV-2 challenge conferred by prior infection or vaccination.

Vaccines have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity and mortality, yet emerging variants challenge their effectiveness. The prevailing approach to updating vaccines targets the antibody response, operating under the presumption that it is the primary defense mechanism following vaccination or infection. This perspective, however, can overlook the role of T cells, particularly when antibody levels are low or absent. Here we show, through studies in mouse models lacking antibodies but maintaining functional B cells and lymphoid organs, that immunity conferred by prior infection or mRNA vaccination can protect against SARS-CoV-2 challenge independently of antibodies. Our findings, using three distinct models inclusive of a novel human/mouse ACE2 hybrid, highlight that CD8+ T cells are essential for combating severe infections, whereas CD4+ T cells contribute to managing milder cases, with interferon-γ having an important function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies.

Memory CD8+ T cell diversity and B cell responses correlate with protection against SARS-CoV-2 following mRNA vaccination.

Understanding immune responses to SARS-CoV-2 messenger RNA (mRNA) vaccines is of great interest, principally because of the poor knowledge about the mechanisms of protection. In the present study, we analyzed longitudinally B cell and T cell memory programs against the spike (S) protein derived from ancestral SARS-CoV-2 (Wuhan-1), B.1.351 (beta), B.1.617.2 (delta) and B.1.1.529 (omicron) variants of concern (VOCs) after immunization with an mRNA-based vaccine (Pfizer). According to the magnitude of humoral responses 3 months after the first dose, we identified high and low responders. Opposite to low responders, high responders were characterized by enhanced antibody-neutralizing activity, increased frequency of central memory T cells and durable S-specific CD8+ T cell responses. Reduced binding antibodies titers combined with long-term specific memory T cells that had distinct polyreactive properties were found associated with subsequent breakthrough with VOCs in low responders. These results have important implications for the design of new vaccines and new strategies for booster follow-up.

Interferon-inducible phospholipids govern IFITM3-dependent endosomal antiviral immunity.

The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.

The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody.

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of the previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryoelectron microscopy (cryo-EM) and X-ray crystallography. We show that mAb J08 has low nanomolar affinity against most VoCs and binds high on the receptor binding domain (RBD) ridge, away from many VoC mutations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy.

Tracking the immune response profiles elicited by the BNT162b2 vaccine in COVID-19 unexperienced and experienced individuals.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection.

Heterogeneity of Latency Establishment in the Different Human CD4+ T Cell Subsets Stimulated with IL-15.

HIV integrates into the host genome, creating a viral reservoir of latently infected cells that persists despite effective antiretroviral treatment. CD4-positive (CD4+) T cells are the main contributors to the HIV reservoir. CD4+ T cells are a heterogeneous population, and the mechanisms of latency establishment in the different subsets, as well as their contribution to the reservoir, are still unclear. In this study, we analyzed HIV latency establishment in different CD4+ T cell subsets stimulated with interleukin 15 (IL-15), a cytokine that increases both susceptibility to infection and reactivation from latency. Using a dual-reporter virus that allows discrimination between latent and productive infection at the single-cell level, we found that IL-15-treated primary human CD4+ T naive and CD4+ T stem cell memory (TSCM) cells are less susceptible to HIV infection than CD4+ central memory (TCM), effector memory (TEM), and transitional memory (TTM) cells but are also more likely to harbor transcriptionally silent provirus. The propensity of these subsets to harbor latent provirus compared to the more differentiated memory subsets was independent of differential expression of pTEFb components. Microscopy analysis of NF-κB suggested that CD4+ T naive cells express smaller amounts of nuclear NF-κB than the other subsets, partially explaining the inefficient long terminal repeat (LTR)-driven transcription. On the other hand, CD4+ TSCM cells display similar levels of nuclear NF-κB to CD4+ TCM, CD4+ TEM, and CD4+ TTM cells, indicating the availability of transcription initiation and elongation factors is not solely responsible for the inefficient HIV gene expression in the CD4+ TSCM subset. IMPORTANCE The formation of a latent reservoir is the main barrier to HIV cure. Here, we investigated how HIV latency is established in different CD4+ T cell subsets in the presence of IL-15, a cytokine that has been shown to efficiently induce latency reversal. We observed that, even in the presence of IL-15, the less differentiated subsets display lower levels of productive HIV infection than the more differentiated subsets. These differences were not related to different expression of pTEFb, and modest differences in NF-κB were observed for CD4+ T naive cells only, implying the involvement of other mechanisms. Understanding the molecular basis of latency establishment in different CD4+ T cell subsets might be important for tailoring specific strategies to reactivate HIV transcription in all the CD4+ T subsets that compose the latent reservoir.

Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b.

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.

COVID-eVax, an electroporated DNA vaccine candidate encoding the SARS-CoV-2 RBD, elicits protective responses in animal models.

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.

DNA aptamers masking angiotensin converting enzyme 2 as an innovative way to treat SARS-CoV-2 pandemic.

All the different coronavirus SARS-CoV-2 variants isolated so far share the same mechanism of infection mediated by the interaction of their spike (S) glycoprotein with specific residues on their cellular receptor: the angiotensin converting enzyme 2 (ACE2). Therefore, the steric hindrance on this cellular receptor created by a bulk macromolecule may represent an effective strategy for the prevention of the viral spreading and the onset of severe forms of Corona Virus disease 19 (COVID-19). Here, we applied a systematic evolution of ligands by exponential enrichment (SELEX) procedure to identify two single strand DNA molecules (aptamers) binding specifically to the region surrounding the K353, the key residue in human ACE2 interacting with the N501 amino acid of the SARS-CoV-2 S. 3D docking in silico experiments and biochemical assays demonstrated that these aptamers bind to this region, efficiently prevent the SARS-CoV-2 S/human ACE2 interaction and the viral infection in the nanomolar range, regardless of the viral variant, thus suggesting the possible clinical development of these aptamers as SARS-CoV-2 infection inhibitors. Our approach brings a significant innovation to the therapeutic paradigm of the SARS-CoV-2 pandemic by protecting the target cell instead of focusing on the virus; this is particularly attractive in light of the increasing number of viral mutants that may potentially escape the currently developed immune-mediated neutralization strategies.

Discovery and antiviral profile of new sulfamoylbenzamide derivatives as HBV capsid assembly modulators.

Chronic hepatitis B (CHB) is a major worldwide public health problem and novel anti-HBV therapies preventing liver disease progression to cirrhosis and hepatocellular carcinoma are urgently needed. Over the last several years, capsid assembly modulators (CAM) have emerged as clinically effective anti-HBV agents which can inhibit HBV replication in CHB patients. As part of a drug discovery program aimed at obtaining novel CAM endowed with high in vitro and in vivo antiviral activity, we identified a novel series of sulfamoylbenzamide (SBA) derivatives. Compound 10, one of the most in vitro potent SBA-derived CAM discovered to date, showed excellent pharmacokinetics in mice suitable for oral dosing. When studied in a transgenic mouse model of hepatic HBV replication, it was considerably more potent than NVR 3-778, the first sulfamoylbenzamide (SBA) CAM that entered clinical trials for CHB, at reducing viral replication in a dose-dependent fashion. We present herein the discovery process, the SAR analysis and the pre-clinical profile of this novel SBA CAM.

The Enigmatic Role of Viruses in Multiple Sclerosis: Molecular Mimicry or Disturbed Immune Surveillance?

Multiple sclerosis (MS) is a T cell driven autoimmune disease of the central nervous system (CNS). Despite its association with Epstein-Barr Virus (EBV), how viral infections promote MS remains unclear. However, there is increasing evidence that the CNS is continuously surveyed by virus-specific T cells, which protect against reactivating neurotropic viruses. Here, we discuss how viral infections could lead to the breakdown of self-tolerance in genetically predisposed individuals, and how the reactivations of viruses in the CNS could induce the recruitment of both autoaggressive and virus-specific T cell subsets, causing relapses and progressive disability. A disturbed immune surveillance in MS would explain several experimental findings, and has important implications for prognosis and therapy.

NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains.

Hepatitis C virus (HCV) RNA is synthesized by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by pre-formed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOR) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPOS) viruses (e.g. H77S.3 and N.2). In luciferase assays, LPOS HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPOR HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNA-dependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPOS H77S.3 and the LPOR H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. Mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPOS and LPOR viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.

IL-10 promotes homeostatic proliferation of human CD8(+) memory T cells and, when produced by CD1c(+) DCs, shapes naive CD8(+) T-cell priming.

IL-10 is an anti-inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8(+) CTL, IL-10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL-10 produced by human APC subsets in T-cell responses. IL-10 production was restricted to CD1c(+) DCs and CD14(+) monocytes. Interestingly, it was differentially regulated, since R848 induced IL-10 in DCs, but inhibited IL-10 in monocytes. Autocrine IL-10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high-affinity peptides. Nevertheless, it completely blocked cross-priming and priming with low-affinity peptides of a self/tumor-antigen. IL-10 also inhibited CD1c(+) DC-induced CD4(+) T-cell priming and enhanced Foxp3 induction, but was insufficient to induce T-cell IL-10 production. CD1c(+) DC-derived IL-10 had also no effect on DC-induced secondary expansions of memory CTL. However, IL-15-driven, TCR-independent proliferation of memory CTL was enhanced by IL-10. We conclude that DC-derived IL-10 selects high-affinity CTL upon priming. Moreover, IL-10 preserves established CTL memory by enhancing IL-15-dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL-10 promotes CTL responses in humans.

DEPDC5 variants increase fibrosis progression in Europeans with chronic hepatitis C virus infection.

UNLABELLED: Chronic hepatitis C virus (HCV) infection may progress to cirrhosis and hepatocellular carcinoma (HCC). Recently, two genetic variants, DEPDC5 rs1012068 and MICA rs2596542, were associated with the onset of HCC in Asian subjects with chronic HCV infection. The aim of the present study was to analyze whether DEPDC5 and MICA genetic variants were associated with liver disease progression in European subjects with chronic HCV infection. In a Northern Italian discovery cohort (n = 477), neither DEPDC5 rs1012068 nor MICA rs2596542 were associated with HCC (n = 150). However, DEPDC5 rs1012068 was independently associated with cirrhosis (n = 300; P = 0.049). The association of rs1012068 with moderate to severe fibrosis was confirmed in an independent cross-sectional German cohort (n = 415; P = 0.006). Furthermore, DEPDC5 rs1012068 predicted faster fibrosis progression in a prospective cohort (n = 247; P = 0.027). Next, we examined the distribution of nonsynonymous DEPDC5 variants in the overall cross-sectional cohort (n = 912). The presence of at least one variant increased the risk of moderate/severe fibrosis by 54% (P = 0.040). To understand the molecular mechanism underlying the genetic association of DEPDC5 variants with fibrosis progression, we performed in vitro studies on immortalized hepatic stellate cells (LX-2). In these cells, down-regulation of DEPDC5 resulted in increased expression of ß-catenin and production of its target matrix metallopeptidase 2 (MMP2), a secreted enzyme involved in fibrosis progression. CONCLUSION: DEPDC5 variants increase fibrosis progression in European subjects with chronic HCV infection. Our findings suggest that DEPDC5 down-regulation may contribute to HCV-related fibrosis by increasing MMP2 synthesis through the ß-catenin pathway.

Transmembrane 6 superfamily member 2 gene E167K variant impacts on steatosis and liver damage in chronic hepatitis C patients.

UNLABELLED: Steatosis and inherited host factors influence liver damage progression in chronic hepatitis C (CHC). The transmembrane 6 superfamily member 2 (TM6SF2) gene E167K variant increases liver fat and risk of progressive steatohepatitis by interfering with lipoprotein secretion. Our aim was to determine whether the E167K variant affects histological severity of steatosis, necroinflammation, and fibrosis in a cross-sectional cohort of 815 Italian therapy-naïve CHC patients. The association with clinically significant fibrosis was replicated in 645 Swiss/German patients. The TM6SF2 E167K variant was genotyped by TaqMan assays, steatosis graded according to the nonalcoholic fatty liver disease activity score, and necroinflammation and fibrosis graded and staged according to Ishak in Italian, and to Metavir in Swiss/German patients. The E167K variant was detected in 69 (9%) Italian patients and was associated with more severe steatosis, independently of confounders (P = 0.038). The association between E167K and steatosis severity was present in patients not infected by genotype 3 (G3) HCV (P = 0.031), but not in those infected by G3 HCV (P = 0.58). Furthermore, the E167K variant was associated with more severe necroinflammation (Ishak grade; adjusted P = 0.037) and nearly associated with more severe fibrosis (Ishak stage; adjusted P = 0.058). At multivariate logistic regression analysis, the E167K variant was independently associated with histologically probable or definite cirrhosis (Ishak stage S6; odds ratio [OR]: 2.19; 95% confidence interval [CI]: 1.18-3.93; P = 0.010). After further conditioning for steatosis and necroinflammation, the E167K variant remained associated with cirrhosis (OR, 3.15; 95% CI: 1.60-5.99; P < 0.001). In Swiss/German patients, the E167K variant was independently associated with clinically significant fibrosis Metavir stage F2-F4 (OR, 1.81; 95% CI: 1.12-3.02; P = 0.016). CONCLUSION: TM6SF2 E167K variant impacts on steatosis severity and is associated with liver damage and fibrosis in patients with CHC.

Oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for HCV replication membrane integrity and cholesterol trafficking.

BACKGROUND & AIMS: Positive-sense RNA viruses remodel intracellular membranes to generate specialized membrane compartments for viral replication. Several RNA viruses, including poliovirus and hepatitis C virus (HCV), require phosphatidylinositol (PI) 4-kinases for their replication. However, it is not known how PI 4-kinases and their product, PI(4)P, facilitate host membrane reorganization and viral replication. In addition, although the HCV replication compartment, known as the membranous web, is believed to be cholesterol enriched, the mechanisms by which this occurs have not been elucidated. We aimed to identify and characterize a PI 4-kinase effector in HCV replication. METHODS: We used a combination of microscopic and biochemical methods to study HCV replication, web morphology, the distribution of intracellular protein and PI(4)P, along with cholesterol trafficking in HCV-infected cells. PI 4-kinase and oxysterol-binding protein (OSBP) were inhibited using RNA interference or small molecules in cells expressing a full-length genotype 1b replicon or infected with the JFH-1 strain of HCV. RESULTS: OSBP was required for HCV replication and membranous web integrity. OSBP was recruited to membranous webs in a PI 4-kinase-dependent manner, and both these factors were found to regulate cholesterol trafficking to the web. We also found OSBP to be required for poliovirus infection but dispensable for dengue virus. CONCLUSIONS: OSBP is a PI 4-kinase effector in HCV infection, and contributes to the integrity and cholesterol enrichment of the membranous web. OSBP might also be a PI 4-kinase effector in poliovirus infection and could be involved in replication of other viruses that require PI 4-kinases.

Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors.

BACKGROUND & AIMS: All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3. METHODS: We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. RESULTS: Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. CONCLUSIONS: DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis.