Regulation of HIV-1 Gag-Pol Expression by Shiftless, an Inhibitor of Programmed -1 Ribosomal Frameshifting.

Programmed -1 ribosomal frameshifting (-1PRF) is a widely used translation recoding mechanism. HIV-1 expresses Gag-Pol protein from the Gag-coding mRNA through -1PRF, and the ratio of Gag to Gag-Pol is strictly maintained for efficient viral replication. Here, we report that the interferon-stimulated gene product C19orf66 (herein named Shiftless) is a host factor that inhibits the -1PRF of HIV-1. Shiftless (SFL) also inhibited the -1PRF of a variety of mRNAs from both viruses and cellular genes. SFL interacted with the -1PRF signal of target mRNA and translating ribosomes and caused premature translation termination at the frameshifting site. Downregulation of translation release factor eRF3 or eRF1 reduced SFL-mediated premature translation termination. We propose that SFL binding to target mRNA and the translating ribosome interferes with the frameshifting process. These findings identify SFL as a broad-spectrum inhibitor of -1PRF and help to further elucidate the mechanisms of -1PRF.

Horizontal transmission of clonal cancer cells causes leukemia in soft-shell clams.

Outbreaks of fatal leukemia-like cancers of marine bivalves throughout the world have led to massive population loss. The cause of the disease is unknown. We recently identified a retrotransposon, Steamer, that is highly expressed and amplified to high copy number in neoplastic cells of soft-shell clams (Mya arenaria). Through analysis of Steamer integration sites, mitochondrial DNA single-nucleotide polymorphisms (SNPs), and polymorphic microsatellite alleles, we show that the genotypes of neoplastic cells do not match those of the host animal. Instead, neoplastic cells from dispersed locations in New York, Maine, and Prince Edward Island (PEI), Canada, all have nearly identical genotypes that differ from those of the host. These results indicate that the cancer is spreading between animals in the marine environment as a clonal transmissible cell derived from a single original clam. Our findings suggest that horizontal transmission of cancer cells is more widespread in nature than previously supposed.

Systematic identification of factors for provirus silencing in embryonic stem cells.

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.

Asymmetric distribution of parental H3K9me3 in S phase silences L1 elements.

In eukaryotes, repetitive DNA sequences are transcriptionally silenced through histone H3 lysine 9 trimethylation (H3K9me3). Loss of silencing of the repeat elements leads to genome instability and human diseases, including cancer and ageing1-3. Although the role of H3K9me3 in the establishment and maintenance of heterochromatin silencing has been extensively studied4-6, the pattern and mechanism that underlie the partitioning of parental H3K9me3 at replicating DNA strands are unknown. Here we report that H3K9me3 is preferentially transferred onto the leading strands of replication forks, which occurs predominantly at long interspersed nuclear element (LINE) retrotransposons (also known as LINE-1s or L1s) that are theoretically transcribed in the head-on direction with replication fork movement. Mechanistically, the human silencing hub (HUSH) complex interacts with the leading-strand DNA polymerase Pol ε and contributes to the asymmetric segregation of H3K9me3. Cells deficient in Pol ε subunits (POLE3 and POLE4) or the HUSH complex (MPP8 and TASOR) show compromised H3K9me3 asymmetry and increased LINE expression. Similar results were obtained in cells expressing a MPP8 mutant defective in H3K9me3 binding and in TASOR mutants with reduced interactions with Pol ε. These results reveal an unexpected mechanism whereby the HUSH complex functions with Pol ε to promote asymmetric H3K9me3 distribution at head-on LINEs to suppress their expression in S phase.

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful against COVID-19 (refs. 1,2). However, because SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here we examined this possibility by in vitro passaging of SARS-CoV-2 in nirmatrelvir using two independent approaches, including one on a large scale. Indeed, highly resistant viruses emerged from both and their sequences showed a multitude of 3CL protease mutations. In the experiment peformed with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Nevertheless, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones showed that these mutations mediated only low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (around 100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next-generation protease inhibitors.

Molecular mechanisms of SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir is a specific antiviral drug that targets the main protease (Mpro) of SARS-CoV-2 and has been approved to treat COVID-191,2. As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations3. The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of Mpro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 Mpro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously3. Such a profile was also observed for ensitrelvir, another clinically relevant Mpro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation Mpro inhibitors.

Upf1 senses 3'UTR length to potentiate mRNA decay.

The selective degradation of mRNAs by the nonsense-mediated decay pathway is a quality control process with important consequences for human disease. From initial studies using RNA hairpin-tagged mRNAs for purification of messenger ribonucleoproteins assembled on transcripts with HIV-1 3' untranslated region (3'UTR) sequences, we uncover a two-step mechanism for Upf1-dependent degradation of mRNAs with long 3'UTRs. We demonstrate that Upf1 associates with mRNAs in a 3'UTR length-dependent manner and is highly enriched on transcripts containing 3'UTRs known to elicit NMD. Surprisingly, Upf1 recruitment and subsequent RNA decay can be antagonized by retroviral RNA elements that promote translational readthrough. By modulating the efficiency of translation termination, recognition of long 3'UTRs by Upf1 is uncoupled from the initiation of decay. We propose a model for 3'UTR length surveillance in which equilibrium binding of Upf1 to mRNAs precedes a kinetically distinct commitment to RNA decay.

CHAF1A/B mediate silencing of unintegrated HIV-1 DNAs early in infection.

Early events of the retroviral life cycle are the targets of many host restriction factors that have evolved to prevent establishment of infection. Incoming retroviral DNAs are transcriptionally silenced before integration in most cell types, and efficient viral gene expression occurs only after formation of the provirus. The molecular machinery for silencing unintegrated retroviral DNAs of HIV-1 remains poorly characterized. Here, we identified the histone chaperones CHAF1A and CHAF1B as essential factors for silencing of unintegrated HIV-1 DNAs. Using RNAi-mediated knockdown (KD) of multiple histone chaperones, we found that KD of CHAF1A or CHAF1B resulted in a pronounced increase in expression of incoming viral DNAs. The function of these two proteins in silencing was independent of their interaction partner RBBP4. Viral DNA levels accumulated to significantly higher levels in CHAF1A KD cells over controls, suggesting enhanced stabilization of actively transcribed DNAs. Chromatin immunoprecipitation assays revealed no major changes in histone loading onto viral DNAs in the absence of CHAF1A, but levels of the H3K9 trimethylation silencing mark were reduced. KD of the H3K9me3-binding protein HP1γ accelerated the expression of unintegrated HIV-1 DNAs. While CHAF1A was critical for silencing HIV-1 DNAs, it showed no role in silencing of unintegrated retroviral DNAs of mouse leukemia virus. Our study identifies CHAF1A and CHAF1B as factors involved specifically in silencing of HIV-1 DNAs early in infection. The results suggest that these factors act by noncanonical pathways, distinct from their histone loading activities, to mediate silencing of newly synthesized HIV-1 DNAs.

Dissociation of EphB2 signaling pathways mediating progenitor cell proliferation and tumor suppression.

Signaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth.

NP220 mediates silencing of unintegrated retroviral DNA.

The entry of foreign DNA into many mammalian cell types triggers the innate immune system, a complex set of responses to prevent infection by pathogens. One aspect of the response is the potent epigenetic silencing of incoming viral DNAs1, including the extrachromosomal DNAs that are formed immediately after infection by retroviruses. These unintegrated viral DNAs are very poorly transcribed in all cells, even in permissive cells, in contrast to the robust expression that is observed after viral integration2-5. The factors that are responsible for this low expression have not yet been identified. Here we performed a genome-wide CRISPR-Cas9 screen for genes that are required for silencing an integrase-deficient MLV-GFP reporter virus to explore the mechanisms responsible for repression of unintegrated viral DNAs in human cells. Our screen identified the DNA-binding protein NP220, the three proteins (MPP8, TASOR and PPHLN1) that comprise the HUSH complex-which silences proviruses in heterochromatin6 and retrotransposons7,8-the histone methyltransferase SETDB1, and other host factors that are required for silencing. Further tests by chromatin immunoprecipitation showed that NP220 is the key protein that recruits the HUSH complex, SETDB1 and the histone deacetylases HDAC1 and HDAC4 to silence the unintegrated retroviral DNA. Knockout of NP220 accelerates the replication of retroviruses. These experiments identify the molecular machinery that silences extrachromosomal retroviral DNA.

Riplet Binds the Zinc Finger Antiviral Protein (ZAP) and Augments ZAP-Mediated Restriction of HIV-1.

The zinc finger antiviral protein (ZAP) is an interferon-stimulated gene (ISG) with potent intrinsic antiviral activity. ZAP inhibits the replication of retroviruses, including murine leukemia virus (MLV) and HIV-1, as well as alphaviruses, filoviruses, and hepatitis B virus, and also the retrotransposition of LINE-1 and Alu retroelements. ZAP operates posttranscriptionally to reduce the levels of viral transcripts available for translation in the cytoplasm, although additional functions might be involved. Recent studies have shown that ZAP preferentially binds viral mRNAs containing clusters of CpG dinucleotides via its four CCCH-type zinc fingers. ZAP lacks enzymatic activity and utilizes other cellular proteins to suppress viral replication. Tripartite motif 25 (TRIM25) and the nuclease KHNYN have been identified as ZAP cofactors. In this study, we identify Riplet, a protein known to play a central role in the activation of the retinoic acid-inducible gene I (RIG-I), as a novel ZAP cofactor. Overexpression of Riplet acts to strongly augment ZAP's antiviral activity. Riplet is an E3 ubiquitin ligase containing three domains, an N-terminal RING finger domain, a central coiled-coil domain, and a C-terminal P/SPRY domain. We show that Riplet interacts with ZAP via its P/SPRY domain and that the ubiquitin ligase activity of Riplet is not required to stimulate ZAP-mediated virus inhibition. Moreover, we show that Riplet interacts with TRIM25, suggesting that both Riplet and TRIM25 may operate as a complex to augment ZAP activity. IMPORTANCE The ZAP is a potent restriction factor inhibiting replication of many RNA viruses by binding directly to viral RNAs and targeting them for degradation. We here identify RIPLET as a cofactor that stimulates ZAP activity. The finding connects ZAP to other innate immunity pathways and suggests oligomerization as a common theme in sensing pathogenic RNAs.

Knockdown screens to knockout HIV-1.

Three recent screens use siRNAs to identify host genes that are critical for HIV-1 replication. These screens have uncovered hundreds of human genes not previously known to be commandeered by the virus during infection. Although some caveats remain, this screening approach opens up a new landscape of viral-host interactions for future exploration.

Yin Yang 1 is a potent activator of human T lymphotropic virus type 1 LTR-driven gene expression via RNA binding.

Yin Yang 1 (YY1) is a DNA-binding transcription factor that either activates or represses gene expression. YY1 has previously been implicated in the transcriptional silencing of many retroviruses by binding to DNA sequences in the U3 region of the viral long terminal repeat (LTR). We here show that YY1 overexpression leads to profound activation, rather than repression, of human T lymphotropic virus type 1 (HTLV-1) expression, while YY1 down-regulation reduces HTLV-1 expression. The YY1 responsive element mapped not to YY1 DNA-binding sites in the HTLV-1 LTR but to the R region. The HTLV-1 R sequence alone is sufficient to provide YY1 responsiveness to a nonresponsive promoter, but only in the sense orientation and only when included as part of the mRNA. YY1 binds to the R region of HTLV-1 RNA in vitro and in vivo, leading to increased transcription initiation and elongation. The findings indicate that YY1 is a potent transactivator of HTLV-1 gene expression acting via binding viral RNA, rather than DNA.

Two lymphoid cell lines potently silence unintegrated HIV-1 DNAs.

Mammalian cells mount a variety of defense mechanisms against invading viruses to prevent or reduce infection. One such defense is the transcriptional silencing of incoming viral DNA, including the silencing of unintegrated retroviral DNA in most cells. Here, we report that the lymphoid cell lines K562 and Jurkat cells reveal a dramatically higher efficiency of silencing of viral expression from unintegrated HIV-1 DNAs as compared to HeLa cells. We found K562 cells in particular to exhibit an extreme silencing phenotype. Infection of K562 cells with a non-integrating viral vector encoding a green fluorescent protein reporter resulted in a striking decrease in the number of fluorescence-positive cells and in their mean fluorescence intensity as compared to integration-competent controls, even though the levels of viral DNA in the nucleus were equal or in the case of 2-LTR circles even higher. The silencing in K562 cells was functionally distinctive. Histones loaded on unintegrated HIV-1 DNA in K562 cells revealed high levels of the silencing mark H3K9 trimethylation and low levels of the active mark H3 acetylation, as detected in HeLa cells. But infection of K562 cells resulted in low H3K27 trimethylation levels on unintegrated viral DNA as compared to higher levels in HeLa cells, corresponding to low H3K27 trimethylation levels of silent host globin genes in K562 cells as compared to HeLa cells. Most surprisingly, treatment with the HDAC inhibitor trichostatin A, which led to a highly efficient relief of silencing in HeLa cells, only weakly relieved silencing in K562 cells. In summary, we found that the capacity for silencing viral DNAs differs between cell lines in its extent, and likely in its mechanism.

Moloney Murine Leukemia Virus p12 Is Required for Histone Loading onto Retroviral DNAs.

During retrovirus infection, a histone-free DNA copy of the viral RNA genome is synthesized and rapidly loaded with nucleosomes de novo upon nuclear entry. The potential role of viral accessory proteins in histone loading onto retroviral DNAs has not been extensively investigated. The p12 protein of Moloney murine leukemia virus (MMLV) is a virion protein that is critical for tethering the incoming viral DNA to host chromatin in the early stages of infection. Infection by virions containing a mutant p12 (PM14) defective in chromatin tethering results in the formation of viral DNAs that do not accumulate in the nucleus. In this report, we show that viral DNAs of these mutants are not loaded with histones. Moreover, the DNA genomes delivered by mutant p12 show prolonged association with viral structural proteins nucleocapsid (NC) and capsid (CA). The histone-poor viral DNA genomes do not become associated with the host RNA polymerase II machinery. These findings provide insights into fundamental aspects of retroviral biology, indicating that tethering to host chromatin by p12 and retention in the nucleus are required to allow loading of histones onto the viral DNA. IMPORTANCE Incoming retroviral DNAs are rapidly loaded with nucleosomal histones upon entry into the nucleus and before integration into the host genome. The entry of murine leukemia virus DNA into the nucleus occurs only upon dissolution of the nuclear membrane in mitosis, and retention in the nucleus requires the action of a viral protein, p12, which tethers the DNA to host chromatin. Data presented here show that the tethering activity of p12 is required for the loading of histones onto the viral DNA. p12 mutants lacking tethering activity fail to acquire histones, retain capsid and nucleocapsid proteins, and are poorly transcribed. The work defines a new requirement for a viral protein to allow chromatinization of viral DNA.

Structural Determinants of Virion Assembly and Release in the C Terminus of the Mason-Pfizer Monkey Virus Capsid Protein.

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid (CA) lattice is located downstream of the CA protein in many retroviral Gags. The HIV-1 Gag protein contains a stretch of 5 amino acid residues termed the "clasp motif," important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of HIV-1 and Mason-Pfizer monkey virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide a comparable function. The importance of the sequences spanning the CA-nucleocapsid (NC) cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study, we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant proteins in vitro and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant viruses in vivo. The mutants revealed major defects in virion assembly and release in HEK 293T and HeLa cells and even larger defects in infectivity. Our data identify the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient retroviral infection. IMPORTANCE The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short "clasp" motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.

Mutations altering acetylated residues in the CTD of HIV-1 integrase cause defects in proviral transcription at early times after integration of viral DNA.

The central function of the retroviral integrase protein (IN) is to catalyze the integration of viral DNA into the host genome to form the provirus. The IN protein has also been reported to play a role in a number of other processes throughout the retroviral life cycle such as reverse transcription, nuclear import and particle morphogenesis. Studies have shown that HIV-1 IN is subject to multiple post-translational modifications (PTMs) including acetylation, phosphorylation and SUMOylation. However, the importance of these modifications during infection has been contentious. In this study we attempt to clarify the role of acetylation of HIV-1 IN during the retroviral life cycle. We show that conservative mutation of the known acetylated lysine residues has only a modest effect on reverse transcription and proviral integration efficiency in vivo. However, we observe a large defect in successful expression of proviral genes at early times after infection by an acetylation-deficient IN mutant that cannot be explained by delayed integration dynamics. We demonstrate that the difference between the expression of proviruses integrated by an acetylation mutant and WT IN is likely not due to altered integration site distribution but rather directly due to a lower rate of transcription. Further, the effect of the IN mutation on proviral gene expression is independent of the Tat protein or the LTR promoter. At early times after integration when the transcription defect is observed, the LTRs of proviruses integrated by the mutant IN have altered histone modifications as well as reduced IN protein occupancy. Over time as the transcription defect in the mutant virus diminishes, histone modifications on the WT and mutant proviral LTRs reach comparable levels. These results highlight an unexpected role for the IN protein in regulating proviral transcription at early times post-integration.

Widespread transmission of independent cancer lineages within multiple bivalve species.

Most cancers arise from oncogenic changes in the genomes of somatic cells, and while the cells may migrate by metastasis, they remain within that single individual. Natural transmission of cancer cells from one individual to another has been observed in two distinct cases in mammals (Tasmanian devils and dogs), but these are generally considered to be rare exceptions in nature. The discovery of transmissible cancer in soft-shell clams (Mya arenaria) suggested that this phenomenon might be more widespread. Here we analyse disseminated neoplasia in mussels (Mytilus trossulus), cockles (Cerastoderma edule), and golden carpet shell clams (Polititapes aureus) and find that neoplasias in all three species are attributable to independent transmissible cancer lineages. In mussels and cockles, the cancer lineages are derived from their respective host species; however, unexpectedly, cancer cells in P. aureus are all derived from Venerupis corrugata, a different species living in the same geographical area. No cases of disseminated neoplasia have thus far been found in V. corrugata from the same region. These findings show that transmission of cancer cells in the marine environment is common in multiple species, that it has originated many times, and that while most transmissible cancers are found spreading within the species of origin, cross-species transmission of cancer cells can occur.

Unintegrated HIV-1 DNAs are loaded with core and linker histones and transcriptionally silenced.

Upon delivery into the nucleus of the host cell, linear double-stranded retroviral DNAs are either integrated into the host genome to form the provirus or act as a target of the DNA damage response and become circularized. Little is known about the chromatinization status of the unintegrated retroviral DNAs of the human immunodeficiency virus type 1 (HIV-1). In this study, we used chromatin immunoprecipitation to investigate the nature of unintegrated HIV-1 DNAs and discovered that core histones, the histone variant H3.3, and H1 linker histones are all deposited onto extrachromosomal HIV-1 DNA. We performed a time-course analysis and determined that the loading of core and linker histones occurred early after virus application. H3.3 and H1 linker histones were also found to be loaded onto unintegrated DNAs of the Moloney murine leukemia virus. The unintegrated retroviral DNAs are potently silenced, and we provide evidence that the suppression of extrachromosomal HIV-1 DNA is histone-related. Unintegrated DNAs were marked by posttranslational histone modifications characteristic of transcriptionally inactive genes: high levels of H3K9 trimethylation and low levels of H3 acetylation. These findings reveal insights into the nature of unintegrated retroviral DNAs.

Cyclin-dependent kinase activity is required for type I interferon production.

Recognition of nucleic acids results in the production of type I IFNs, which activate the JAK/STAT pathway and promote the expression of IFN-stimulated genes. In a search for modulators of this pathway, we discovered an unexpected requirement for cyclin-dependent kinases (CDK) in the production of type I IFN following nucleic acid sensing and virus infection. Inhibition of CDK activity or knockdown of CDK levels leads to a striking block in STAT activation and IFN-stimulated gene expression. CDKs are not required for the initial nucleic acid sensing leading to IFN-ß mRNA induction, nor for the response to exogenous IFN-α/ß, but are critical for IFN-ß release into culture supernatants, suggesting a posttranscriptional role for CDKs in type I IFN production. In the absence of CDK activity, we demonstrate a translational block specific for IFN-ß, in which IFN-ß mRNA is removed from the actively translating polysomes, while the distribution of other cellular mRNAs or global translation rates are unaffected. Our findings reveal a critical role for CDKs in the translation of IFN-ß.