Anti-SARS-CoV-2 receptor-binding domain antibody evolution after mRNA vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B cell responses that continue to evolve for at least a year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern1. As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested1,2. Here we examine memory B cell evolution five months after vaccination with either Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccine in a cohort of SARS-CoV-2-naive individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge five months after vaccination of naive individuals express antibodies that are similar to those that dominate the initial response. While individual memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination, the overall neutralizing potency of plasma is greater following vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines will increase plasma neutralizing activity but may not produce antibodies with equivalent breadth to those obtained by vaccinating convalescent individuals.

Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies.

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasmas, including plasmas from individuals from whom some of the antibodies were isolated. While the binding of polyclonal plasma antibodies are affected by mutations across multiple RBD epitopes, the plasma-escape maps most resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is skewed towards a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

Affinity maturation of SARS-CoV-2 neutralizing antibodies confers potency, breadth, and resilience to viral escape mutations.

Antibodies elicited by infection accumulate somatic mutations in germinal centers that can increase affinity for cognate antigens. We analyzed 6 independent groups of clonally related severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) Spike receptor-binding domain (RBD)-specific antibodies from 5 individuals shortly after infection and later in convalescence to determine the impact of maturation over months. In addition to increased affinity and neutralization potency, antibody evolution changed the mutational pathways for the acquisition of viral resistance and restricted neutralization escape options. For some antibodies, maturation imposed a requirement for multiple substitutions to enable escape. For certain antibodies, affinity maturation enabled the neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection.

More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year.

Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies 1,2 . Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines 3,4 . In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naïve individuals 2,5-8 . The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern 4,9 . In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies.

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies.

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

Dynamic regulation of TFH selection during the germinal centre reaction.

The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells1,2. Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide-major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction.

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-21-4. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection5,6. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors5-8. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease 2019 (COVID-19) including two novel mRNA-based vaccines 1,2 . These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known 3-6 . Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) binding titers 3,5,6 . Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection 7,8 . However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.

Evolution of antibody immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

Evolution of Antibody Immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with development of variable levels of antibodies with neutralizing activity that can protect against infection in animal models. Antibody levels decrease with time, but the nature and quality of the memory B cells that would be called upon to produce antibodies upon re-infection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection. We find that IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by five-fold in pseudotype virus assays. In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response. Analysis of intestinal biopsies obtained from asymptomatic individuals 4 months after coronavirus disease-2019 (COVID-19) onset, using immunofluorescence, or polymerase chain reaction, revealed persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 volunteers. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

Enhanced SARS-CoV-2 neutralization by dimeric IgA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of COVID-19. Although potent immunoglobulin G (IgG) antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might affect the initial viral spread and transmissibility from the mucosa. Here, we characterize the IgA response to SARS-CoV-2 in a cohort of 149 convalescent individuals after diagnosis with COVID-19. IgA responses in plasma generally correlated with IgG responses. Furthermore, clones of IgM-, IgG-, and IgA-producing B cells were derived from common progenitor cells. Plasma IgA monomers specific to SARS-CoV-2 proteins were demonstrated to be twofold less potent than IgG equivalents. However, IgA dimers, the primary form of antibody in the nasopharynx, were, on average, 15 times more potent than IgA monomers against the same target. Thus, dimeric IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.

Enhanced SARS-CoV-2 Neutralization by Secretory IgA in vitro.

SARS-CoV-2 primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of the illness. Although potent IgG antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might impact the initial viral spread and transmissibility from the mucosa. Here we characterize the IgA response to SARS-CoV-2 in a cohort of 149 individuals. IgA responses in plasma generally correlate with IgG responses and clones of IgM, IgG and IgA producing B cells that are derived from common progenitors are evident. Plasma IgA monomers are 2-fold less potent than IgG equivalents. However, IgA dimers, the primary form in the nasopharynx, are on average 15 times more potent than IgA monomers. Thus, secretory IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies.

Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-CoV, and mild coronaviruses, and how avidity effects contributed to increased binding/neutralization of IgGs over Fabs. Electron microscopy reconstructions of polyclonal plasma Fab-spike complexes showed recognition of both S1A and RBD epitopes. A 3.4Å cryo-EM structure of a neutralizing monoclonal Fab-S complex revealed an epitope that blocks ACE2 receptor-binding on "up" RBDs. Modeling suggested that IgGs targeting these sites have different potentials for inter-spike crosslinking on viruses and would not be greatly affected by identified SARS-CoV-2 spike mutations. These studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals.

During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml-1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Protein Amounts of the MYC Transcription Factor Determine Germinal Center B Cell Division Capacity.

High-affinity B cell selection in the germinal center (GC) is governed by signals delivered by follicular helper T (Tfh) cells to B cells. Selected B cells undergo clonal expansion and affinity maturation in the GC dark zone in direct proportion to the amount of antigen they capture and present to Tfh cells in the light zone. Here, we examined the mechanisms whereby Tfh cells program the number of GC B cell divisions. Gene expression analysis revealed that Tfh cells induce Myc expression in light-zone B cells in direct proportion to antigen capture. Conditional Myc haplo-insufficiency or overexpression combined with cell division tracking showed that MYC expression produces a metabolic reservoir in selected light-zone B cells that is proportional to the number of cell divisions in the dark zone. Thus, MYC constitutes the GC B cell division timer that when deregulated leads to emergence of B cell lymphoma.

Transcriptional repression of c-Jun's E3 ubiquitin ligases contributes to c-Jun induction by UV.

UV radiation is a major environmental carcinogen. The oncoprotein c-Jun that is required for development of skin cancer is stabilized by UV radiation. The mechanism leading to its stabilization after exposure to UV is not known. The lack of knowledge was particularly sharpened, after the discovery that JNK, the most potent positive regulator of c-Jun, activates Itch, an E3-ligase of c-Jun and JunB. In this study we demonstrate that the expression of all three E3 ubiquitin ligases of c-Jun is down-regulated by UV. The levels of Itch/AIP4 and Fbw7alpha transcripts are reduced following UV exposure in every cell line examined. Repression of hCOP1 and its associated protein hDET1, which is required for c-Jun degradation, is cell type dependent. Expression of Fbw7alpha is down-regulated by UVC or UVB, independently of the p53, MAPK and the PKC pathways but the repression is inhibited in the absence of active Fbw7 proteins suggesting that a target protein of Fbw7 is involved in Fbw7 expression/repression. The repression does not require protein synthesis and UV does not change Fbw7 mRNA stability. The characteristics of Fbw7alpha repression perfectly match with those of c-Jun induction. Unlike UV, ionizing radiation does not repress Fbw7alpha and does not induce c-Jun. In addition, the repression kinetics correlates tightly with the kinetics of c-Jun induction by UV. Moreover, abrogation of Fbw7 UV-responsiveness abolishes c-Jun induction by UV, and knockdown of Fbw7 results in elevated basal expression of c-Jun but reduced UV-dependent induction thus, proving the essential role of this repression in c-Jun induction by UV.