Cytomegalovirus-mediated activation of pyrimidine biosynthesis drives UDP–sugar synthesis to support viral protein glycosylation

Human cytomegalovirus (HCMV) induces numerous changes to the host metabolic network that are critical for high-titer viral replication. We find that HCMV infection substantially induces de novo pyrimidine biosynthetic flux. This activation is important for HCMV replication because inhibition of pyrimidine biosynthetic enzymes substantially decreases the production of infectious virus, which can be rescued through medium supplementation with pyrimidine biosynthetic intermediates. Metabolomic analysis revealed that pyrimidine biosynthetic inhibition considerably reduces the levels of various UDP–sugar metabolites in HCMV-infected, but not mock-infected, cells. Further, UDP–sugar biosynthesis, which provides the sugar substrates required for glycosylation reactions, was found to be induced during HCMV infection. Pyrimidine biosynthetic inhibition also attenuated the glycosylation of the envelope glycoprotein B (gB). Both glycosylation of gB and viral growth were restored by medium supplementation with either UDP–sugar metabolites or pyrimidine precursors. These results indicate that HCMV drives de novo-synthesized pyrimidines to UDP–sugar biosynthesis to support virion protein glycosylation. The importance of this link between pyrimidine biosynthesis and UDP–sugars appears to be partially shared among diverse virus families, because UDP–sugar metabolites rescued the growth attenuation associated with pyrimidine biosynthetic inhibition during influenza A and vesicular stomatitis virus infection, but not murine hepatitis virus infection. In total, our results indicate that viruses can specifically modulate pyrimidine metabolic flux to provide the glycosyl subunits required for protein glycosylation and production of high titers of infectious progeny.A variety of evolutionarily divergent viruses have been shown to activate specific metabolic activities upon infection (1–3). These virally induced metabolic activities can be targeted for antiviral therapy. The most common metabolic-based antivirals include those targeting divergent nucleotide metabolism and are used to treat hepatitis B virus, HIV, human cytomegalovirus (HCMV), and herpes simplex virus infections (4, 5). Increasing evidence has identified additional nonnucleotide metabolic activities that are both specifically induced by viral infection and important for viral replication (6–9). Despite the importance of these activities, in most cases, little is known about how viruses induce these activities or how they contribute to viral infection.HCMV, a member of the betaherpesvirus family, is a widespread pathogen that causes serious disease in immunosuppressed individuals, including cancer patients, transplant recipients, and AIDS patients (10). Additionally, congenital HCMV infection occurs in 1–2% of all live births (11) and can result in multiple system abnormalities, including central nervous system damage (12). HCMV is a double-stranded DNA virus that contains a ∼235-kb genome that encodes >200 ORFs. The genome is encapsulated in a protein capsid that is surrounded by a tegument protein layer. Collectively, this structure is enclosed in a phospholipid envelope, which contains a number of viral glycoproteins that mediate virus attachment and entry (13).We have previously demonstrated that HCMV infection is responsible for numerous changes to the host cell metabolic network (14, 15). These changes include induction of many branches of central carbon metabolism, including glycolysis and the tricarboxylic acid cycle (15). Additionally, HCMV infection results in an expansion of pyrimidine metabolite pools (14). De novo pyrimidine biosynthesis is the main source of pyrimidines during cellular replication, whereas the pyrimidine salvage pathway provides a smaller amount of pyrimidines to quiescent cells and cells in G0 (16, 17). The de novo pathway is primarily regulated through its rate-limiting enzyme, carbamoyl phosphate synthetase–aspartate transcarbamylase–dihydroorotase (CAD). CAD catalyzes the first three steps of the pathway, including the first committed step (18, 19). CAD is a ∼250-kDa protein that possesses three enzymatic activities and multimerizes in vivo (20, 21). CAD is heavily regulated by posttranslational modifications, which alter the sensitivity by which CAD is allosterically activated and inhibited and, in turn, induce or inhibit de novo pyrimidine biosynthesis, respectively (16, 22).De novo pyrimidine biosynthesis also provides pyrimidines for synthesis of UDP–sugars, which are widely used as substrates to feed cellular glycosylation reactions. The UDP–sugars, including UDP–glucose and UDP–N-acetyl-glucosamine (UDP–GlcNAc), along with GDP–mannose, are required for building the necessary precursor oligosaccharide structure that forms immediately before N-linked glycosylation. Additionally, UDP–GlcNAc, but not UDP–glucose, is required for the formation of O-linked glycosyl groups (23). The HCMV viral envelope contains a number of glycoproteins that are critical for HCMV replication (24), but little is known about how HCMV infection may impact the cellular glycosylation machinery.Here, we show that de novo pyrimidine biosynthetic flux is induced upon HCMV infection and that inhibition of de novo pyrimidine biosynthesis reduces HCMV replication, indicating that induction of pyrimidine biosynthesis is necessary for high-titer viral replication. Further, we find that HCMV-infected cells require pyrimidine biosynthesis to maintain UDP–sugar pools and proper glycosylation of the gB virion protein. This link between pyrimidine biosynthesis and the UDP–sugars is important to HCMV infection, because UDP–GlcNAc or UDP–glucose supplementation rescues viral growth in the face of pyrimidine biosynthetic inhibition. Further, the importance of this metabolic link was also observed during influenza A and vesicular stomatitis virus (VSV) infection, suggesting that it could be a common feature of viral infection.     

STING facilitates nuclear import of herpesvirus genome during infection

Once inside the host cell, DNA viruses must overcome the physical barrier posed by the nuclear envelope to establish a successful infection. The mechanism underlying this process remains unclear. Here, we show that the herpesvirus exploits the immune adaptor stimulator of interferon genes (STING) to facilitate nuclear import of the viral genome. Following the entry of the viral capsid into the cell, STING binds the viral capsid, mediates capsid docking to the nuclear pore complex via physical interaction, and subsequently enables accumulation of the viral genome in the nucleus. Silencing STING in human cytomegalovirus (HCMV)-susceptible cells inhibited nuclear import of the viral genome and reduced the ensuing viral gene expression. Overexpressing STING increased the host cell’s susceptibility to HCMV and herpes simplex virus 1 by improving the nuclear delivery of viral DNA at the early stage of infection. These observations suggest that the proviral activity of STING is conserved and exploited by the herpesvirus family. Intriguingly, in monocytes, which act as latent reservoirs of HCMV, STING deficiency negatively regulated the establishment of HCMV latency and reactivation. Our findings identify STING as a proviral host factor regulating latency and reactivation of herpesviruses.

Human cytomegalovirus (HCMV) belongs to the β-herpesvirus subfamily and establishes lifelong latent infection in 60 to 90% of the population worldwide. The inability of the immune response to clear the virus and the absence of a vaccine renders HCMV a serious global health burden (1). HCMV is the most common cause of congenital viral infection during pregnancy and also increases the morbidity and mortality among immunosuppressed transplant patients and AIDS patients (2, 3).DNA viruses must sequentially overcome two physical barriers, the plasma membrane and the nuclear envelope, to replicate in the nucleus (4). The herpesvirus double-stranded DNA (dsDNA) genome is tightly packed inside an icosahedral capsid structure measuring 130 nm in diameter (5, 6). The nuclear pore complex (NPC) allows the transport of macromolecules up to 39 nm in diameter; thus, nuclear entry of herpesviruses with a capsid diameter that is larger than this diffusion limit is a critical step for successful viral infection (7). Herpesviruses employ an uncoating strategy whereby the capsid undergoes conformational change without disruption of the capsid structure, open the portal, and release the viral DNA into the nucleus via NPC. Several host cell surface receptors determining HCMV cell tropism and permissiveness have been identified (8–14); however, the host factor(s) involved in capsid uncoating and nuclear genome import is (are) yet to be uncovered.In the event of improper translocation of the viral genome, the genome is exposed to the cytoplasm. Then, host factors recognize viral DNA as a foreign antigen and elicit antiviral immune response resulting in degradation of the viral genome by cytosolic nucleases (15). Pressure-driven viral DNA release into the host nucleus accounts for only 50% of DNA ejection into the cellular environment, and small polycationic molecules can block viral genome ejection from the capsid, according to recent studies (16, 17). These findings offer two interesting perspectives: the presence of unknown host factors that regulate uncoating and the possibility of developing antiviral agents that target this uncoating process.Although a primary lytic HCMV infection induces robust innate and adaptive immune responses, HCMV is not completely cleared in vivo but, rather, persists in the host cell, becoming a lifelong potential threat (18). HCMV has contrived a strategy to establish latency in the host cell, such as CD34⁺ hematopoietic progenitor cell (HPC) and CD14⁺ monocyte, to survive. Although several entry receptors in fibroblasts and endothelial and epithelial cells have been identified (8–14), only a few host genes or signaling pathways in monocytic cells required for HCMV infection have been researched. For example, EGFR and c-Src signaling must be active to lead to cytoplasmic trafficking, nuclear translocation, and infection in monocytes (19, 20). However, host factors that regulate the HCMV latency and reactivation after HCMV enters the monocytes are largely unknown.Stimulator of interferon (IFN) genes (STING) is an endoplasmic reticulum–resident membrane protein ubiquitously expressed in both immune cells and nonimmune cells (21, 22). Recognition of cytosolic dsDNA leads to the synthesis of cyclic GMP–AMPs (cGAMPs) by activating a cyclic GMP–AMP synthase (cGAS). cGAMPs bind to STING, inducing activation of TBK1 and IRF3 signaling cascades (23). The canonical IFN-dependent role of STING in antiviral immunity has been extensively investigated. Interestingly, several recent reports suggest a noncanonical proviral function of STING in viral infection (24, 25). A potential proviral role of STING in HCMV infection is largely unknown.Here, we demonstrate a proviral function of STING during herpesvirus infection. We show that ectopic expression of STING in STING-deficient cells converts an otherwise insusceptible cell to an HCMV- and herpes simplex virus 1 (HSV-1)–susceptible cell, implying that this STING function is evolutionarily conserved. Furthermore, loss of STING significantly represses the establishment of HCMV latency and viral reactivation in monocytic cells, suggesting that STING is a host factor that regulates the herpesvirus life cycle.     

Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.Human cytomegalovirus (HCMV) is an important pathogen in transplant patients (1–5), and its infection can lead to invasive end-organ diseases, such as pneumonitis and hepatitis, as well as vascular pathology contributing to graft failure (4, 6, 7). HCMV is also the most common cause of in utero viral infections in North America and Europe, affecting 0.5–2% of newborns annually (8–10). Congenital HCMV infection can lead to symptomatic diseases at birth and also cause developmental disabilities in children (10, 11). Maternal seropositivity before conception protects against congenital transmission (12, 13), and both maternal humoral and cellular immunity are likely to contribute to the protection (14–16). Antibodies in particular are important for preventing congenital infection, serving as the first line of defense against maternal infection. It may also play a role in preventing transmission to the fetus, supported by the results of a small, nonrandomized study in pregnant women with primary HCMV infection, in which the passive immunity of monthly infusions of HCMV hyperimmune human IgG (HCMV-HIG) (200 mg/kg maternal weight) was ∼60% effective in protecting against congenital HCMV infection (17, 18). These studies suggest that it is feasible to develop a vaccine for preventing congenital HCMV infection and its sequelae. However, despite the fact that the Institute of Medicine has identified development of an effective vaccine for prevention of congenital HCMV as a top priority since 1999 (19), progress toward this goal has only been incremental (8, 20, 21). One of the hurdles to the efforts is our limited understanding of component of natural immunity associated with protection against HCMV infection.HCMV is a large, complex virus, with a genome capable of encoding >150 proteins (22–26). Because of the strict species specificity, options of animal models for HCMV research are limited (27). Thus, the functions of most HCMV antigens in viral infection in vivo and their roles as targets for host immunity are poorly understood. Furthermore, culture systems of single cell types have limitations for studying HCMV pathogenesis. Immunohistochemistry studies showed that HCMV can infect varieties of cells in vivo, including endothelial, epithelial cells, fibroblasts, and leukocytes (28–36). Many HCMV end-organ diseases, such as pneumonitis and gastroenteritis, are due to infection of the epithelial/endothelial cells in the affected organ (35–39). However, common laboratory strains, such as AD169 and Towne, were culture-adapted in fibroblast cells, with genomic mutations (22, 24, 40) and, more importantly, have lost their tropism to endothelial and epithelial cells, in contrast to pathogenic clinical isolates (32, 33, 41, 42).Loss of viral tropism to endothelial and epithelial cells was mapped to various mutations in the viral UL131-128 locus, and these mutations abrogated the expression of the pentameric glycoprotein H (gH) complex, composed of gH, gL, UL128, UL130, and UL131 proteins, a determinant for viral tropism to endothelial and epithelial cells (42–44). Because the pentameric gH complex is missing in common laboratory strains (42, 43), its importance in viral tropism, viral pathogenesis, and vaccine design was not fully appreciated until recently (42, 45). With this understanding, it is not surprising that Towne virus and recombinant glycoprotein B (gB) vaccines, although with ∼50% efficacy against primary infection in the clinic (46–49), induced poor neutralizing titers against viral infection of epithelial cells, in contrast to immune sera from HCMV-seropositive donors (50, 51). Thus, missing the pentameric gH complex is likely a deficiency in antigen composition for both vaccines (50). Studies of monoclonal antibodies (mAbs) isolated from HCMV-seropositive donors or polyclonal IgG enriched for antigen specificity supported the hypothesis that the pentameric gH complex, not gB, appears to be important for neutralizing activity in human subjects with natural infection (52).We recently described an experimental vaccine virus in which expression of the pentameric gH complex was restored (53). Unlike the parental AD169 virus and the recombinant gB vaccine, this virus can elicit high levels of neutralizing antibodies in rabbits and rhesus macaques (53). To support clinical development of this vaccine centered its concept on the pentameric gH complex, we established a comprehensive panel of 45 mAbs from a single rabbit that received vaccination. Of the 45 mAbs, 25 had neutralizing activity against viral entry in epithelial cells, including 11 elite neutralizers with ≥10-fold greater potency than HCMV-HIG. Biochemical analysis demonstrated that all elite neutralizers preferentially bound to the virus expressing the pentameric gH complex, and the majority of elite neutralizers (8 of 11) specifically recognized a recombinant form of the pentameric gH complex. Interestingly, binding affinity for intact virions was not correlated with neutralizing activity. Moreover, genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. In addition, neutralizing antibodies had longer complementarity-determining region 3 (CDR3) for both heavy and light chains than those of antibodies with no neutralizing activity. These data establish the importance of the pentameric gH complex as the primary target for potent neutralizing antibodies by vaccination, and support development of an experimental HCMV vaccine featuring the pentameric gH complex.     

Antibody-driven design of a human cytomegalovirus gHgLpUL128L subunit vaccine that selectively elicits potent neutralizing antibodies

The use of neutralizing antibodies to identify the most effective antigen has been proposed as a strategy to design vaccines capable of eliciting protective B-cell immunity. In this study, we analyzed the human antibody response to cytomegalovirus (human cytomegalovirus, HCMV) infection and found that antibodies to glycoprotein (g)B, a surface glycoprotein that has been developed as a HCMV vaccine, were primarily nonneutralizing. In contrast, most of the antibodies to the complex formed by gH, gL, protein (p)UL128, pUL130, and pUL131 (the gHgLpUL128L pentamer) neutralized HCMV infection with high potency. Based on this analysis, we developed a single polycistronic vector encoding the five pentamer genes separated by “self-cleaving” 2A peptides to generate a stably transfected CHO cell line constitutively secreting high levels of recombinant pentamer that displayed the functional antigenic sites targeted by human neutralizing antibodies. Immunization of mice with the pentamer formulated with different adjuvants elicited HCMV neutralizing antibody titers that persisted to high levels over time and that were a hundred- to thousand-fold higher than those found in individuals that recovered from primary HCMV infection. Sera from mice immunized with the pentamer vaccine neutralized infection of both epithelial cells and fibroblasts and prevented cell-to-cell spread and viral dissemination from endothelial cells to leukocytes. Neutralizing monoclonal antibodies from immunized mice showed the same potency as human antibodies and targeted the same as well as additional sites on the pentamer. These results illustrate with a relevant example a general and practical approach of analytic vaccinology for the development of subunit vaccines against complex pathogens.Human cytomegalovirus (HCMV) is a ubiquitously distributed member of the Herpesviridae family that establishes a lifelong infection and represents a major threat for human health. Primary infection during pregnancy is the most frequent cause of congenital birth defects, with an overall 0.6% incidence, whereas severe infections develop in immunocompromised patients (1, 2). In addition, HCMV has been proposed as an agent associated with immune senescence (3) and atherosclerosis (4).HCMV has a broad cell tropism and exploits multiple glycoprotein complexes present on the virion envelope for binding and fusion with host cells. Some glycoproteins (g), such as gM/gN and gB, are used to infect several cell types, whereas glycoprotein complexes containing gH and gL mediate cell type-specific virus entry (5, 6). A pentameric complex comprising gH, gL, protein (p)UL128, pUL130, and pUL131 [gHgLpUL128locus (L)] was shown to be required by clinical HCMV isolates to infect endothelial, epithelial, and myeloid cells (7–10). In vitro cultured HCMV viruses with mutations in the UL128–131 locus lose tropism for endothelial and epithelial cells but retain the expression of the gHgL-containing complex, which is sufficient to infect fibroblasts (11).Because of the high incidence rate of HCMV infections and its impact on public health, considerable efforts have been made in the last decade to develop treatments or vaccines capable of preventing HCMV infection (12). The major target populations for a HCMV vaccine are seronegative women of childbearing age, whereas infants represent another potential population contributing to viral dissemination (13). In addition, patients on a list for organ transplantation (especially those with HCMV-seronegative who are at risk for life-threatening HCMV disease) would benefit from a HCMV vaccine. The administration of the HCMV-attenuated Towne vaccine prevented the development of disease in kidney transplant recipients, although it did not prevent infection (14).The abundant virion protein gB was shown to elicit vigorous T-cell and antibody responses and represents the basis of most vaccines developed so far (15). However, in recent phase II trials, a MF59-adjuvanted gB vaccine showed modest efficacy in preventing infection (16) and reducing duration of viremia in transplant recipients (17). These findings may be explained by the finding that most antibodies induced by the vaccines lack virus-neutralizing activity (18), whereas those that neutralized did not block efficiently infection of epithelial cells (19). Therefore, a HCMV vaccine capable of eliciting neutralizing antibodies that prevent the infection of multiple cellular targets and block viral dissemination is considered a high priority (20).Passively administered polyclonal antibodies isolated from seropositive donors were suggested to be effective in preventing infection of the fetus (21). These findings were not confirmed in a recent randomized study where the same antibody preparation showed a modest, not significant, effect on the rate of congenital HCMV infection, possibly due to the low level of neutralizing antibodies contained in Ig preparation (22).We previously isolated from HCMV immune donors antibodies that bound to conformational epitopes on the gHgLpUL128L pentameric complex and were extraordinarily potent in neutralizing HCMV infection of epithelial, endothelial, and myeloid cells (23). The pentamer-specific antibodies neutralized viral infection at picomolar concentrations and were a thousand-fold more potent than antibodies to gB, gH, or gMgN complex (23). More recently, we showed that an early antibody response to the pentamer was associated with lack of viral transmission to the fetus from HCMV-infected pregnant mothers, suggesting that pentamer-specific antibodies are responsible for the inhibition of viral spread in vivo (24).In this study, we report a systematic analysis of the human antibody response to HCMV infection, which indicates that the gHgLpUL128L pentamer is the target of the most effective neutralizing antibodies. Based on this information, we developed a novel process to produce in a secreted form a recombinant pentamer vaccine from a mammalian CHO cell line stably transfected by a single polycistronic vector encoding the five different HCMV pentamer genes separated by autonomous “self-cleaving” 2A peptides. We found that this vaccine can elicit in mice titers of neutralizing antibodies 100–1,000-fold higher than those induced by natural infection. These antibodies neutralized infection of both epithelial cells and fibroblasts and prevented viral dissemination from endothelial cells to leukocytes.     

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ₁34.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ₁34.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ₁34.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ₁34.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.

Herpes simplex viruses (HSV) are human pathogens that switch between lytic and latent infections intermittently (1, 2). This is a lifelong source of infectious viruses (1, 2), in which immediate early proteins drive the onset of HSV replication. Among them, ICP0 enables viral gene expression or reactivation from latency (2–4), which involves chromatin remodeling of the HSV genome, resulting in de novo virus production. In this process, the accessory factor γ₁34.5 of HSV is thought to govern viral protein synthesis (5, 6). It has long been known that γ₁34.5 precludes translation arrest mediated by double-stranded RNA–dependent protein kinase PKR (7–9). The γ₁34.5 protein has also been shown to dampen intracellular nucleic acid sensing, inhibit autophagy, and facilitate virus nuclear egress (10–17). In experimental animal models, wild-type HSV, but not HSV that lacks the γ₁34.5 gene, replicates competently, penetrates from the peripheral tissues to the nervous system and reactivates from latency (18–23). Despite these observations, active HSV replication or reactivation from latency is not readily reconciled by the currently known functions of the γ₁34.5 protein (8–13, 16, 17).Several lines of work demonstrate that tripartite motif (TRIM) proteins regulate innate immune signaling and cell intrinsic resistance to virus infections (24, 25). These host factors typically work as E3 ubiquitin ligases that can synthesize degradative or nondegradative ubiquitination on viral or host proteins. A number of TRIM proteins, for example TRIM5α, TRIM19, TRIM21, TRIM22, and TRIM43, act at different steps of virus replication and subsequently inhibit viral production (26–32). Recent evidence indicates that TRIM23 limits the replication of certain RNA viruses and DNA viruses, including HSV-1 (33). In doing so, TRIM23 recruits TANK-binding kinase 1 (TBK1) to autophagosomes, thus promoting TBK1-mediated phosphorylation and activation of the autophagy receptor p62 and ultimately leading to autophagy. It is unknown whether TRIM23 plays an additional role(s) in HSV infection.Here, we report that ICP0 expression is regulated by the γ₁34.5 protein and TRIM23 during HSV-1 infection. We show that TRIM23 facilitates the proteasomal degradation of ICP0, whereas viral γ₁34.5 maintains steady-state ICP0 expression by preventing K27-linked TRIM23 autoubiquitination that is required for TRIM23 activation. The γ₁34.5 protein also interacts with and stabilizes ICP0, enabling productive infection. Furthermore, we provide evidence that TRIM23 binds to ICP0 and induces its K11-linked polyubiquitination, which triggers K48-linked polyubiquitin-dependent proteasomal degradation of ICP0. These insights establish a model of posttranslational networks in which virus- and host-mediated mechanisms regulate immediate early protein ICP0 stability and thereby lytic HSV replication.     

SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

SARS-CoV-2 is a novel beta-coronavirus that is closely related to two other highly pathogenic human coronaviruses, SARS-CoV and MERS-CoV (1). The spike (S) proteins of SARS-CoV-2 and SARS-CoV mediate entry into target cells, and both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor (2–6). The spike protein of SARS-CoV-2 is also responsible for induction of neutralizing antibodies, thus playing a critical role in host immunity to viral infection (7–10).Similar to HIV and other class I viral fusion proteins, SARS-CoV-2 spike is synthesized as a precursor that is subsequently cleaved and highly glycosylated; these properties are critical for regulating viral fusion activation, native spike structure, and evasion of host immunity (11–15). However, distinct from SARS-CoV, yet similar to MERS-CoV, the spike protein of SARS-CoV-2 is cleaved by furin into S1 and S2 subunits during the maturation process in producer cells (6, 16, 17). S1 is responsible for binding to the ACE2 receptor, whereas S2 mediates viral membrane fusion (18, 19). SARS-CoV-2 spike can also be cleaved by additional host proteases, including transmembrane serine protease 2 (TMPRSS2) on the plasma membrane and several cathepsins in the endosome, which facilitate viral membrane fusion and entry into host cells (20–22).Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact (23–26). The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases (27), resulting in efficient transfer of virus to neighboring cells (24). Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV) (28–32). Low levels of neutralizing antibodies, as well as a deficiency in type I IFNs, have been reported for SARS-CoV-2 (18, 33–37) and may have contributed to the COVID-19 pandemic and disease progression (38–43).In this work, we evaluated cell-to-cell transmission of SARS-CoV-2 in the context of cell-free infection and in comparison with SARS-CoV. Results from this in vitro study reveal the heretofore unrecognized role of cell-to-cell transmission that potentially impacts SARS-CoV-2 spread, pathogenesis, and shielding from antibodies in vivo.     

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.Human cytomegalovirus (HCMV) is a member of the β-herpesvirus subfamily with >60% seropositivity in adults worldwide (1). HCMV infection is typically asymptomatic, but can cause severe disease or death in immunocompromised solid organ and hematopoietic stem cell transplant recipients. In addition, HCMV can infect the placenta and cross this barrier to infect developing fetuses, causing severe birth defects (2). Given the severity and importance of this disease, obtaining an effective vaccine is considered a public health priority (3).The ability of HCMV to cause disease in a wide range of organs and tissue types is reflected at the cellular level by the virus infecting epithelial cells, endothelial cells, fibroblasts, dendritic cells, hepatocytes, neurons, macrophages, and leukocytes (4). Similar to other herpesviruses, the envelope glycoproteins gB and gH/gL form the conserved fusion machinery required for viral entry (5, 6). Recent structural and mutagenesis analysis suggested that gB is responsible for mediating virus and host membrane fusion during viral entry (7, 8). The role of gH/gL in fusion is less clear because crystal structures of herpes simplex virus 2 (HSV-2), pseudo-rabies virus (PrV), and Epstein–Barr virus (EBV) gH/gL did not reveal any similarity to known viral fusion proteins (9–11). It has been proposed that gH/gL is involved in the entry process through activation of gB (12). In addition to gB and gH/gL, most herpesviruses encode additional glycoproteins that are able to interact with gH/gL and are capable of either mediating binding to specific cellular receptors or regulating the activity of the gH/gL–gB complex (5, 6).HCMV entry into both epithelial and endothelial cells requires a pentameric glycoprotein complex (Pentamer) formed between gH/gL and the UL128, UL130, and UL131A proteins (13, 14). Mutations in the UL131A–UL128 gene locus are sufficient to eliminate epithelial/endothelial tropism and occur spontaneously within only a few passages of wild-type (WT) HCMV in fibroblasts (15, 16). In addition, Pentamer cell surface overexpression interferes with HCMV entry into epithelial cells, but not into fibroblasts, suggesting the presence of a cell-type-specific Pentamer receptor (17).HCMV entry into fibroblasts is mediated by the gH/gL/gO complex at the cell surface at neutral pH (18–21). gO is a highly glycosylated protein and has been shown to covalently interact with gH/gL (22, 23). It has been proposed that gO might function as a molecular chaperone to promote gH/gL incorporation, but not gH/gL/gO, into the virion (21). However, it has been recently demonstrated that gH/gL/gO and Pentamer are much more abundant on the HCMV envelope than gH/gL alone (24).Highly potent HCMV-neutralizing monoclonal antibodies were isolated from the memory B-cell repertoire of HCMV-immune donors and shown to bind the Pentamer. These antibodies were capable of neutralizing HCMV infection of epithelial/endothelial cells, but not fibroblasts (25, 26). In addition, several studies have demonstrated that the Pentamer is the main target of the neutralizing humoral response to HCMV infection in epithelial/endothelial cells (27–29). Consistent with these observations, immunization with the Pentamer has been shown to elicit a strong neutralizing antibody response in mouse, rabbit, and rhesus macaque models (30–32). Together these data indicate that the Pentamer represents a key antigenic target for vaccine development against HCMV infection.Here we report the purification and biochemical characterization of HCMV gH/gL, gH/gL/gO, and Pentamer. In addition, we describe the architecture of these complexes by electron microscopy (EM) and characterize their interaction with MSL-109, a previously described HCMV-neutralizing antibody isolated from the spleen of a HCMV-seropositive individual (33, 34). Our data provide new insights into the structure and function of the HCMV gH/gL/gO and Pentamer complexes.     

SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell–cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2′ fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2′ cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2′ cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has exceeded 240 million cases across the globe, but the molecular mechanisms of viral infection and host pathogenesis remain elusive. The SARS-CoV-2 spike (S) glycoprotein is a class I fusion protein decorated on the viral lipid envelope and is a key determinant of viral entry (1). The SARS-CoV-2 spike monomer contains two fragments: The amino terminus S1 subunit contains a receptor binding domain (RBD) (2–5), which recognizes the host receptor angiotensin-converting enzyme 2 (ACE2) for initial docking, while the carboxyl terminus S2 subunit catalyzes the fusion of viral and cell membranes (6, 7), enabling the subsequent release of viral RNA genome and downstream replication within the infected cells (8). Although many studies have captured the stationary phases of spike binding to human ACE2 (9–11), key molecular and cellular processes downstream of receptor recognition have not been explored.Spike can be proteolytically processed (12). SARS-CoV-2 spike encodes a polybasic cleavage site at its S1/S2 junction, and is posttranslationally processed by the endopeptidase furin (13, 14); cleaved S1 and S2 subunits remain noncovalently attached and fusion competent (15). Furin-cleaved S1 also exposes a C-terminal motif recognized by the host receptor neuropilin-1 (NRP1) (16, 17), which can facilitate SARS-CoV-2 entry. Although spike protein is autoprocessed, additional proteolytic cleavage event within the S2 subunit is proposed to be responsible for the subsequent membrane fusion (18, 19). This cleavage can be mediated at the plasma membrane by the type II transmembrane serine proteases (TMPRSS2) (20–23), or processed by the lysosomal cathepsins during the endocytosis of viral particles (24). Secreted tissue proteases, such as elastase and trypsin, can also facilitate this cleavage event and promote infection (25). As a result, this proteolytic event within the S2 subunit induces the release of a highly conserved hydrophobic region, known as the fusion peptide (18), which subsequently anchors the target host cell membrane (6, 26). A conformational reconfiguration within the S2 subunit then pulls the viral and host membranes into close proximity, allowing lipid membranes to fuse (7, 27–29). The unilateral change of the S2 subunit is of the utmost importance during viral entry, but molecular events regulating the spike processing and activation have not been demonstrated.Cells infected with SARS-CoV-2 drive the fusion with adjacent ACE2-expressing cells, producing morphologically distinct multinuclear giant cells, also known as syncytia (2, 30, 31). Spike-mediated syncytia have been reported in the postmortem lung samples of severe COVID-19 patients (32, 33). Apart from virus to cell transmission, spike-driven syncytia formation may provide an additional route for cell–cell transmission of SARS-CoV-2. Here, by using a cell–cell fusion system, in complement with a pseudoviral particle infection model, we study the functional and molecular requirements of spike activation. Through analyzing the prefusion and postfusion spike protein products, we show that proteolytic cleavage at the S2′ site is triggered by human cell receptor recognition in a range of immortalized cell lines and humanized primary cells. Generation of the S2′ fragment specifically requires spike recognition of functional host ACE2 and is conserved in the several variants of concern. We highlight that arginine 815, but not arginine residues at the S1/S2 cleavage site, is indispensable for the S2′ cleavage and syncytia formation in wild type (WT), as well as the more infectious Alpha, Beta, and Delta spike variants. Hence, these data highlight that both receptor recognition and proteolytic event at the S2′ site are functionally important for spike-mediated membrane fusion and SARS-CoV-2 infection.     

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15–gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

Bacteriophages (phages) are viruses that infect bacteria and are considered to be the most abundant entities on Earth. Members of the order Caudovirales are double-stranded DNA (dsDNA) tailed phages (1, 2) that can infect a wide variety of hosts, and they are present in many different environments (3). These viruses possess a tail protein complex that is assembled at one special vertex of their icosahedral capsid, known as the portal vertex (4). Connector or portal proteins create an entry channel to the viral capsid and serve as a docking point for the tail complex (4, 5). Tailed phages generally show a common infection strategy and thus have strong structural homologies, although the protein machinery responsible for host adsorption has specific characteristics depending on each phage family (3). Some phages of the Podoviridae family present a short, noncontractile tail that cannot traverse the complex bacterial wall of gram-negative bacteria (6), whereas others do not present tail machinery at all. These latter viruses have developed an alternative mechanism to cross the bacterial envelope using internal capsid proteins or membranes that are able to assemble tubular structures during infection (7–11).Bacteriophage T7 is a well characterized member of the Podoviridae family that infects Escherichia coli. The viral particle is composed of a 55-nm icosahedral capsid and a 23-nm short noncontractile tail (12). The most remarkable structure of the T7 viral particle is the internal core, a cylindrical structure that is ∼290-Å long and ∼170-Å wide located on top of the connector, which stabilizes the packaged DNA inside the capsid (13). This complex is made up of three proteins: gp14 (20.8 kDa), gp15 (84.2 kDa), and gp16 (144 kDa) (9, 13). These internal core proteins are not essential for morphogenesis of the viral capsid, but they are required to translocate the viral genome during the T7 infection process (11, 14, 15). A lytic transglycosylase motif present in gp16 is essential during infection at temperatures below 20 °C to overcome the highly cross-linked peptidoglycan (16–18).As is the case for most phages, T7 first binds reversibly to a primary receptor that allows correct tail orientation in relation to the bacterial surface. Then, an irreversible interaction takes place with the rough lipopolysaccharide (LPS) (19), which causes conformational changes in the tail leading to the opening of the channel. Later, a tubular conduit is assembled, probably composed of the core proteins, and crosses the bacterial wall (9, 11), although it is not clear how this is accomplished. One hypothesis was proposed by Hu et al. (11) in a study using cryo-electron tomography, in which they described the presence of transient tubular structures during T7 infection. They proposed that the core complex formed by gp14, gp15, and gp16 could disassemble after adsorption and pass through the open tail channel in a completely or partially unfolded state (11). According to this hypothesis, partially unfolded gp14 would be ejected through the channel of the tail complex and then refolded to form a pore in the outer membrane of E. coli, which allows unfolded gp15 and gp16 to cross (11, 18). Once in the periplasm, gp15 and gp16 oligomerize as a tubular structure that spans the periplasm and internal membrane and reaches the cytoplasm. When the channel formed by gp14, gp15, and gp16 is complete, translocation of the T7 genome into the bacteria cytoplasm can take place.Here, we report two cryo-electron microscopy (cryo-EM) structures of the T7 core assemblies: gp15 alone (505 kDa) and that of the complex formed by gp15 and gp16 (gp15–gp16 complex; 1.365 MDa). The gp15 protein alone forms a tubular structure in vitro, although its carboxyl-terminal half is disordered. The gp15–gp16 complex is also a tubular structure, but this time with a fully folded gp15. Although only a small fragment of gp16 is observed in the structure of the complex, the solved model comprises the transglycosylase domain. In this article, we have shown that these bacteriophage proteins, which form part of the mature virus in the fully structured core complex with eightfold symmetry (13, 15, 20, 21), are able to unfold during the infection process, exit the phage, and reassemble into a hexameric tubular structure whose size is compatible with the translocation of viral DNA across the bacterial envelope.     

Host cytoskeletal vimentin serves as a structural organizer and an RNA-binding protein regulator to facilitate Zika viral replication

Emerging microbe infections, such as Zika virus (ZIKV), pose an increasing threat to human health. Investigations on ZIKV replication have revealed the construction of replication complexes (RCs), but the role of cytoskeleton in this process is largely unknown. Here, we investigated the function of cytoskeletal intermediate filament protein vimentin in the life cycle of ZIKV infection. Using advanced imaging techniques, we uncovered that vimentin filaments undergo drastic reorganization upon viral protein synthesis to form a perinuclear cage-like structure that embraces and concentrates RCs. Genetic removal of vimentin markedly disrupted the integrity of RCs and resulted in fragmented subcellular dispersion of viral proteins. This led to reduced viral genome replication, viral protein production, and release of infectious virions, without interrupting viral binding and entry. Furthermore, mass spectrometry and RNA-sequencing screens identified interactions and interplay between vimentin and hundreds of endoplasmic reticulum (ER)-resident RNA-binding proteins. Among them, the cytoplasmic-region of ribosome receptor binding protein 1, an ER transmembrane protein that directly binds viral RNA, interacted with and was regulated by vimentin, resulting in modulation of ZIKV replication. Together, the data in our work reveal a dual role for vimentin as a structural element for RC integrity and as an RNA-binding-regulating hub during ZIKV infection, thus unveiling a layer of interplay between Zika virus and host cell.

Zika virus (ZIKV), a mosquito-borne enveloped RNA virus that belongs to the Flaviviridae family, has gained notoriety recently, due to its explosive outbreaks and association with serious clinical diseases, such as Guillain-Barré syndrome in adults and microcephaly in newborns (1–4). Currently, no ZIKV-specific therapies or prophylactic vaccines are available (5). ZIKV genome is a positive-sense, single-stranded RNA [ssRNA(+)] (6). The viral replication occurs on the surface of the endoplasmic reticulum (ER), where the double-strand RNA (dsRNA) is synthesized from viral genomic ssRNA(+) and transcribed into new proteins (7, 8). The viral genome is translated into a polyprotein, which is proteolytically processed into three structural proteins (capsid [C], precursor membrane [prM], and envelop [Env]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), by both host and viral proteases (9, 10). ZIKV infection can rewire cellular components to establish viral replication complexes (RCs), which increase the local concentration of viral and cellular factors for efficient viral replication (11–13). However, the function of rewired cytoskeleton network remains elusive in ZIKV infection.Vimentin is the most abundant intermediate filaments (IFs), which generally surrounds the nucleus and extends throughout the cytoplasm, providing help to important biological processes, such as organelle positioning, cell migration, and cell signaling (14). Except for acting as an integrator of cellular mechanical functions, the highly dynamic nature of vimentin filaments enables it to respond rapidly to pathological stimuli (15). Several studies have observed the phenomenon of vimentin network rearrangement in viral infections and proposed that the role of intact vimentin scaffold could contribute to the viral life cycle (16–21). However, evidence for the dynamic changes of vimentin IFs during ZIKV infection and its contribution to RCs integrity and stability remain understudied.In addition to providing a structural scaffold, there is evidence indicating that cytoskeletal proteins also regulate translational apparatus (22). For example, ribosomes can physically associate with microtubules (MTs) and F-actin in different cells (23, 24). Disorganization of F-actin by cytochalasin D impairs local protein synthesis in isolated axoplasmic nerve fibers (25). The interaction between keratin IFs and the Y subunit of eukaryotic elongation factor-1 (eEF1BY) plays an essential role in protein synthesis (26). MTs can bind to the cytoplasmic tail of RRBP1 and take part in ER organization and neuronal polarity (27, 28). However, information on the spatial and functional relationship between vimentin IFs and the translational machinery, especially in the context of virus infection, remain completely unknown.In this study, we investigated the function of vimentin IFs in ZIKV infection. By monitoring spatial-temporal responses of the cellular vimentin network throughout various steps of the ZIKV life cycle, we demonstrated that ZIKV infection induces massive rearrangements of cytoplasmic vimentin. When vimentin protein was genetically depleted from cells, distribution of viral proteins is scattered within infected cells, indicating its “organizer” role. Viral RNA replication, protein synthesis, and virion release are subsequently reduced. Using mass spectrometry (MS) and RNA-sequencing (RNA-seq) analysis, we discovered many intimate interactions between vimentin and RNA-binding proteins (RBPs), and that vimentin facilitates ZIKV RNA replication by interacting with and regulating the level and subcellular distribution of RRBP1, indicating its “regulator” role. Thus, our work establishes important connections among vimentin filaments dynamics, ZIKV RCs, and cellular RBPs in highly effective infection.     

Thermodynamics of formation of coffinite,USiO4

Coffinite, USiO₄, is an important U(IV) mineral, but its thermodynamic properties are not well-constrained. In this work, two different coffinite samples were synthesized under hydrothermal conditions and purified from a mixture of products. The enthalpy of formation was obtained by high-temperature oxide melt solution calorimetry. Coffinite is energetically metastable with respect to a mixture of UO₂ (uraninite) and SiO₂ (quartz) by 25.6 ± 3.9 kJ/mol. Its standard enthalpy of formation from the elements at 25 °C is −1,970.0 ± 4.2 kJ/mol. Decomposition of the two samples was characterized by X-ray diffraction and by thermogravimetry and differential scanning calorimetry coupled with mass spectrometric analysis of evolved gases. Coffinite slowly decomposes to U₃O₈ and SiO₂ starting around 450 °C in air and thus has poor thermal stability in the ambient environment. The energetic metastability explains why coffinite cannot be synthesized directly from uraninite and quartz but can be made by low-temperature precipitation in aqueous and hydrothermal environments. These thermochemical constraints are in accord with observations of the occurrence of coffinite in nature and are relevant to spent nuclear fuel corrosion.In many countries with nuclear energy programs, spent nuclear fuel (SNF) and/or vitrified high-level radioactive waste will be disposed in an underground geological repository. Demonstrating the long-term (10⁶–10⁹ y) safety of such a repository system is a major challenge. The potential release of radionuclides into the environment strongly depends on the availability of water and the subsequent corrosion of the waste form as well as the formation of secondary phases, which control the radionuclide solubility. Coffinite (1), USiO₄, is expected to be an important alteration product of SNF in contact with silica-enriched groundwater under reducing conditions (2–8). It is also found, accompanied by thorium orthosilicate and uranothorite, in igneous and metamorphic rocks and ore minerals from uranium and thorium sedimentary deposits (2, 4, 5, 8–16). Under reducing conditions in the repository system, the uranium solubility (very low) in aqueous solutions is typically derived from the solubility product of UO₂. Stable U(IV) minerals, which could form as secondary phases, would impart lower uranium solubility to such systems. Thus, knowledge of coffinite thermodynamics is needed to constrain the solubility of U(IV) in natural environments and would be useful in repository assessment.In natural uranium deposits such as Oklo (Gabon) (4, 7, 11, 12, 14, 17, 18) and Cigar Lake (Canada) (5, 13, 15), coffinite has been suggested to coexist with uraninite, based on electron probe microanalysis (EPMA) (4, 5, 7, 11, 13, 17, 19, 20) and transmission electron microscopy (TEM) (8, 15). However, it is not clear whether such apparent replacement of uraninite by a coffinite-like phase is a direct solid-state process or occurs mediated by dissolution and reprecipitation.The precipitation of USiO₄ as a secondary phase should be favored in contact with silica-rich groundwater (21) [silica concentration >10⁻⁴ mol/L (22, 23)]. Natural coffinite samples are often fine-grained (4, 5, 8, 11, 13, 15, 24), due to the long exposure to alpha-decay event irradiation (4, 6, 25, 26) and are associated with other minerals and organic matter (6, 8, 12, 18, 27, 28). Hence the determination of accurate thermodynamic data from natural samples is not straightforward. However, the synthesis of pure coffinite also has challenges. It appears not to form by reacting the oxides under dry high-temperature conditions (24, 29). Synthesis from aqueous solutions usually produces UO₂ and amorphous SiO₂ impurities, with coffinite sometimes being only a minor phase (24, 30–35). It is not clear whether these difficulties arise from kinetic factors (slow reaction rates) or reflect intrinsic thermodynamic instability (33). Thus, there are only a few reported estimates of thermodynamic properties of coffinite (22, 36–40) and some of them are inconsistent. To resolve these uncertainties, we directly investigated the energetics of synthetic coffinite by high-temperature oxide melt solution calorimetry to obtain a reliable enthalpy of formation and explored its thermal decomposition.     

Fog and rain in the Amazon

The diurnal and seasonal water cycles in the Amazon remain poorly simulated in general circulation models, exhibiting peak evapotranspiration in the wrong season and rain too early in the day. We show that those biases are not present in cloud-resolving simulations with parameterized large-scale circulation. The difference is attributed to the representation of the morning fog layer, and to more accurate characterization of convection and its coupling with large-scale circulation. The morning fog layer, present in the wet season but absent in the dry season, dramatically increases cloud albedo, which reduces evapotranspiration through its modulation of the surface energy budget. These results highlight the importance of the coupling between the energy and hydrological cycles and the key role of cloud albedo feedback for climates over tropical continents.Tropical forests, and the Amazon in particular, are the biggest terrestrial CO₂ sinks on the planet, accounting for about 30% of the total net primary productivity in terrestrial ecosystems. Hence, the climate of the Amazon is of particular importance for the fate of global CO₂ concentration in the atmosphere (1). Besides the difficulty of estimating carbon pools (1–3), our incapacity to correctly predict CO₂ fluxes in the continental tropics largely results from inaccurate simulation of the tropical climate (1, 2, 4, 5). More frequent and more intense droughts in particular are expected to affect the future health of the Amazon and its capacity to act as a major carbon sink (6–8). The land surface is not isolated, however, but interacts with the weather and climate through a series of land−atmosphere feedback loops, which couple the energy, carbon, and water cycles through stomata regulation and boundary layer mediation (9).Current General Circulation Models (GCMs) fail to correctly represent some of the key features of the Amazon climate. In particular, they (i) underestimate the precipitation in the region (10, 11), (ii) do not reproduce the seasonality of either precipitation (10, 11) or surface fluxes such as evapotranspiration (12), and (iii) produce errors in the diurnal cycle and intensity of precipitation, with a tendency to rain too little and too early in the day (13, 14). In the more humid Western part of the basin, surface incoming radiation, evapotranspiration, and photosynthesis all tend to peak in the dry season (15–17), whereas GCMs simulate peaks of those fluxes in the wet season (10, 11). Those issues might be related to the representation of convection (1, 2, 4, 5, 13, 14) and vegetation water stress (6–8, 15–17) in GCMs.We here show that we can represent the Amazonian climate using a strategy opposite to GCMs in which we resolve convection and parameterize the large-scale circulation (Methods). The simulations lack many of the biases observed in GCMs and more accurately capture the differences between the dry and wet season of the Amazon in surface heat fluxes and precipitation. Besides top-of-the-atmosphere insolation, the simulations require the monthly mean temperature profile as an input. We demonstrate that this profile, whose seasonal cycle itself is a product of the coupled ocean−land−atmosphere dynamics, mediates the seasonality of the Amazonian climate by modulating the vertical structure of the large-scale circulation in such a way that thermal energy is less effectively ventilated in the rainy season.