A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates.     

Protection against Lethal Influenza with a Viral Mimic

Despite countermeasures against influenza virus that prevent (vaccines) and treat (antivirals) infection, this upper respiratory tract human pathogen remains a global health burden, causing both seasonal epidemics and occasional pandemics. More potent and safe new vaccine technologies would contribute significantly to the battle against influenza and other respiratory infections. Using plasmid-based reverse genetics techniques, we have developed a single-cycle infectious influenza virus (sciIV) with immunoprotective potential. In our sciIV approach, the fourth viral segment, which codes for the receptor-binding and fusion protein hemagglutinin (HA), has been removed. Thus, upon infection of normal cells, although no infectious progeny are produced, the expression of other viral proteins occurs and is immunogenic. Consequently, sciIV is protective against influenza homologous and heterologous viral challenges in a mouse model. Vaccination with sciIV protects in a dose- and replication-dependent manner, which is attributed to both humoral responses and T cells. Safety, immunogenicity, and protection conferred by sciIV vaccination were also demonstrated in ferrets, where this immunization additionally blocked direct and aerosol transmission events. All together, our studies suggest that sciIV may have potential as a broadly protective vaccine against influenza virus.     

A Unique Role for the Host ESCRT Proteins in Replication of Tomato bushy stunt virus

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.     

The Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41 Harbors Lipid Raft Association Determinants

The molecular basis for localization of the human immunodeficiency virus type 1 envelope glycoprotein (Env) in detergent-resistant membranes (DRMs), also called lipid rafts, still remains unclear. The C-terminal cytoplasmic tail of gp41 contains three membrane-interacting, amphipathic α-helical sequences, termed lentivirus lytic peptide 2 (LLP-2), LLP-3, and LLP-1, in that order. Here we identify determinants in the cytoplasmic tail which are crucial for Env''s association with Triton X-100-resistant rafts. Truncations of LLP-1 greatly reduced Env localization in lipid rafts, and the property of Gag-independent gp41 localization in rafts was conserved among different strains. Analyses of mutants containing single deletions or substitutions in LLP-1 showed that the α-helical structure of the LLP-1 hydrophobic face has a more-critical role in Env-raft associations than that of the hydrophilic face. With the exception of a Pro substitution for Val-833, all Pro substitution and charge-inverting mutants showed wild-type virus-like one-cycle viral infectivity, replication kinetics, and Env incorporation into the virus. The intracellular localization and cell surface expression of mutants not localized in lipid rafts, such as the TM844, TM813, 829P, and 843P mutants, were apparently normal compared to those of wild-type Env. Cytoplasmic subdomain targeting analyses revealed that the sequence spanning LLP-3 and LLP-1 could target a cytoplasmic reporter protein to DRMs. Mutations of LLP-1 that affected Env association with lipid rafts also disrupted the DRM-targeting ability of the LLP-3/LLP-1 sequence. Our results clearly demonstrate that LLP motifs located in the C-terminal cytoplasmic tail of gp41 harbor Triton X-100-resistant raft association determinants.Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), are unusual in possessing a long cytoplasmic domain (∼150 amino acids) in their envelope (Env) transmembrane (TM) glycoprotein compared to those of other retroviruses (20 to 50 amino acids). The cytoplasmic domain of HIV-1 TM protein gp41, which encompasses residues 706 to 856, has multiple functions during the virus life cycle, including viral replication, infectivity, transmission, and cytopathogenicity. Truncations of the HIV-1 cytoplasmic domains may modulate cell-cell fusion properties of the Env protein, presumably due to alterations in the levels of cell surface Env expression and conformation of the Env ectodomain (23, 81). The cytoplasmic domain is characterized by the presence of three structurally conserved, amphipathic α-helical segments, located at residues 828 to 856, 770 to 795, and 786 to 813 and referred to as lentivirus lytic peptide 1 (LLP-1), LLP-2, and LLP-3, respectively, at its C terminus (Fig. ​(Fig.1A).1A). The LLP-1 and LLP-2 sequences were shown to be inserted into viral membranes by a photoinduced chemical reaction (73). These LLP motifs have been implicated in a variety of functions, such as cell surface expression (12), Env fusogenicity (30), and Env incorporation into a virus (47, 56), as well as Env protein stability (33) and multimerization (34).Open in a separate windowFIG. 1.(A) Schematic representation of the gp41 cytoplasmic domain and truncation mutants examined in this study. The cytoplasmic tail of gp41 contains a tyrosine-based endocytic YSPL signal located at residue 712, a hydrophilic region, a diaromatic YW motif, and three amphipathic α-helices, termed LLP-2, LLP-3, and LLP-1, at its C terminus. The amino acid sequence from residues 806 to 856 of the WT HXB2 Env is presented in single amino acid code, and the C-terminal dileucine motif is underlined in the sequence. Truncation mutants (TMs) generating stop codons immediately downstream of the indicated amino acids and their respective sequences are also shown. (B) pHXB2R3-based mutant proviruses used in this study. All mutants were generated by a PCR overlap cloning strategy, and the mutation sites are indicated. A dash or dot indicates that the residue in that position of the mutant provirus sequence is identical to or absent from that of the WT provirus sequence, respectively. The substituted amino acids in the mutant proviruses are also indicated.Gag and Env carry specific intracellular localization signals governing the site(s) of virus assembly/budding and release into the extracellular milieu. Env trafficking to the plasma membrane is regulated by the conserved C-terminal dileucine motif and the endocytic, membrane-proximal, tyrosine-based GY₇₁₂SPL signal in the cytoplasmic tail of gp41 (Fig. ​(Fig.1A)1A) and by their respective interactions with the clathrin adaptor proteins, AP1 and AP2 (4, 9, 21, 49, 65, 77). A diaromatic motif, Y₈₀₂W₈₀₃, was shown to bind to TIP47, a protein required for the retrograde transport of mannose-6-phosphate receptors from late endosomes to the trans-Golgi network, and this interaction was involved in the retrograde transport of Env to the trans-Golgi network (8). Alterations of these intracellular localization signals may affect viral infectivity, Env assembly into the virus, and viral replication (8, 20). Likewise, Gag also contains important sequences required for its trafficking to and assembly at the plasma membrane. The matrix (MA) protein, p17, contains a myristoyl group and a cluster of basic amino acids, while p6 contains a late domain which interacts with the components of the endosomal sorting complex required for transport (ESCRT) pathway to mediate Gag trafficking to the virion assembly/budding site (for reviews, see references 25, 45, 57, and 59). It is well documented that the specific interaction between the cytoplasmic domain of gp41 and the trimeric MA protein in infected cells facilitates recruitment of the Env into virus assembly/budding sites on target membranes (for reviews, see references 18, 24, and 46). TIP47 was demonstrated to act as an adaptor to bridge the gp41 cytoplasmic domain and Gag, which allows the physical encounter between Gag and Env, resulting in efficient Env incorporation into the virus during the viral assembly/budding process (39).Lipid rafts, also called detergent-resistant membranes (DRMs), are highly specialized membrane microdomains present in both the plasma and endosomal membranes of eukaryotic cells. These dynamic microdomains are characterized by their detergent insolubility, light density on a sucrose gradient, and enrichment of cholesterol, glycosphingolipids, and glycosylphosphatidylinositol (GPI)-linked proteins that are anchored in the membrane by their attached GPI moieties (1). HIV-1 utilizes lipid rafts to efficiently enter host cells (40, 74, 80) and selectively assembles and buds from lipid rafts on the surfaces of infected cells (27, 36, 48, 50, 54). Also, the HIV-1 Env protein was detected in lipid raft membranes (48, 54, 64). Lipid rafts are thought to facilitate Env-Gag interactions, to concentrate viral Env glycoproteins, and to promote multimerization of intracellular viral components (for a review, see reference 51). However, what governs Env transport to and localization in lipid rafts is a long-standing question.Although the mechanisms by which proteins associate with lipid rafts are not fully understood, determinants for targeting of signal proteins to DRMs have been identified. These include a GPI anchor (2, 61) and an N-terminal Met-Gly-Cys in which Gly is myristylated and Cys is palmitoylated (43, 71). The latter includes certain dually acylated heterotrimeric guanine nucleotide-binding protein (G protein) α subunits (44). In addition, acylation by palmitoylation also serves as a signal to target signaling molecules to lipid rafts (for reviews, see references 11 and 60). Some Env proteins of membrane-enveloped viruses are known to be associated with lipid rafts (35, 41, 54, 69, 79), and acylation of viral Env proteins, in particular, palmitoylation, is important for targeting these Env proteins to lipid rafts (for reviews, see references 58 and 70).It is generally believed that the association of HIV-1 Env with lipid rafts requires a palmitoylation signal(s) located in the cytoplasmic tail of gp41 (6, 64). Nevertheless, the two cytoplasmic palmitoylated Cys residues in the HXB2 strain Env protein are not conserved among HIV-1 isolates, and some isolates do not even contain cysteine residues in their cytoplasmic tail (32). In accordance with this notion, we previously demonstrated that the two cytoplasmic palmitoylated Cys residues in T-cell (T)- and macrophage (M)-tropic Env proteins do not play an obvious role in the virus life cycle, including Env''s association with lipid rafts (13), suggesting that other factors may substitute for cytoplasmic palmitoylation to promote Env localization in lipid rafts. Clapham''s group showed that mutations in MA or the cytoplasmic tail that prevent Env from incorporating into the virus and impair virus infectivity also interfere with Env''s association with lipid rafts (7), indicating that the Gag-Env interaction drives efficient Env association with lipid rafts, which in turn modulates Env budding and assembly onto viral particles. In contrast to their findings, we previously also noted that the Env protein of the HXB2 strain expressed without Gag is still located in lipid rafts (13), providing compelling evidence for the proposal that the Env per se contains sufficient information for its sequestration into lipid rafts.To further understand the nature of Env''s association with lipid rafts, in the present study we show that sequestering Env in Triton X-100-resistant lipid rafts is an intrinsic property of Env and is independent of Gag-Env interactions. Additionally, the LLP motifs, in particular the α-helical structure of the hydrophobic face of LLP-1, play a crucial role in Env''s localization in lipid rafts. Except for the 833P mutant of Env, which is unstable and degrades (33), all Pro-substituted mutants not located in lipid rafts exhibited wild-type (WT)-like phenotypes of intracellular localization, cell surface expression, incorporation into virions, and viral replication capacity. Importantly, the α-helix of the hydrophobic face of LLP-1 is also critical for the raft-targeting ability of the LLP-3/LLP-1 sequence. Our study depicts, for the first time, the critical role of the α-helix of the gp41 cytoplasmic domain in mediating Env''s association with and targeting to Triton X-100-resistant lipid rafts.     

Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.     

A Scientometric Evaluation of the Chagas Disease Implementation Research Programme of the PAHO and TDR

The Special Programme for Research and Training in Tropical Diseases (TDR) is an independent global programme of scientific collaboration cosponsored by the United Nations Children''s Fund, the United Nations Development Program, the World Bank, and the World Health Organization. TDR''s strategy is based on stewardship for research on infectious diseases of poverty, empowerment of endemic countries, research on neglected priority needs, and the promotion of scientific collaboration influencing global efforts to combat major tropical diseases. In 2001, in view of the achievements obtained in the reduction of transmission of Chagas disease through the Southern Cone Initiative and the improvement in Chagas disease control activities in some countries of the Andean and the Central American Initiatives, TDR transferred the Chagas Disease Implementation Research Programme (CIRP) to the Communicable Diseases Unit of the Pan American Health Organization (CD/PAHO).This paper presents a scientometric evaluation of the 73 projects from 18 Latin American and European countries that were granted by CIRP/PAHO/TDR between 1997 and 2007. We analyzed all final reports of the funded projects and scientific publications, technical reports, and human resource training activities derived from them. Results about the number of projects funded, countries and institutions involved, gender analysis, number of published papers in indexed scientific journals, main topics funded, patents inscribed, and triatomine species studied are presented and discussed.The results indicate that CIRP/PAHO/TDR initiative has contributed significantly, over the 1997–2007 period, to Chagas disease knowledge as well as to the individual and institutional-building capacity.     

Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase

Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.     

Formation of Syncytia Is Repressed by Tetraspanins in Human Immunodeficiency Virus Type 1-Producing Cells

In vitro propagation studies have established that human immunodeficiency virus type 1 (HIV-1) is most efficiently transmitted at the virological synapse that forms between producer and target cells. Despite the presence of the viral envelope glycoprotein (Env) and CD4 and chemokine receptors at the respective surfaces, producer and target cells usually do not fuse with each other but disengage after the viral particles have been delivered, consistent with the idea that syncytia, at least in vitro, are not required for HIV-1 spread. Here, we tested whether tetraspanins, which are well known regulators of cellular membrane fusion processes that are enriched at HIV-1 exit sites, regulate syncytium formation. We found that overexpression of tetraspanins in producer cells leads to reduced syncytium formation, while downregulation has the opposite effect. Further, we document that repression of Env-induced cell-cell fusion by tetraspanins depends on the presence of viral Gag, and we demonstrate that fusion repression requires the recruitment of Env by Gag to tetraspanin-enriched microdomains (TEMs). However, sensitivity to fusion repression by tetraspanins varied for different viral strains, despite comparable recruitment of their Envs to TEMs. Overall, these data establish tetraspanins as negative regulators of HIV-1-induced cell-cell fusion, and they start delineating the requirements for this regulation.The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is incorporated into released virus particles and enables the virus to attach to and fuse with target cells in order to initiate the infectious cycle. Before Env mediates the fusion of viral and cellular membranes, i.e., while it is still incorporated in the plasma membrane of the infected cell, it drives the adhesion between virus producer cell and target cells, which gives rise to the formation of the so-called virological synapse (VS) (21, 24, 35, 36). The VS shares certain characteristics with the immunological synapse, including an accumulation of specific cellular membrane proteins and lipids (see, e.g., reference 5), and it provides efficient and secure transfer of virus particles from infected to uninfected cells (8). Importantly, the two adhering cells, like the pre- and postsynaptic cells that form an immunological synapse, typically do not fuse during such cell-to-cell transfer events. At first glance this seems surprising, as HIV-1 Env, unlike many other viral envelope proteins, can induce membrane fusion at physiological pH. Also, adhesion of producer and target cell, which can be initiated when the uropod of the infected cell contacts the uninfected cell (8), followed by reorganization of the cytoskeleton (25) and formation of full-fledged synapses, can extend over minutes (see, e.g., reference 20). This process should allow enough time to trigger cell-cell fusion. However, it is now well established that newly synthesized Env is efficiently internalized upon its arrival at the host cell plasma membrane, unless it is recruited into budding structures by viral Gag (see, e.g., reference 11; also discussed in references 3 and 6). Further, and likely also contributing to the prevention of producer-target cell fusion, immature Gag at the host cell plasma membrane represses Env-driven fusion, and this repression is lost only once Gag is processed in released virions (9, 22, 23, 31, 50). Finally, because syncytia are clearly not required for the transmission of virus from cell to cell in vitro and are possibly detrimental to virus spread in vivo, we hypothesize that HIV-1 cooperates with cellular membrane proteins to prevent cell-cell fusion.Members of a group of cellular proteins known as tetraspanins play an important role as regulators of cellular fusion processes, including myotube formation and fertilization (28, 30, 44; reviewed in, e.g., reference 17). As membrane organizers, these proteins homo- and heteromultimerize and associate with other cellular proteins to form variably sized but discrete microdomains, the so-called tetraspanin-enriched microdomains (TEMs) (29) (also called TERMs [1] or TEAs [12]). Knowledge of the molecular mechanisms through which tetraspanins regulate the fusion of cellular membranes is still lacking, though the available evidence strongly suggests (i) that these proteins are not themselves fusogens but rather that they coordinate the fusion activity of other cellular proteins and (ii) that they can act both as positive and negative regulators of cellular fusion processes. For instance, several in vivo studies unequivocally showed that CD9 expression in oocytes is essential for sperm-egg fusion (27, 28, 30), but CD9 and CD81 ablation in monocytes enhances the formation of multinucleated phagocytes that are involved in immune defense against certain microbes (45). Interestingly, the same two tetraspanins are also known to regulate virus-induced fusion processes. CD9 is involved in regulating cell-cell fusion driven by canine distemper virus, as the anti-CD9 antibody K41 inhibits syncytium formation by this virus (42), and CD81 is a necessary cofactor for infection of cells by hepatitis C virus (see, e.g., references 2 and 52). Finally, tetraspanins on uninfected (target) cells inhibit HIV-1-induced cell-cell fusion (14). This fusion regulation is likely due to interactions of CD9 and CD81 with CD4 and coreceptors at the surface of target cells, though the tetraspanin CD63 has also been implicated in the trafficking of CXCR4 to the plasma membrane (51).Because tetraspanins in HIV-1-producing cells are enriched at budding sites (4, 10, 13, 15, 33, 46, 49) and at the VS (26), we hypothesized that they regulate Env-driven fusion at the VS. Here, we document that tetraspanins in HIV-1-producing cells can indeed restrict syncytium formation. We also define some of the requirements for this fusion inhibition, thus laying the necessary groundwork for future mechanistic analyses. In addition, the characterization of cell-cell fusion regulation parameters in this study will allow the fusion-inhibitory activities to be distinguished from other regulatory functions exerted by tetraspanins, such as the modulation of virion infectivity and the regulation of cell-to-cell transmission of HIV-1.     

HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.     

A Novel Link between Fic (Filamentation Induced by cAMP)-mediated Adenylylation/AMPylation and the Unfolded Protein Response

The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP''s ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling.     

Conserved and Variable Features of Gag Structure and Arrangement in Immature Retrovirus Particles

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.Retrovirus assembly is driven by the oligomerization of Gag, a multidomain protein, including an N-terminal membrane binding domain (MA), a two-domain structural component (CA), and an RNA binding domain (NC). The Gag proteins of all orthoretroviruses, including the alpha-, beta-, and lentiretroviruses discussed here, share this conserved modular architecture (Fig. ​(Fig.1).1). Despite very weak sequence conservation, the tertiary structures of MA, CA, and NC are conserved among retroviruses. Outside these conserved domains the Gag proteins of different retroviruses exhibit substantial variability. Other domains may be present or absent, and the length and sequence of linker peptides may also vary (12) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Modular architecture of the full-length Gag proteins of HIV, M-PMV, and RSV. White rectangles illustrate Gag polyprotein cleavage products. The extent of the constructs used in the electron microscopic analysis is specified under each protein as a black rectangle. Gray triangles specify cleavage sites. Residue numbers are counted from the beginning of Gag.Oligomerization of Gag in an infected cell leads to the formation of roughly spherical immature virus particles, where Gag is arranged in a radial fashion with the N-terminal MA domain associated with a surrounding lipid bilayer, and the more C-terminal NC pointing toward the center of the particle (15, 44, 46). Subsequent multiple cleavages of Gag by the viral protease lead to a rearrangement of the virus. NC and the RNA condense in the center of the particle, CA assembles into a capsid or shell around the nucleoprotein, and MA remains associated with the viral membrane. This proteolytic maturation is required to generate an infectious virion (2). In contrast to the mature CA lattice, which has been extensively studied (11, 16, 36), the Gag lattice in immature particles is incompletely understood.Gag itself contains all of the necessary determinants for particle assembly. For example, the expression of Gag alone in an insect cell expression system is sufficient to generate viruslike particles (3, 17, 22, 38). Retroviral Gag proteins also can be assembled in vitro in the presence of nucleic acids to form spherical particles (9, 19, 39, 43, 47). The arrangement of Gag within these in vitro-assembled Gag particles is indistinguishable from that found in immature virus particles (6), and the in vitro assembly systems have proved valuable for unraveling the principles of virus assembly (18, 28, 29, 39). Multiple layers of interaction promote the assembly of Gag in vivo, including MA-membrane-MA interactions, CA-CA interactions, and NC-RNA-NC interactions. An extensive body of literature has explored which regions of Gag are required for assembly and which can be replaced or deleted without compromising assembly. MA-membrane-MA interactions contribute but are not essential. NC-RNA-NC interactions appear to function to nonspecifically link Gag molecules together and can be replaced both in vivo and in vitro by other interaction domains such as leucine zippers (4, 13, 20, 32, 48). The C-terminal domain of CA (referred to here as C-CA) and the stretch of amino acids immediately following this domain (termed the spacer peptide [SP] region) are critical for assembly and sensitive to mutation (1, 22, 27, 30).We set out to understand how the substantial sequence variation among Gag proteins in different retroviruses is manifested in structural differences in the immature Gag lattice. To do this, we studied three retroviruses from different genera: the lentivirus human immunodeficiency virus type 1 (HIV-1), the betaretrovirus Mason-Pfizer monkey virus (M-PMV), and the alpharetrovirus Rous sarcoma virus (RSV). These retroviruses are those for which in vitro assembly was first established and has been most extensively studied (6, 19, 24, 28, 29, 35, 43, 47).The domain structures of the three retroviruses differ most substantially upstream of CA. Both M-PMV and RSV have domains located between MA and CA that are absent in HIV (Fig. ​(Fig.1).1). In M-PMV there are 198 residues forming the pp24 and p12 domains; in RSV there are 84 residues forming the p2a, p2b, and p10 domains. The three retroviruses have different requirements for regions upstream of CA during assembly. The C-terminal 25 residues of p10 are essential for proper immature RSV assembly, both in vitro and in vivo, and these residues are inferred to interact directly with N-CA to stabilize the hexamer by forming contacts between adjacent N-CA domains (35). An equivalent assembly domain has not been described for other retroviruses. Within M-PMV p12 is the so-called internal scaffolding domain that is not essential for assembly in vitro (43) but is required for particle assembly when the precursor is expressed under the control of the M-PMV promoter (41). It is a key domain for the membrane-independent assembly of immature capsids (40).In HIV, five residues upstream of CA must be present for assembly of immature virus-like spherical particles in vitro, although larger upstream extensions, including part of MA, are required for efficient assembly of regular particles, both for HIV and RSV. For HIV, if the entire MA domain is included, in vitro assembly requires the presence of inositol penta- or hexakis phosphate (8). If no sequences upstream of CA are present, the in vitro particles in both of these viruses adopt a mature-type tubular morphology (10, 18). It has been hypothesized that cleavage at the N terminus of N-CA during maturation leads to the N-terminal residues of CA folding back into the N-CA structure to form a β-hairpin. The β-hairpin is important for assembly of the mature CA lattice, whereas its absence is important for immature assembly (23, 42). These requirements explain why, in HIV and RSV, immature Gag lattice-like structures are formed only if regions upstream of CA are present (18). In M-PMV, an immature Gag lattice can be produced when the regions upstream of CA are deleted if this is combined with mutations (such as deleting the initial proline of CA), which prevent β-hairpin formation (43).During maturation, HIV and RSV Gag proteins are cleaved twice between CA and NC to release a small peptide called SP1 or SP. In RSV the most N-terminal of these two cleavages can occur at one of two possible positions such that the released peptide is either 9 or 12 amino acids long (33). In M-PMV only one cleavage occurs between CA and NC, and no short peptide is produced. The region between the final helix of CA and the Zn fingers has been proposed to adopt a helical bundle architecture in HIV and RSV based on bioinformatic prediction, on mutational analysis, and on structural studies (1, 22, 27, 45). In all three viruses, C-CA and the residues immediately downstream are critical for assembly and are sensitive to mutation. C-CA contains the major homology region, a group of residues that are highly conserved across the retroviruses.Cryo-electron tomography (cET) studies of immature virus particles (6, 45) have resolved the electron density of the HIV Gag lattice in three dimensions at low resolution. Using these methods, we have also described the three-dimensional architecture of in vitro-assembled HIV Gag particles (6). In immature viruses and in vitro-assembled particles, Gag is seen to adopt an 8 nm hexameric lattice, as was predicted from previous Fourier analysis of two-dimensional images (7, 46). The hexameric lattice is interrupted by irregularly shaped holes and cracks in the lattice (6, 45). A similar observation has been made using AFM of in vitro-assembled particles of M-PMV Gag (26). These holes and cracks allow an otherwise planar hexameric lattice to form the surface of an approximately spherical particle.The radial positions of the MA, CA, and NC domains had been assigned previously from cryo-electron micrographs (44, 46). Based on these assignments and the shape of the density, the position and relative orientations of CA domains can be modeled into the low-resolution structure of the HIV lattice (6, 45). Density ascribed to the N-terminal domain of CA (N-CA) forms rings around large holes at the 6-fold symmetry positions in the lattice. Below this layer, at the expected radius of the C-CA, are 2-fold densities, interpreted as corresponding to dimers of C-CA. These densities are linked by rodlike densities, which descend into the NC-nucleic acid layer.HIV is the only retrovirus for which the arrangement of Gag in the immature particle has been described in three dimensions. Prior to this work, important open questions were therefore: which features of the arrangement of Gag are conserved between genera and therefore reflect general principles of Gag-Gag interactions, and which features are specific to certain genera? We have applied subtomogram averaging of cryo-electron tomograms to generate reconstructions of in vitro-assembled Gag particles from HIV, M-PMV, and RSV. These allow identification of the general and variable features of the arrangement of Gag and the architecture of immature retroviruses.     

CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca²⁺ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca²⁺-regulated proteins that—in mature sperm—are involved in flagellar bending.