Role of the ATP-Binding Domain of the Human Papillomavirus Type 11 E1 Helicase in E2-Dependent Binding to the Origin

Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37 degrees C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.     

A C-Terminal Helicase Domain of the Human Papillomavirus E1 Protein Binds E2 and the DNA Polymerase α-Primase p68 Subunit

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase α-primase (polα-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polα-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polα-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.     

Functional Interaction between the Bovine Papillomavirus Virus Type 1 Replicative Helicase E1 and Cyclin E-Cdk2

We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in Xenopus egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from Xenopus eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication.     

Functional Mapping of the DNA Binding Domain of Bovine Papillomavirus E1 Protein

Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1(179-191)) and HR3 (E1(241-252)), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 alpha-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).     

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A_C). Caf1A_C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A_C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A_C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.     

A Conserved Domain in the Coronavirus Membrane Protein Tail Is Important for Virus Assembly

Coronavirus membrane (M) proteins play key roles in virus assembly, through M-M, M-spike (S), and M-nucleocapsid (N) protein interactions. The M carboxy-terminal endodomain contains a conserved domain (CD) following the third transmembrane (TM) domain. The importance of the CD (SWWSFNPETNNL) in mouse hepatitis virus was investigated with a panel of mutant proteins, using genetic analysis and transient-expression assays. A charge reversal for negatively charged E₁₂₁ was not tolerated. Lysine (K) and arginine (R) substitutions were replaced in recovered viruses by neutrally charged glutamine (Q) and leucine (L), respectively, after only one passage. E₁₂₁Q and E₁₂₁L M proteins were capable of forming virus-like particles (VLPs) when coexpressed with E, whereas E₁₂₁R and E₁₂₁K proteins were not. Alanine substitutions for the first four or the last four residues resulted in viruses with significantly crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. All recovered viruses with alanine substitutions in place of SWWS residues had second-site, partially compensating, changes in the first TM of M. Alanine substitution for proline had little impact on the virus. N protein coexpression with some M mutants increased VLP production. The results overall suggest that the CD is important for formation of the viral envelope by helping mediate fundamental M-M interactions and that the presence of the N protein may help stabilize M complexes during virus assembly.Coronaviruses are widespread, medically important respiratory and enteric pathogens of humans and a wide range of animals. New human coronaviruses (HCoV), including severe acute respiratory syndrome CoV (SARS-CoV), HCoV-NL63, and HCoV-HKU1, were recently identified (40, 47). The potential for emergence of other new viruses and the zoonotic nature of some coronaviruses strongly warrants understanding old and new viruses. Understanding vital interactions that take place during virus assembly and conserved domains (CDs) that mediate these interactions can provide insight toward identification of targets for development of antiviral therapeutics and vaccines.Coronaviruses are enveloped positive-stranded RNA viruses that belong to the Coronaviridae family in the Nidovirales order. The virion envelope contains at least three structural proteins, the membrane (M), spike (S), and envelope (E) proteins. The genomic RNA is encapsidated by the N phosphoprotein to form a helical nucleocapsid. The S glycoprotein is the viral attachment protein that facilitates infection through fusion of viral and cellular membranes and is the major target of neutralizing antibodies (13). The M glycoprotein is the most abundant component of the viral envelope and plays required, key roles in virus assembly (9, 20, 31, 33, 41). The E protein is a minor component of the viral envelope that plays an important, not clearly defined, role(s) during virus assembly and release (2, 5, 41).Coronavirus M proteins are divergent in their amino acid content, but all share the same overall basic structural characteristics. The proteins have three TM domains, flanked by a short amino-terminal glycosylated domain and a long carboxy-terminal tail located outside and inside the virion, respectively (14) (Fig. ​(Fig.11 A). M localizes in the Golgi region when expressed alone (20, 22). M molecules interact with each other and also with the spike and nucleocapsid during virus assembly (8-10, 23, 31, 33). M-M interactions constitute the overall scaffold for the viral envelope. The S protein and a small number of E molecules are interspersed in the M protein lattice in mature virions. Previous studies from a number of labs implicated multiple M domains and residues as being important for coronavirus assembly (6, 8, 9, 17, 43). Coronaviruses assemble and bud at intracellular membranes in the region of the endoplasmic reticulum (ER) Golgi intermediate compartment (ERGIC) (22, 39). Coexpression of only the M and the E proteins is sufficient for virus-like particle (VLP) assembly for most coronaviruses (2, 41).Open in a separate windowFIG. 1.M protein conserved domain and mutants. (A) A linear schematic of the M protein illustrating the relative positions of the three TM domains (black boxes) and the position of the CD in the tail. (B) Alignment of CDs from representative coronaviruses. Full-length amino acid sequences from transmissible gastroenteritis virus (TGEV), feline coronavirus (FeCoV), human coronavirus 229E, human coronavirus NL63, mouse hepatitis virus (MHV), bovine coronavirus (BCoV), human coronavirus OC43, porcine hemagglutinating encephalomyelitis virus (HEV), human coronavirus HKU1, SARS-CoV, infectious bronchitis virus (IBV), and turkey coronavirus (TCoV) were aligned by using CLUSTAL W (25). (C) Mutations introduced into the MHV CD, with + and − symbols used to indicate VLP production and virus recovery for each mutant.The long intravirion (cytoplasmic) tail of M consists of an amphipathic domain following the third TM and a short hydrophilic region at the carboxyl end of the tail (Fig. ​(Fig.1A).1A). The amphipathic domain appears to be closely associated with the membrane (34). At the amino terminus of the amphipathic domain, there is a highly conserved 12-amino-acid domain (SWWSFNPETNNL), consisting of residues 114 to 125 in the mouse hepatitis virus (MHV) A59 M protein (Fig. ​(Fig.1B)1B) (19). These residues are almost identically conserved across the entire Coronaviridae family. Because of the crucial role that M plays in virus assembly and the high conservation of this domain, we hypothesized that it is functionally important for virus assembly. To test this, a series of changes were introduced in the CD. The functional impact of the changes was studied in the context of the virus by genetic analysis and the ability of the mutant M proteins to participate in VLP assembly. The results show that the CD is functionally important for M protein to participate in virus assembly. The domain may help mediate important lateral interactions between M molecules. The results suggest that the N protein helps stabilize M complexes during virus assembly.     

The Papillomavirus E1 Protein Forms a DNA-Dependent Hexameric Complex with ATPase and DNA Helicase Activities

The E1 protein from bovine papillomavirus has site-specific DNA binding activity, DNA helicase activity, and DNA-dependent ATPase activity consistent with the properties of an initiator protein. Here we have identified and characterized a novel oligomeric form of E1 that is associated with the ATPase and DNA helicase activities and whose formation is strongly stimulated by single-stranded DNA. This oligomeric form corresponds to a hexamer of E1.     

A型流感病毒NS1蛋白羧基端PL结构域影响NS1在细胞核内的定位

A型流感病毒NS1蛋白羧基端4个氨基酸可以与PDZ结构域(the domain of PSD95,Dig and ZO-1)相结合,称为PL结构域(PDZ ligand domain)．对不同亚型或毒株的流感病毒而言,其NS1蛋白PL结构域的组成存在比较大的差异．有研究发现这种差异能够影响NS1与宿主细胞蛋白的相互作用进而影响病毒的致病力．为进一步探讨PL结构域对NS1蛋白生物学特性的影响,首先构建出4种不同亚型流感病毒(H1N1、H3N2、H5N1、H9N2)来源的NS1绿色荧光蛋白表达质粒．在此基础上,对野生型H3N2病毒NS1表达质粒进行人工改造,将其PL结构域缺失或者替换为其他亚型流感病毒的PL结构域,制备出4种重组NS1蛋白表达质粒．通过比较上述不同NS1蛋白在HeLa细胞中的定位情况发现,只有野生型H3N2病毒的NS1蛋白可以定位于核仁当中,而野生型H1N1、H5N1、H9N2病毒的NS1蛋白以及PL结构域缺失或替代的H3N2病毒NS1蛋白都不能定位于核仁．而通过比较上述NS1蛋白在流感病毒易感的MDCK细胞中的定位,进一步发现所有这些蛋白均不定位于核仁．上述结果表明：PL结构域的不同可以明显影响NS1蛋白在HeLa细胞核内的定位和分布,这有可能造成其生物学功能的差异．同时,NS1蛋白在细胞核内的定位还与宿主细胞的来源有着密切关系．     

Crystal Structure of the Conserved Amino Terminus of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus Gripped by an Antibody

We report the crystal structure of the M2 ectodomain (M2e) in complex with a monoclonal antibody that binds the amino terminus of M2. M2e extends into the antibody binding site to form an N-terminal β-turn near the bottom of the paratope. This M2e folding differs significantly from that of M2e in complex with an antibody that binds another part of M2e. This suggests that M2e can adopt at least two conformations that can elicit protective antibodies.     

Papillomavirus Assembly Requires Trimerization of the Major Capsid Protein by Disulfides between Two Highly Conserved Cysteines

We have used viruslike particles (VLPs) of human papillomaviruses to study the structure and assembly of the viral capsid. We demonstrate that mutation of either of two highly conserved cysteines of the major capsid protein L1 to serine completely prevents the assembly of VLPs but not of capsomers, whereas mutation of all other cysteines leaves VLP assembly unaffected. These two cysteines form intercapsomeric disulfides yielding an L1 trimer. Trimerization comprises about half of the L1 molecules in VLPs but all L1 molecules in complete virions. We suggest that trimerization of L1 is indispensable for the stabilization of intercapsomeric contacts in papillomavirus capsids.     

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

Mutations in the apically located Na⁺-K⁺-2Cl⁻ co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet the mechanisms underlying the regulation of mutated NKCC2 proteins in renal cells have not been investigated. Here, we identified a trihydrophobic motif in the distal COOH terminus of NKCC2 that was required for endoplasmic reticulum (ER) exit and surface expression of the co-transporter. Indeed, microscopic confocal imaging showed that a naturally occurring mutation depriving NKCC2 of its distal COOH-terminal region results in the absence of cell surface expression. Biotinylation assays revealed that lack of cell surface expression was associated with abolition of mature complex-glycosylated NKCC2. Pulse-chase analysis demonstrated that the absence of mature protein was not caused by reduced synthesis or increased rates of degradation of mutant co-transporters. Co-immunolocalization experiments revealed that these mutants co-localized with the ER marker protein-disulfide isomerase, demonstrating that they are retained in the ER. Cell treatment with proteasome or lysosome inhibitors failed to restore the loss of complex-glycosylated NKCC2, further eliminating the possibility that mutant co-transporters were processed by the Golgi apparatus. Serial truncation of the NKCC2 COOH terminus, followed by site-directed mutagenesis, identified hydrophobic residues ¹⁰⁸¹LLV¹⁰⁸³ as an ER exit signal necessary for maturation of NKCC2. Mutation of ¹⁰⁸¹LLV¹⁰⁸³ to AAA within the context of the full-length protein prevented NKCC2 ER exit independently of the expression system. This trihydrophobic motif is highly conserved in the COOH-terminal tails of all members of the cation-chloride co-transporter family, and thus may function as a common motif mediating their transport from the ER to the cell surface. Taken together, these data are consistent with a model whereby naturally occurring premature terminations that interfere with the LLV motif compromise co-transporter surface delivery through defective trafficking.The Na-K-2Cl co-transporter, NKCC2, provides the major route for sodium/chloride transport across the apical plasma membrane of the thick ascending limb (TAL)³ of the kidney (1). This co-transporter is critical for salt reabsorption, acid-base regulation, and divalent mineral cation metabolism (2). The prominent importance of NKCC2 in renal functions is evidenced by the effect of loop diuretics, which as pharmacologic inhibitors of NKCC2, are extensively used in the treatment of edematous states (2). Even more impressive, inactivating mutations of the NKCC2 gene in humans causes Bartter syndrome type 1 (BS1), a life-threatening renal tubular disorder for which the diagnosis is usually made in the antenatal-neonatal period, due to the presence of polyhydramnios, premature delivery, salt loss, hypokalemia, metabolic alkalosis, hypercalciuria, and nephrocalcinosis (3). Without appropriate treatment, patients with BS1 will not survive the early neonatal period (4). In congruence with the severity of the symptoms and the uniformity of the clinical picture, functional analysis of diverse NKCC2 mutants consistently revealed a loss of function effect of the tested mutations (5, 6). However, regulatory characterizations of mutants NKCC2 were limited to Xenopus laevis oocytes. Indeed, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty of expressing the co-transporter protein in mammalian cells (7, 8). As a consequence, our knowledge of the molecular mechanisms underlying membrane trafficking of mutated NKCC2 proteins in mammalian cells is nil. Increasing our understanding of the molecular determinants underlying NKCC2 expression in renal cells is essential for elucidating the pathophysiology of BS1 and for improving the available treatments (9, 10). Undeniably, only analysis of the expression such NKCC2 of mutants in renal cells would definitively establish their cellular fate.NKCC2 belongs to the superfamily of electroneutral cation-coupled chloride (CCC) co-transporters (SLC12A) (1). The cation-chloride co-transporters (CCCs) family comprises two principal branches of homologous membrane proteins. One branch includes the Na⁺-dependent chloride co-transporters composed of the Na⁺-K⁺-2Cl⁻ co-transporters (NKCC1 and NKCC2) and the Na⁺-Cl⁻ co-transporter (NCC). The second branch includes the Na⁺-independent K⁺-Cl⁻ co-transporters composed of at least four different isoforms: KCC1 KCC2, KCC3, and KCC4 (11). Within the families, the CCCs share 25–75% amino acid identity. All of these co-transporters exhibit similar hydropathy profiles with 12 transmembrane-spanning domains, an amino terminus of variable length, and a long cytoplasmic carboxyl terminus. Because the COOH-terminal domain of NKCC2 is the predominant cytoplasmic region, it is likely to be a major factor in the trafficking of the NKCC2 protein. Moreover, there have been several reports demonstrating that COOH-terminal residues are important for correct protein targeting (12–14). Occasionally, COOH-terminal mutations are known to cause genetic disorders (15–17). Although studies of other ion transporters support the importance of the COOH-terminal signals in protein stability, maturation, surface delivery, and ER export (18–22), little is known about the role of COOH-terminal signals in the biogenesis of NKCC2.We were recently able to express NKCC2 protein in mammalian cells (23), providing therefore a powerful tool to study and understand the molecular mechanisms underlying the co-transporter expression and regulation in renal cells. This allowed us, in this study, to take the advantage of the existence of natural mutants altering the COOH-terminal tail of the co-transporter to investigate the role of the COOH terminus in the biogenesis of NKCC2 and to explore possible mechanisms implicated in BS1. The results demonstrate the importance of the COOH terminus in normal maturation of the NKCC2 protein. Indeed, we identified a motif of three hydrophobic residues, ¹⁰⁸¹LLV¹⁰⁸³, highly conserved in the COOH-terminal tails of all members of the CCC family, that controls the rate of ER export and thus of surface expression of NKCC2. Loss of the motif disrupts glycosylation and plasma membrane localization of NKCC2. Therefore, we propose abnormal trafficking as a common BS1 mechanism associated with mutations depriving NKCC2 of its COOH terminus.