RNA polymerase II nascent transcripts are capped during pausing before elongation. Here we report that hSPT5, the human homolog of yeast elongation factor SPT5, interacts directly with the capping enzyme. hSPT5 stimulated capping enzyme guanylylation and mRNA capping by severalfold. Although RNA 5-triphosphatase activity was unaffected, binding to this domain in the full-length enzyme is likely involved in the stimulation, as hSPT5 did not increase the activity of the guanylyltransferase fragment. Consistent with capping enzyme binding, TFIIH-phosphorylated CTD stimulated guanylylation, and this increase was not additive with hSPT5.

During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.

Mammalian capping enzymes are bifunctional proteins with both RNA 5-triphosphatase and guanylyltransferase activities. The N-terminal 237-aa triphosphatase domain contains (I͞V)HCXXGXXR(S͞T)G, a sequence corresponding to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Analysis of point mutants of mouse RNA 5-triphosphatase identified the motif Cys and Arg residues and an upstream Asp as required for activity. Like PTPs, this enzyme was inhibited by iodoacetate and VO 4 3؊ and independent of Mg 2؉ , providing additional evidence for phosphate removal from RNA 5 ends by a PTP-like mechanism. The full-length, 597-aa mouse capping enzyme and the C-terminal guanylyltransferase fragment (residues 211-597), unlike the triphosphatase domain, bound poly (U) and were nuclear in transfected cells. GpppN, which is present on most eukaryotic mRNAs (1), plays crucial roles in enhancing mRNA processing (2-5), stability (6-9), nuclear transport (10, 11), and translation (1,(12)(13)(14). Cap formation is catalyzed by the cotranscriptional, sequential action of three enzymes. The ␥-phosphate on the nascent RNA terminus is first removed by RNA 5Ј-triphosphatase (RTP) to generate a 5Ј diphosphorylated end; GMP derived from GTP is transferred by RNA guanylyltransferase to the 5Ј end to form a 5Ј-5Ј triphosphate structure, GpppN-; and the guanine N 7 position subsequently is methylated by RNA (guanine-7-) methyltransferase (1,4,15).Although this pathway was identified more than 20 years ago (16, 17), cellular cap-forming enzymes have been cloned and sequenced only more recently (18)(19)(20)(21)(22)(23)(24)(25)(26). Capping in yeast species is mediated by separately encoded RTP and guanylyltransferase proteins (18), while higher eukaryotes contain bifunctional capping enzymes with N-terminal RTP and Cterminal guanylyltransferase (22,27,28). Despite this difference in genomic organization, capping function apparently has been conserved between unicellular and metazoan organisms. Yeast strains lacking RTP or guanylyltransferase, which are both essential, were complemented for growth by the mammalian counterparts (22,23,26).In mammalian capping enzyme, the C-terminal guanylyltransferase domain contains several motifs characteristic of the nucleotidyl transferase superfamily (19,22,28,29). These include the highly conserved KXDG motif, which is required for formation of the capping enzyme intermediate by phosphoamide linkage of GMP to the -amino group of the motif Lys (4). Ala substitution of this Lys eliminated guanylylation (22). The mammalian capping enzyme N-terminal 5Ј-triphosphatase domain also contains a conserved motif, (I͞ V)HCXXGXXR(S͞T)G, which is characteristic of protein tyrosine phosphatases (PTPs) that form a motif cysteinylphosphate (Cys-P) intermediate as an essential part of their catalytic mechanism (30-32). This similarity was first described in Caenorhabditis elegans RTP where replacement of the motif Cys eliminated 5Ј-triphosphatase activity (27). This suggested a similar mechanism fo...

LP2086 is a family of outer membrane lipoproteins fromNeisseria meningitidis, which elicits bactericidal antibodies and are currently undergoing human clinical trials in a bivalent formulation where each antigen represents one of the two known LP2086 subfamilies. Here we report the NMR structure of the recombinant LP2086 variant B01, a representative of the LP2086 subfamily B. The structure reveals a novel fold composed of two domains: a "taco-shaped" N-terminal ␤-sheet and a C-terminal ␤-barrel connected by a linker. The structure in micellar solution is consistent with a model of LP2086 anchored to the outer membrane bilayer through its lipidated N terminus. A long flexible chain connects the folded part of the protein to the lipid anchor and acts as spacer, making both domains accessible to the host immune system. Antibodies broadly reactive against members from both subfamilies have been mapped to the N terminus. A surface of subfamily-defining residues was identified on one face of the protein, offering an explanation for the induction of subfamily-specific bactericidal antibodies.Neisseria meningitidis is a Gram-negative bacterial pathogen, which colonizes the upper respiratory tract, occasionally invading the bloodstream, causing sepsis, and crossing the blood-brain barrier, resulting in meningitis. Despite the availability of effective antibiotic treatment, the rapid progression of meningococcal disease still results in substantial morbidity and mortality (1). Five meningococcal serogroups, categorized according to the chemical structure of the bacterial capsular polysaccharides, A, B, C, Y, and W135, account for most of the disease (2). Although a vaccine against four of the five major serogroups of meningococci is currently available, a vaccine for the prevention of serogroup B disease is still an unmet clinical need (3). The development of vaccines against serogroup B meningococci has focused on subcapsular antigens, in order to avoid the risk of autoimmunity arising from structural similarities between the capsular polysaccharides and the sialic acidmodified surface of developing human brain (1,4,5).Recently, a new family of lipidated outer membrane proteins, LP2086, was identified as a potential vaccine target (6). Members of the LP2086 family have been divided into two subfamilies, subfamily A and B, based on their genetic variation (6, 7). Since recombinant LP2086 (rLP2086) 3 elicits a bactericidal response that is largely subfamily-specific, a bivalent vaccine containing one protein from each subfamily will offer protection against serogroup B meningococci (6, 8 -11). LP2086 lipoproteins are lipidated at the N-terminal Cys with a tripalmitoyl lipid tail, which anchors the protein to the bacterial membrane (12). More recently, LP2086 was found to induce serum resistance via binding with human Factor H, a key regulator of the alternative complement pathway that prevents autologous complement attack (13).Our work seeks to understand the structural elements of LP2086 responsible for inducing the subf...

No abstract