Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused widespread outbreaks of debilitating human disease in the past five years. CHIKV invasion of susceptible cells is mediated by two viral glycoproteins, E1 and E2, which carry the main antigenic determinants and form an icosahedral shell at the virion surface. Glycoprotein E2, derived from furin cleavage of the p62 precursor into E3 and E2, is responsible for receptor binding, and E1 for membrane fusion. In the context of a concerted multidisciplinary effort to understand the biology of CHIKV, here we report the crystal structures of the precursor p62-E1 heterodimer and of the mature E3-E2-E1 glycoprotein complexes. The resulting atomic models allow the synthesis of a wealth of genetic, biochemical, immunological and electron microscopy data accumulated over the years on alphaviruses in general. This combination yields a detailed picture of the functional architecture of the 25 MDa alphavirus surface glycoprotein shell. Together with the accompanying report on the structure of the Sindbis virus E2-E1 heterodimer at acidic pH (ref. 3), this work also provides new insight into the acid-triggered conformational change on the virus particle and its inbuilt inhibition mechanism in the immature complex.

2Dengue disease is caused by four different flavivirus 1 serotypes, which infect 390 million people yearly with 25% symptomatic cases 2 and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (DENV--2) 3,4 . Structural studies so far have characterized only epitopes recognized by serotype specific human antibodies 5,6 . We recently isolated human antibodies potently neutralizing all four DENV serotypes 7 . Here we describe the X--ray structures of four of these broadly neutralizing antibodies (bnAbs) in complex with the envelope glycoprotein E from DENV--2, revealing that the recognition determinants are at a serotype conserved site at the E dimer interface, including the exposed main chain of the E fusion loop 8 and the two conserved glycan chains.This "E--dimer dependent epitope" (EDE) is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell 9 , explaining its conservation across serotypes and highlighting an Achilles heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.Exposed at the surface of infectious mature DENV particles, protein E is the sole target of neutralizing antibodies. It displays an icosahedral arrangement in which 90 E dimers completely coat the viral surface 10,11 and which is sensitive to the environmental pH. Upon entry of DENV into cells via receptor--mediated endocytosis, the acidic 3 endosomal environment triggers an irreversible fusogenic conformational change in E that leads to fusion of viral and endosomal membranes 1 . The structure of the isolated E dimer has been determined by X--ray crystallography using the soluble ectodomain (sE) 8,12 . Protein E is relatively conserved, displaying about 65% amino acid sequence identity when comparing the most distant DENV serotypes. In particular, there are two conserved N--linked glycosylation sites at positions N67 and N153. To examine its interaction with the antibodies, we selected four highly potent bnAbs identified in the accompanying work: 747(4) A11 and 747 B7 (EDE2 group, requiring glycosylation at position N153 for efficient binding) and 752--2 C8 and 753(3) C10 (EDE1 group, binding regardless of the glycosylation at N153) 7 -referred to as A11, B7, C8 and C10 from hereon. The EDE2 bnAbs were isolated from the same patient (who had a secondary infection with DENV--2), and are somatic variants of the same IgG clone, derived from the IGHV3--74 and IGLV2--23 germ lines. The heavy chain has a very long (26 amino acids, IMGT convention) complementarity--determining region 3 (CDR H3). The EDE1 bnAbs were isolated from different patients and derive from (EDE1 C8, the patient appeared to have a primary infection of undetermined serotype) and IGHV1--3* and IGLV2--14 (EDE1 C10, from a patient with secondary DENV--1 infecti...

In bacteria, regulatory phosphorylation of proteins at serine and/or threonine residues by Ser/Thr protein kinase (STPK) is an emerging theme in prokaryotic signaling, particularly since the prediction of the occurrence of several STPKs from genome sequencing and sequence surveys. Here we show that protein PknH possesses an autokinase activity and belongs to the large STPK family found in Mycobacterium tuberculosis. Evidence is presented that PknH can also phosphorylate EmbR, a protein suspected to modulate the level of arabinosyltransferase activity involved in arabinan biosynthesis of arabinogalactan, a key molecule of the mycobacterial cell wall. Interestingly, EmbR possesses an FHA (forkhead-associated) domain, a newly described phosphoprotein recognition domain, which plays an essential role in PknH-EmbR interaction and phosphorylation of EmbR by PknH. It is demonstrated that mutation of each of three particular residues of this FHA domain, Arg312, Ser326, and Asn348, totally abolishes the PknH-mediated phosphorylation of EmbR, thus highlighting the critical role of this domain in the direct interaction between EmbR and PknH.