A guide to the crystallographic analysis of icosahedral viruses

Determining the structure of an icosahedral virus crystal by X-ray diffraction follows very much the same course as conventional protein crystallography. The major differences arise from the relatively large sizes of the particles, which significantly affect the data collection process, data processing and management, and later, the refinement of a model. Most of the other differences are due to the high 5 3 2 point group symmetry of icosahedral viruses. This alters dramatically the means by which initial phases are obtained by molecular substitution, extended to higher resolution by electron density averaging and density modification, and the refinement of the structure in the light of high non-crystallographic symmetry. In this review, we attempt to lead the investigator through the various steps involved in solving the structure of a virus crystal. These steps include the purification of viruses, their crystallization, the recording of X-ray diffraction data, and its reduction to structure amplitudes. It further addresses the problems attending phase determination and ultimately the refinement of a model. Finally, we describe the unique properties of virus crystals and the factors that influence their physical and diffraction properties

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses

Structural basis of Na+-independent and cooperative substrate/product antiport in CaiT

Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer, and is essential for life. Here we report the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3-Å and from Escherichia coli (EcCaiT) at 3.5-Å resolution. CaiT belongs to the family of betaine/carnitine/choline transporters (BCCT), which are mostly Na+ or H+ dependent, whereas EcCaiT is Na+ and H+ independent. The three-dimensional architecture of CaiT resembles that of the Na+-dependent transporters LeuT and BetP, but in CaiT a methionine sulphur takes the place of the Na+ ion to coordinate the substrate in the central transport site, accounting for Na+-independent transport. Both CaiT structures show the fully open, inward-facing conformation, and thus complete the set of functional states that describe the alternating access mechanism. EcCaiT contains two bound butyrobetaine substrate molecules, one in the central transport site, the other in an extracellular binding pocket. In the structure of PmCaiT, a tryptophan side chain occupies the transport site, and access to the extracellular site is blocked. Binding of both substrates to CaiT reconstituted into proteoliposomes is cooperative, with Hill coefficients up to 1.7, indicating that the extracellular site is regulatory. We propose a mechanism whereby the occupied regulatory site increases the binding affinity of the transport site and initiates substrate translocation.