Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P 2 . There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P 2 binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2′-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1′-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1′-acyl chains of PI(4,5)P 2 and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.

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SUMMARY The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Herein, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic a-helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-p interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface.

Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason-Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C-and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.capsid assembly ͉ dynein motor ͉ matrix protein structure ͉ retrovirus ͉ transport G ag polyproteins are major structural subunits of immature retroviral capsids and contain the determinants that mediate interactions with viral genomic RNA as well as particle assembly. The molecular mechanisms that control the accumulation of Gag molecules at the sites of assembly vary among retroviruses. Based on the assembly site, retroviruses have been shown to follow two major morphogenic pathways (1). While alpharetroviruses, gammaretroviruses, and lentiviruses (C-type retroviruses) assemble immature capsids at the inner side of the plasma membrane, the capsids of betaretroviruses (B/D-type) are formed in the cytoplasm. It has been shown that MasonPfizer monkey virus (M-PMV), which is the prototype of the D-type retroviruses, assembles at the pericentriolar region of an infected cell (2). Numerous studies have demonstrated that the matrix protein (MA), located at the N terminus of the Gag polyprotein, is responsible for targeting the polyprotein precursors to the site of assembly and for mediating transport of immature retroviral particles to the plasma membrane where budding occurs (3). A subtle difference in the regulation of the transport process has been suggested, as the results from several laboratories indicate that the destination of polyprotein precursors can be altered by mutations within MA. Amino acid substitutions in several domains of HIV-1 MA dramatically reduced the efficiency of particle production and redirected the majority of them to cytoplasmic vacuoles (4). Similarly, a substitution of basic for acidic residues in helix A of HIV-1 MA caused relocation of virus assembly to intracellular locations and produced normally budded noninfectious virions (5). Mutation of the N-terminal polybasic region of Moloney murine leukemia virus (Mo-MuLV) MA redirected virus assembly to the cytoplasm, suggesting a role of tryptophan residues in the intracellular transport (6).The N terminus of MA from most retroviruses, including M-PMV, is myristoylat...

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Upon host infection, Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) into the cytosol of infected macrophages, leading to host cell death by necroptosis. TNT hydrolyzes NAD + in the absence of any exogenous cofactor, thus classifying it as a β-NAD + glycohydrolase. However, TNT lacks sequence similarity with other NAD + hydrolyzing enzymes and lacks the essential motifs involved in NAD + binding and hydrolysis by these enzymes. In this study, we used NMR to examine the enzymatic activity of TNT and found that TNT hydrolyzes NADP + as fast as NAD + but does not cleave the corresponding reduced dinucleotides. This activity of TNT was not inhibited by ADP-ribose or nicotinamide, indicating low affinity of TNT for these reaction products. A selection assay for nontoxic TNT variants in Escherichia coli identified four of six residues in the predicted NAD + -binding pocket and four glycine residues that form a cradle directly below the NAD + -binding site, a conserved feature in the TNT protein family. Site-directed mutagenesis of residues near the predicted NAD + -binding site revealed that Phe 727 , Arg 757 , and Arg 780 are essential for NAD + hydrolysis by TNT. These results identify the NAD + -binding site of TNT. Our findings also show that TNT is an NAD + glycohydrolase with properties distinct from those of other bacterial glycohydrolases. Because many of these residues are conserved within the TNT family, our findings provide insights into understanding the function of the >300 TNT homologs.

Targeting of the Gag polyprotein to the plasma membrane (PM) for assembly is a critical event in the late phase of immunodeficiency virus type-1 (HIV-1) infection. Gag binding to the PM is mediated by interactions between the myristoylated matrix (MA) domain and PM lipids. Despite the extensive biochemical and in vitro studies of Gag and MA binding to membranes over the last two decades, the discovery of the role of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in Gag binding to the PM has sparked a string of studies aimed at elucidating the molecular mechanism of retroviral Gag–PM binding. Electrostatic interactions between a highly conserved basic region of MA and acidic phospholipids have long been thought to be the main driving force for Gag–membrane interactions. However, recent studies suggest that the mechanism is rather complex since other factors such as the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. Here we summarize the current understanding of HIV-1 Gag–membrane interactions at the molecular and structural levels and briefly discuss the underlying forces governing interactions of other retroviral MA proteins with the PM.

bCronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and Pub-MLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.