The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virionassociated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.

The plus-strand RNA genome of flavivirus contains a 5 terminal cap 1 structure (m 7 GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA 3 m 7 GpppA 3 m 7 GpppAm. The 2-O methylation can be uncoupled from the N-7 methylation, since m 7 GpppA-RNA can be readily methylated to m 7 GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2-O cap methylations require distinct buffer conditions and different side chains within the K 61 -D 146 -K 182 -E 218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.Eukaryotic mRNAs possess a 5Ј cap structure that is cotranscriptionally formed in the nucleus. mRNA capping is essential for mRNA stability and efficient translation (13, 39). Most animal viruses that replicate in cytoplasm encode their own capping machinery to produce capped RNAs. RNA capping generally consists of three steps in which the 5Ј triphosphate end of nascent RNA transcript is first hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase, then capped with GMP by an RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by an RNA guanine-methyltransferase (N-7 MTase) (15). Additionally, the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2Ј-OH position by a nucleoside 2Ј-O MTase, to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (13). Both N-7 and 2Ј-O MTases use S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-L-homocysteine (SAH) as a by-product. The order of capping and methylation varies among cellular and viral RNAs (13).The genus Flavivirus comprises approximately 70 viruses, many of which are important human pathogens, including four serotypes of dengue virus (DENV), yellow fever virus (YFV), St. Louis encephalitis virus, and West Nile virus (WNV) (23).The flaviviru...

Most eukaryotic and viral mRNAs possess a 5Ј cap that is important for mRNA stability and efficient translation (11). The cap consists of an inverted guanosine, methylated at the N-7 position, linked to the first transcribed RNA nucleotide by a unique 5Ј-5Ј triphosphate bridge (m 7 GpppN; cap 0 structure) (32). The process of RNA capping generally consists of three steps, in which the 5Ј triphosphate end of the nascent RNA transcript is first hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase, then capped with GMP by an RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by an RNA guanine-methyltransferase (N-7 MTase) (13). Additionally, the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2Ј-OH position by a nucleoside 2Ј-O MTase to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (11). Both N-7 and 2Ј-O MTases use S-adenosyl-Lmethionine (AdoMet) as a methyl donor and generate Sadenosyl-L-homocysteine (AdoHcy) as a by-product. The order of the capping and methylation steps is variable among cellular and viral RNAs (11).Many members of the Flavivirus genus are arthropod-borne human pathogens, including West Nile virus (WNV), Yellow fever virus, four serotypes of Dengue virus (DENV), Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, and Tick-borne encephalitis virus (4). The flavivirus genome is a single-stranded, plus-sense RNA of about 11,000 nucleotides that contains a type 1 cap at its 5Ј end (5, 35) and terminates with 5Ј-CU OH -3Ј (35) (see Fig. 1A). 5Ј and 3Ј untranslated regions flank a single open reading frame which encodes a polyprotein that is co-and posttranslationally processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane [prM] or membrane [M], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (4). Since flaviviruses replicate in the cytoplasm, they are expected to encode their own capping enzymes, rather than to use the host's capping apparatus located in the nucleus. Alternatively, since all host proteins have to be synthesized in the cytoplasm, it is possible that cellular capping components could be retained in the cytoplasm for viral RNA capping through specific interactions with a viral protein. Of the four enzymes required for flavivirus m 7 GpppAm-cap formation, only the RNA triphosphatase and 2Ј-O MTase have been mapped to NS3 (19,36) and NS5 (8), respectively, whereas the guanylyltransferase and N-7 MTase remain to be identified. The crystal structure of a ternary complex comprising the DENV-2 MTase domain, AdoHcy, and a GTP analogue suggested that, during 2Ј-O methylation, a specific cap-binding site holds the guanine cap to register the ribose 2Ј-OH of the first transcribed adenosine in close proximity to the AdoMet CH 3 donor (2, 8). Structure and sequence alignments of DENV, vaccinia virus VP39, and other 2Ј-O MTases indicate that a conserved K-D-K-E ...

Viral and host factors influence the rate of HIV-1 disease progression. For HIV-1 to fuse, a CD4+ cell must express a co-receptor that the virus can use. The chemokine receptors CCR5 and CXCR4 are used by R5 and X4 viruses, respectively. Most new infections involve transmission of R5 viruses, but variants can arise later that also use CXCR4 (R5-X4 or X4 viruses). This is associated with an increased rate of CD4+ T-cell loss and poor prognosis. The ability of host cells to support HIV-1 entry also influences progression. The absence of CCR5 in approximately 1% of the Caucasian population, due to homozygosity for a 32-nucleotide deletion in the coding region (delta32-CCR5 allele), very strongly protects against HIV-1 transmission. Heterozygosity for the delta32-CCR5 allele delays progression typically by 2 years. A recent study showed that a conservative substitution (V64I) in the coding region of CCR2 also has a significant impact on disease progression, but not on HIV-1 transmission. This was unexpected, since CCR2 is rarely used as a co-receptor in vitro and the V64I change is in a transmembrane region. Because a subsequent study did not confirm this effect on progression to disease, we analyzed CCR2-V64I using subjects in the Chicago MACS. We show that CCR2-V64I is indeed protective against disease progression and go on to show that the CCR2-V64I allele is in complete linkage disequilibrium with a point mutation in the CCR5 regulatory region.

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na + or H + gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na + -coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs + (a Na + congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na + -binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate-and cationbinding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na + -induced drug export. Based on our structural and functional analyses, we suggest that Na + triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.cation coordination | substrate recognition | membrane protein | multidrug resistance | monobody

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by coupling the efflux of drugs to the influx of Na+ or H+. Known structures of Na+-coupled, extracellular-facing MATE transporters from the NorM subfamily revealed twelve membrane-spanning segments related by a quasi-twofold rotational symmetry and a multidrug-binding cavity situated near the membrane surface. Here we report the crystal structure of an H+-coupled MATE transporter from Bacillus halodurans and the DinF subfamily at 3.2 Å-resolution, unveiling a surprisingly asymmetric arrangement of twelve transmembrane helices. We also identified a membrane-embedded substrate-binding chamber by combining crystallographic and biochemical analyses. Our studies further suggested a direct competition between H+ and substrate during DinF-mediated transport, and how a MATE transporter alternates between its extracellular- and intracellular-facing conformations to propel multidrug extrusion. Collectively, our results demonstrated hitherto unrecognized mechanistic diversity among MATE transporters.

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Fragments of foreign antigens associated with class I molecules of the major histocompatibility complex (MHC) are presented at the cell surface to elicit an immune response. This presentation requires the coordinated expression of several genes contained in the MHC, including those encoding the MHC class I heavy chain, the proteins LMP-2 and LMP-7, which are involved in the proteasomal degradation of cytosolic antigens into peptide fragments that are destined for association with MHC class I molecules, and TAP-1 and TAP-2, which transport these fragments across the membrane of the endoplasmic reticulum at the start of their journey to the cell surface. In many virus-transformed cell lines and spontaneous tumours, these genes are simultaneously repressed. However, the key factor(s) that are essential for their expression and repression have not been identified. Here we report that the proto-oncogene product PML induces expression of LMP-2, LMP-7, TAP-1 and TAP-2 in an MHC-class I-negative, recurrent tumour, leading to the re-expression of cell-surface MHC in tumours and to rejection of the tumours. PML also regulates MHC expression in untransformed fibroblasts. We conclude that malfunction of PML may enable a tumour to evade the immune defence of its host.