Generation of infectious arenavirus-like particles requires the virus RING finger Z protein and surface glycoprotein precursor (GPC) and the correct processing of GPC into GP1, GP2, and a stable signal peptide (SSP). Z is the driving force of arenavirus budding, whereas the GP complex (GPc), consisting of hetero-oligomers of SSP, GP1, and GP2, forms the viral envelope spikes that mediate receptor recognition and cell entry. Based on the roles played by Z and GP in the arenavirus life cycle, we hypothesized that Z and the GPc should interact in a manner required for virion formation. Here, using confocal microscopy and coimmunoprecipitation assays, we provide evidence for subcellular colocalization and biochemical interaction, respectively, of Z and the GPc. Our results from mutation-function analysis reveal that Z myristoylation, but not the Z late (L) or RING domain, is required for Z-GPc interaction. Moreover, Z interacted directly with SSP in the absence of other components of the GPc. We obtained similar results with Z and GPC from the prototypical arenavirus lymphocytic choriomeningitis virus and the hemorrhagic fever arenavirus Lassa fever virus.

The eukaryotic translation initiation factor eIF4E, a potent oncogene, is highly regulated. One class of eIF4E regulators, including eIF4G and the 4E-binding proteins (4E-BPs), interact with eIF4E using a conserved YXXXXLΦ-binding site. The structural basis of this interaction and its regulation are well established. Really Interesting New Gene (RING) domain containing proteins, such as the promyelocytic leukemia protein PML and the arenaviral protein Z, represent a second class of eIF4E regulators that inhibit eIF4E function by decreasing eIF4E's affinity for its m 7 G cap ligand. To elucidate the structural basis of this inhibition, we determined the structure of Z and studied the Z-eIF4E complex using NMR methods. We show that Z interacts with eIF4E via a novel binding site, which has no homology with that of eIF4G or the 4E-BPs, and is different from the RING recognition site used in the ubiquitin system. Z and eIF4G interact with distinct parts of eIF4E and differentially alter the conformation of the m 7 G cap-binding site. Our results provide a molecular basis for how PML and Z RINGs reduce the affinity of eIF4E for the m 7 G cap and thereby act as key inhibitors of eIF4E function. Furthermore, our findings provide unique insights into RING protein interactions.Lassa Fever Virus-Z | NMR | promyelocytic leukemia protein

In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO 4 ) repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Data base mining revealed 12 candidate NST genes in the L. major genome, including LPG2 as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L) and several pseudogenes. Gene knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although the single lpg5A ؊ and lpg5B ؊ mutants produced PGs, an lpg5A ؊ /5B ؊ double mutant was completely deficient. PG synthesis was restored in the lpg5A ؊ /5B ؊ mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, since the lpg5A ؊ mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the lpg5B ؊ mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally.

Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(␤1,4)Man(␣1-PO 4 )]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2 ؊ L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2 ؊ , instead resembling the L. major lipophosphoglycan-deficient lpg1 ؊ mutant. Metacyclic lpg5A ؊ /lpg5B ؊ promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A ؊ /lpg5B ؊ mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2 ؊ Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.

The glycoprotein of lymphocytic choriomeningitis virus (LCMV) contains nine potential N-linked glycosylation sites. We investigated the function of these N-glycosylations by using alanine-scanning mutagenesis. All the available sites were occupied on GP1 and two of three on GP2. N-linked glycan mutations at positions 87 and 97 on GP1 resulted in reduction of expression and absence of cleavage and were necessary for downstream functions, as confirmed by the loss of GP-mediated fusion activity with T87A, S97A mutants. In contrast, T234A and E379N/A381T mutants impaired GP-mediated cell fusion without altered expression or processing. Infectivity via virus-like particles required glycans and a cleaved glycoprotein. Glycosylation at the first site within GP2, not normally utilized by LCMV, exhibited increased VLP-infectivity. We also confirmed the role of the N-linked glycan at position 173 in the masking of the neutralizing epitope GP-1D. Taken together, our results indicated a strong relationship between fusion and infectivity.

/5B-mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2 -in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2 -promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage.

We examined herpes simplex virus (HSV)-infected human HEp-2 cells or porcine cells that express herpes virus entry mediator (HVEM) for virus and receptor protein interactions. Antibody to HVEM, or its viral ligand gD, coimmunoprecipitated several similar proteins. A prominent 110-kDa protein that coprecipitated was identified as gH. The HVEM/gD/gH complex was detected with mild or stringent cell lysis conditions. It did not form in cells infected with HSV-1(KOS)Rid1 virus or with null virus lacking gD, gH, or gL. Thus, in cells a complex forms through physical associations of HVEM, gD, and at least gH.Herpes simplex virus (HSV) enters into cells through attachments that lead to fusion of the virus envelope with the plasma membrane (12). This involves multiple virus envelope and host cell proteins (reviewed in reference 29). Herpes virus entry mediator (HVEM or HveA), isolated because it mediated HSV-1(KOS) entry into Chinese hamster ovary (CHO) cells (22), is a member of the tumor necrosis factor receptor family that binds protein ligands lymphotoxin-␣ and LIGHT (13,28). Biochemical analyses in vitro indicate that HVEM binds to soluble forms of glycoprotein D (gD) of HSV (4,6,7,37). Several structural studies map the amino acid contacts of purified gD when it is bound to HVEM (4,6,7,37,38). However, what occurs in cells has not been determined.How HSV glycoproteins bind cellular receptors to lead to pH-independent infection of susceptible cells is not yet clear. Binding of a viral ligand such as gD to a cellular attachment receptor, such as HVEM, is required for stable attachment and to alter protein conformation for events of entry (5,25,28).The HSV envelope contains at least 10 integral membrane glycoproteins (29). Their organization in the virus envelope and interactions among the glycoproteins and with cell receptors during infection are actively investigated (14,20,23,25,28,31,34,36). Reports in the literature differ regarding the physical interactions detected among HSV glycoproteins (14,15,26). Handler et al. found homo-and hetero-oligomeric complexes of viral envelope proteins from cross-linking of purified virions or virus exposed to cells (14, 15). However, others found no evidence of complex formation among the essential glycoproteins gB, gD, and gH/gL in virions (26).To further explore HSV receptors and viral proteins during infection, we took advantage of porcine cells that were previously well characterized as poorly susceptible to HSV due to the lack of a stable attachment receptor (25, 32). They provide a highly tractable system to explore interactions of HSV with individual or combinations of entry receptors. The porcine cell line SK6-A7 was stably transformed to constitutively express human HVEM (22). HVEM RNA was detected by reverse transcription-PCR (Fig. 1A). HVEM protein was detected in the cell surface by fluorescence-activated cell sorting (FACS) using polyclonal antibodies R-140 and R-95 made against a truncated HVEM, 200tHVEM (Fig. 1E to H). As shown with HB1-9 as one representative cel...

The precise role of Leishmania glycoconjugate molecules including phosphoglycans (PGs) and lipophosphoglycan (LPG) on host cellular responses is still poorly defined. Here, we investigated the interaction of Leishmania major LPG2 null mutant (lpg2 ؊ ), which lacks both PGs and LPG, with dendritic cells (DCs) and the subsequent early immune response in infected mice. Surprisingly, the absence of phosphoglycans did not influence expression pattern of major histocompatibility complex class II (MHC II), CD40, CD80, and CD86 on DCs in vitro and in vivo. However, lpg2 ؊ L. major induced significantly higher production of interleukin12p40 (IL-12p40) by infected bone marrow-derived DCs (BMDCs) than wild-type (WT) parasites in vitro. Furthermore, the production of IL-12p40 by draining lymph node cells from lpg2 ؊ mutant-infected mice was higher than those from WT L. major-infected mice. In model antigen presentation experiments, DCs from lpg2