Vaccinia virus A56/K2 fusion regulatory protein interacts with the A16 and G9 subunits of the entry fusion complex

Deletion of the A56R or K2L gene of vaccinia virus (VACV) results in the spontaneous fusion of infected cells to form large multinucleated syncytia. A56 and K2 polypeptides bind to one another (A56/K2) and together are required for interaction with the VACV entry fusion complex (EFC); this association has been proposed to prevent the fusion of infected cells. At least eight viral polypeptides comprise the EFC, but no information has been available regarding their interactions either with each other or with A56/K2. Utilizing a panel of recombinant VACVs designed to repress expression of individual EFC subunits, we demonstrated that A56/K2 interacted with two polypeptides: A16 and G9. Both A16 and G9 were required for the efficient binding of each to A56/K2, suggesting that the two polypeptides interact with each other within the EFC. Such an interaction was established by the copurification of A16 and G9 from infected cells under conditions in which a stable EFC complex failed to assemble and from detergent-treated lysates of uninfected cells that coexpressed A16 and G9. A recombinant VACV that expressed G9 modified with an N-terminal epitope tag induced the formation of syncytia, suggesting partial interference with the functional interaction of A56/K2 with the EFC during infection. These data suggest that A16 and G9 are physically associated within the EFC and that their interaction with A56/K2 suppresses spontaneous syncytium formation and possibly "fuse-back" superinfection of cells.     

Identification of a GP64 subdomain involved in receptor binding by budded virions of the baculovirus Autographica californica multicapsid nucleopolyhedrovirus

Enveloped virus entry into host cells is typically initiated by an interaction between a viral envelope glycoprotein and a host cell receptor. For budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, the envelope glycoprotein GP64 is involved in host cell receptor binding, and GP64 is sufficient to mediate low-pH-triggered membrane fusion. To better define the role of GP64 in receptor binding, we generated and characterized a panel of antisera against subdomains of GP64. Eight subdomain-specific antisera were generated, and their reactivities with GP64 proteins and neutralization of virus infectivity and binding were examined. Antibodies directed against the N-terminal region of GP64 (amino acids 21 to 159) showed strong neutralization of infectivity and effectively inhibited binding of (35)S-labeled budded virions to Sf9 cells. In addition, we generated virions displaying truncated GP64 constructs. A construct displaying the N-terminal 274 amino acids (residues 21 to 294) of the ectodomain was sufficient to mediate virion binding. Additional studies of antisera directed against small subdomains revealed that an antiserum against a 40-amino-acid region (residues 121 to 160) neutralized virus infectivity. Site-directed mutagenesis was subsequently used for functional analysis of that region. Recombinant viruses expressing GP64 proteins with single amino acid substitutions within amino acids 120 to 124 and 142 to 148 replicated to high titers, suggesting that those amino acids were not critical for receptor binding or other important GP64 functions. In contrast, GP64 proteins with single amino acid substitutions of residues 153 and 156 were unable to substitute for wild-type GP64 and did not rescue a gp64 knockout virus. Further analysis showed that these substitutions substantially reduced binding of recombinant virus to Sf9 cells. Thus, the amino acid region from positions 21 to 159 was identified as a putative receptor binding domain, and amino acids 153 and 156 appear to be important for receptor binding.     

Characterization of a Newly Identified 35-Amino-Acid Component of the Vaccinia Virus Entry/Fusion Complex Conserved in All Chordopoxviruses

The original annotation of the vaccinia virus (VACV) genome was limited to open reading frames (ORFs) of at least 65 amino acids. Here, we characterized a 35-amino-acid ORF (O3L) located between ORFs O2L and I1L. ORFs similar in length to O3L were found at the same genetic locus in all vertebrate poxviruses. Although amino acid identities were low, the presence of a characteristic N-terminal hydrophobic domain strongly suggested that the other poxvirus genes were orthologs. Further studies demonstrated that the O3 protein was expressed at late times after infection and incorporated into the membrane of the mature virion. An O3L deletion mutant was barely viable, producing tiny plaques and a 3-log reduction in infectious progeny. A mutant VACV with a regulated O3L gene had a similar phenotype in the absence of inducer. There was no apparent defect in virus morphogenesis, though O3-deficient virus had low infectivity. The impairment was shown to be at the stage of virus entry, as cores were not detected in the cytoplasm after virus adsorption. Furthermore, O3-deficient virus did not induce fusion of infected cells when triggered by low pH. These characteristics are hallmarks of a group of proteins that form the entry/fusion complex (EFC). Affinity purification experiments demonstrated an association of O3 with EFC proteins. In addition, the assembly or stability of the EFC was impaired when expression of O3 was repressed. Thus, O3 is the newest recognized component of the EFC and the smallest VACV protein shown to have a function.Vaccinia virus (VACV), the best-studied member of the poxvirus family of cytoplasmic DNA viruses, encodes ∼200 genes, some of which are still uncharacterized (27). The focus of the present study is VACV O3L, a short 35-amino-acid open reading frame (ORF) that was recognized by homology to a 41-amino-acid ORF in molluscum contagiosum virus (37) but not previously investigated. Here, we show that O3L is conserved in all chordopoxviruses, expressed late in infection, and involved in cell entry.Considerable information regarding VACV entry has been obtained during the past several years (28). There are two related infectious forms of VACV: the mature virion (MV) and the enveloped virions (EV). The MV is comprised of a lipoprotein membrane enclosing a nucleoprotein core, whereas the EV has an additional outer membrane that must be disrupted before fusion can occur (24). The MV can enter cells either by fusion at the plasma membrane (7) or by a low-pH-mediated endosomal route involving macropinocytosis (20, 26, 44). Regardless of which route is used, the ability of VACV to enter cells depends on a large number of proteins in the MV membrane that form or are associated with the entry/fusion complex (EFC) (39). Using genetic and biochemical methods, 11 entry/fusion proteins have been identified: A16 (33), A21 (43), A28 (40), F9 (4), G3 (21), G9 (32), H2 (38), I2 (31), J5 (39), L1 (3), and L5 (42). Eight of these proteins (A16, A21, A28, G3, G9, H2, J5, and L5) comprise the EFC, which depends on multiple interactions for assembly or stability. Although the structure of the EFC remains to be elucidated, there is evidence for direct interactions between A28 and H2 (30) and between A16 and G9 (50). An additional role for A16 and G9 involves an interaction with the A56/K2 heterodimer, which is present on the surface of infected cells, to prevent spontaneous cell-cell fusion and superinfection by progeny virus (45, 46, 48-50). Binding of L1 to an unidentified cell receptor has been suggested (16). Roles in membrane fusion have also been considered for A17 and A27 (23).Here we provide physical and functional evidence that O3 (VACWR069.5) is an integral component of the EFC and participates in virus entry and membrane fusion. With just 35 amino acids, O3 is the smallest VACV protein with a defined function.     

The myristate moiety and amino terminus of vaccinia virus l1 constitute a bipartite functional region needed for entry

Vaccinia virus (VACV) L1 is a myristoylated envelope protein which is required for cell entry and the fusion of infected cells. L1 associates with members of the entry-fusion complex (EFC), but its specific role in entry has not been delineated. We recently demonstrated (Foo CH, et al., Virology 385:368-382, 2009) that soluble L1 binds to cells and blocks entry, suggesting that L1 serves as the receptor-binding protein for entry. Our goal is to identify the structural domains of L1 which are essential for its functions in VACV entry. We hypothesized that the myristate and the conserved residues at the N terminus of L1 are critical for entry. To test our hypothesis, we generated mutants in the N terminus of L1 and used a complementation assay to evaluate their ability to rescue infectivity. We also assessed the myristoylation efficiency of the mutants and their ability to interact with the EFC. We found that the N terminus of L1 constitutes a region that is critical for the infectivity of VACV and for myristoylation. At the same time, the nonmyristoylated mutants were incorporated into mature virions, suggesting that the myristate is not required for the association of L1 with the viral membrane. Although some of the mutants exhibited altered structural conformations, two mutants with impaired infectivity were similar in conformation to wild-type L1. Importantly, these two mutants, with changes at A4 and A5, undergo myristoylation. Overall, our results imply dual differential roles for myristate and the amino acids at the N terminus of L1. We propose a myristoyl switch model to describe how L1 functions.     

The N- and C-terminal domains of the NS1 protein of influenza B virus can independently inhibit IRF-3 and beta interferon promoter activation

The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only approximately 20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of alpha/beta interferon (IFN-alpha/beta) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-alpha/beta synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-3 nuclear translocation and IFN-beta promoter activation. A mutational analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity correlated with IFN-beta promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation, as determined by its nuclear translocation, and of IFN-alpha/beta synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-3- and IFN-induced antiviral host responses to virus infection.     

Interaction of rotaviruses with Hsc70 during cell entry is mediated by VP5

Rotavirus infection seems to be a multistep process in which the viruses are required to interact with several cell surface molecules to enter the cell. The virus spike protein VP4, which is cleaved by trypsin into two subunits, VP5 and VP8, is involved in some of these interactions. We have previously shown that the neuraminidase-sensitive rotavirus strain RRV initially attaches to a sialic acid-containing cell molecule through the VP8 subunit of VP4 and subsequently interacts with integrin alpha2beta1 through VP5. After these initial contacts, the virus interacts with at least two additional proteins located at the cell surface, the integrin alphavbeta3 and the heat shock cognate protein Hsc70. In this work, we have shown that rotavirus RRV and its neuraminidase-resistant variant nar3 interact with Hsc70 through a VP5 domain located between amino acids 642 and 658 of the protein. This conclusion is based on the observation that a recombinant protein comprising the 300 carboxy-terminal amino acids of VP5 binds specifically to Hsc70 and a synthetic peptide containing amino acids 642 to 658 competes with the binding of the RRV and nar3 viruses to the heat shock protein. The VP5 peptide also competed with the binding to Hsc70 of the recombinant VP5 protein, and an antibody to Hsc70 reduced the binding of the recombinant protein to the surface of MA104 cells. The fact that the synthetic peptide blocks the infectivity of rotaviruses RRV and nar3 but not their binding to cells indicates that the interaction of VP5 with Hsc70 most probably occurs at a postattachment step during the virus entry process.     

TnGV增强蛋白在AcMNPV中的表达和活性分析

为了研究杆状病毒增强蛋白的活性基团 ,本文构建了分别表达三种N端部分缺失的粉纹夜蛾颗粒体病毒增强蛋白的重组杆状病毒 ,这三种蛋白在N端分别缺失了 15 0 ,186和 2 5 0个氨基酸。用重组病毒感染Tn 5B1 4细胞 ,成功地表达了这三种蛋白 ,并得到了纯化的蛋白质。通过体外降解围食膜的方法检测这些部分缺失的增强蛋白的活性 ,结果证实这三种蛋白均失去了增强蛋白的降解围食膜粘蛋白的活性。这一结果表明 ,增强蛋白的N端对其降解围食膜粘蛋白的功能是必需的     

The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly

The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.     

GRAB proteins,novel members of the NAC domain family,isolated by their interaction with a geminivirus protein

Geminiviruses encode a few proteins and depend on cellular factors to complete their replicative cycle. As a way to understand geminivirus-host interactions, we have searched for cellular proteins which interact with viral proteins. By using the yeast two-hybrid technology and the wheat dwarf geminivirus (WDV) RepA protein as a bait, we have isolated a family of proteins which we termed GRAB (for Geminivirus Rep A-binding). We report here the molecular characterization of two members, GRAB1 and GRAB2. We have found that the 37 C-terminal amino acids of RepA are required for interaction with GRAB proteins. This region contains residues conserved in an equivalent region of the RepA proteins encoded by other viruses of the WDV subgroup. The N-terminal domain of GRAB proteins is necessary and sufficient to interact with WDV RepA. GRAB proteins contain an unique acidic C-terminal domain while their N-terminal domain, of ca. 170 amino acids, are highly conserved in all of them. Interestingly, this conserved N-terminal domain of GRAB proteins exhibits a significant amino acid homology to the NAC domain present in proteins involved in plant development and senescence. GRAB1 and GRAB2 mRNAs are present in cultured cells and roots but are barely detectable in leaves. GRAB expression inhibits WDV DNA replication in cultured wheat cells. Our studies highlight the importance that the pathway(s) mediated by GRAB proteins, as well as by other NAC domain-containing proteins, might have on geminivirus DNA replication in connection to plant growth, development and senescence pathways.     

Virion Incorporation of Human Immunodeficiency Virus Type 1 Nef Is Mediated by a Bipartite Membrane-Targeting Signal: Analysis of Its Role in Enhancement of Viral Infectivity

The nef gene of primate immunodeficiency viruses is essential for high-titer virus replication and AIDS pathogenesis in vivo. In tissue culture, Nef is not required for human immunodeficiency virus (HIV) infection but enhances viral infectivity. We and others have shown that Nef is incorporated into HIV-1 particles and cleaved by the viral proteinase. To determine the signal for Nef incorporation and to analyze whether virion-associated Nef is responsible for enhancement of infectivity, we generated a panel of nef mutants and analyzed them for virion incorporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV particles. Incorporation was reconstituted by targeting the respective proteins to the plasma membrane by using a heterologous signal. Mutational analysis revealed that both myristoylation and an N-terminal cluster of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domain of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These results indicate that Nef incorporation into HIV-1 particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef correlated with enhanced infectivity of the respective viruses in a single-round replication assay. However, the phenotypes of HIV mutants with reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or different mechanisms may be involved in this system.     

Retention of viral infectivity after extensive mutation of the highly conserved immunodominant domain of the feline immunodeficiency virus envelope.

In lentiviruses, including human immunodeficiency virus and feline immunodeficiency virus (FIV), the principal immunodominant domain (PID) of the transmembrane glycoprotein elicits a strong humoral response in infected hosts. The PID is marked by the presence of two cysteines that delimit a sequence, composed of five to seven amino acids in different lentiviruses, which is highly conserved among isolates of the same lentiviral species. While the conservation of the sequence suggests the presence of functional constraints, the conservation of the immunodominance among divergent lentiviruses raises the hypothesis of a selective advantage for the infecting virus conferred by the host humoral response against this domain. We and others have previously shown that an appropriate structure of the PID is required for the production of a functional envelope. In the present work, we analyzed virological functions and immune reactivity of the envelope after random mutagenesis of the PID of FIV. We obtained nine mutant envelopes which were correctly processed and retained fusogenic ability. Mutation of the two C-terminal residues of the PID sequence between the cysteines in a molecular clone of FIV abolished infectivity. In contrast, three molecular clones containing extensive mutations in the four N-terminal amino acids were infectious. However, the mutations affected PID reactivity with sera from infected cats. Our results suggest that functional constraints, although existent, are not sufficient to account for PID sequence conservation. Such conservation may also result from positive selection by anti-PID antibodies which enhance infection.     

Mutations in the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein impair the incorporation of Env proteins into mature virions.

In-frame stop codons were introduced into the coding region of human immunodeficiency virus type 1 (HIV-1) transmembrane protein (gp41). Truncation of 147 amino acids from the carboxyl terminus of gp41 (TM709) significantly decreased the stability and cell surface expression of the viral Env proteins, while truncation of 104 amino acids (TM752) did not. Truncation of 43 or more amino acids from the carboxyl terminus of gp41 generated mutant viruses which were noninfectious in several human CD4+ T lymphoid cell lines and fresh peripheral blood mononuclear cells. Analysis of the noninfectious mutant virions revealed significantly reduced incorporation of the Env proteins compared with the wild-type virions. Comparable amounts of Env proteins were detected on the surfaces of wild-type- and TM752-transfected cells, suggesting that the structures of gp41 required for efficient incorporation of Env proteins were disrupted in mutant TM752. Truncation of the last 12 amino acids (TM844) from the carboxyl terminus of gp41 did not significantly affect the assembly and release of virions or the incorporation of Env proteins into mature virions. However, the TM844 virus had dramatically decreased infectivity compared with the wild-type virus. This suggests that the cytoplasmic domain of gp41 also plays a role in other steps of virus replication.     

Membrane association of greasy grouper nervous necrosis virus protein A and characterization of its mitochondrial localization targeting signal

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. The betanodavirus greasy grouper (Epinephelus tauvina) nervous necrosis virus (GGNNV) is a positive-RNA virus with one of the smallest genomes among RNA viruses replicating in fish cells. To understand the localization of GGNNV replication complexes, we generated polyclonal antisera against protein A, the GGNNV RNA-dependent RNA polymerase. Protein A was detected at 5 h postinfection in infected sea bass cells. Biochemical fractionation experiments revealed that GGNNV protein A sedimented with intracellular membranes upon treatment with an alkaline pH and a high salt concentration, indicating that GGNNV protein A is tightly associated with intracellular membranes in infected cells. Confocal immunofluorescence microscopy and bromo-UTP incorporation studies identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. In addition, protein A fused with green fluorescent protein (GFP) was detected in the mitochondria in transfected cells and was demonstrated to be tightly associated with intracellular membranes by biochemical fractionation analysis and membrane flotation assays, indicating that protein A alone was sufficient for mitochondrial localization in the absence of RNA replication, nonstructural protein B, or capsid proteins. Three sequence analysis programs showed two regions of hydrophobic amino acid residues, amino acids 153 to 173 and 229 to 249, to be transmembrane domains (TMD) that might contain a membrane association domain. Membrane fraction analysis showed that the major domain is N-terminal amino acids 215 to 255, containing the predicted TMD from amino acids 229 to 249. Using GFP as the reporter by systematically introducing deletions of these two regions in the constructs, we further confirmed that the N-terminal amino acids 215 to 255 of protein A function as a mitochondrial targeting signal.     

Evidence that the algI/algJ gene cassette, required for O acetylation of Pseudomonas aeruginosa alginate, evolved by lateral gene transfer

Pseudomonas aeruginosa strains, isolated from chronically infected patients with cystic fibrosis, produce the O-acetylated extracellular polysaccharide, alginate, giving these strains a mucoid phenotype. O acetylation of alginate plays an important role in the ability of mucoid P. aeruginosa to form biofilms and to resist complement-mediated phagocytosis. The O-acetylation process is complex, requiring a protein with seven transmembrane domains (AlgI), a type II membrane protein (AlgJ), and a periplasmic protein (AlgF). The cellular localization of these proteins suggests a model wherein alginate is modified at the polymer level after the transport of O-acetyl groups to the periplasm. Here, we demonstrate that this mechanism for polysaccharide esterification may be common among bacteria, since AlgI homologs linked to type II membrane proteins are found in a variety of gram-positive and gram-negative bacteria. In some cases, genes for these homologs have been incorporated into polysaccharide biosynthetic operons other than for alginate biosynthesis. The phylogenies of AlgI do not correlate with the phylogeny of the host bacteria, based on 16S rRNA analysis. The algI homologs and the gene for their adjacent type II membrane protein present a mosaic pattern of gene arrangement, suggesting that individual components of the multigene cassette, as well as the entire cassette, evolved by lateral gene transfer. AlgJ and the other type II membrane proteins, although more diverged than AlgI, contain conserved motifs, including a motif surrounding a highly conserved histidine residue, which is required for alginate O-acetylation activity by AlgJ. The AlgI homologs also contain an ordered series of motifs that included conserved amino acid residues in the cytoplasmic domain CD-4; the transmembrane domains TM-C, TM-D, and TM-E; and the periplasmic domain PD-3. Site-directed mutagenesis studies were used to identify amino acids important for alginate O-acetylation activity, including those likely required for (i) the interaction of AlgI with the O-acetyl precursor in the cytoplasm, (ii) the export of the O-acetyl group across the cytoplasmic membrane, and (iii) the transfer of the O-acetyl group to a periplasmic protein or to alginate. These results indicate that AlgI belongs to a family of membrane proteins required for modification of polysaccharides and that a mechanism requiring an AlgI homolog and a type II membrane protein has evolved by lateral gene transfer for the esterification of many bacterial extracellular polysaccharides.     

Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain.

Upon insertion of a signal-anchor protein into the endoplasmic reticulum membrane, either the C-terminal or the N-terminal domain is translocated across the membrane. Charged residues flanking the transmembrane domain are important determinants for this decision, but are not necessarily sufficient to generate a unique topology. Using a model protein that is inserted into the membrane to an equal extent in either orientation, we have tested the influence of the size and the folding state of the N-terminal domain on the insertion process. A small zinc finger domain or the full coding sequence of dihydrofolate reductase were fused to the N-terminus. These stably folding domains hindered or even prevented their translocation. Disruption of their structure by destabilizing mutations largely restored transport across the membrane. Translocation efficiency, however, did not depend on the size of the N-terminal domain within a range of 40-237 amino acids. The folding behavior of the N-terminal domain is thus an important factor in the topogenesis of signal-anchor proteins.     

A Short N-Proximal Region in the Large Envelope Protein Harbors a Determinant That Contributes to the Species Specificity of Human Hepatitis B Virus

Infection by hepatitis B virus (HBV) is mainly restricted to humans. This species specificity is likely determined at the early phase of the viral life cycle. Since the envelope proteins are the first viral factors to interact with the cell, they represent attractive candidates for controlling the HBV host range. To investigate this assumption, we took advantage of the recent discovery of a second virus belonging to the primate Orthohepadnavirus genus, the woolly monkey HBV (WMHBV). A recombinant plasmid was constructed for the expression of all WMHBV envelope proteins. In additional constructs, N-terminal sequences of the WMHBV large envelope protein were substituted for their homologous HBV counterparts. All wild-type and chimeric WMHBV surface proteins were properly synthesized by transfected human hepatoma cells, and they were competent to replace the original HBV proteins for the production of complete viral particles. The resulting pseudotyped virions were evaluated for their infectious capacity on human hepatocytes in primary culture. Virions pseudotyped with wild-type WMHBV envelope proteins showed a significant loss of infectivity. By contrast, infectivity was completely restored when the first 30 residues of the large protein originated from HBV. Analysis of smaller substitutions within this domain limited the most important region to a stretch of only nine amino acids. Reciprocally, replacement of this motif by WMHBV residues in the context of the HBV L protein significantly reduced infectivity of HBV. Hence this short region of the L protein contributes to the host range of HBV.     

Identification of the beta-dystroglycan binding epitope within the C-terminal region of alpha-dystroglycan.

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post-translationally cleaved to form two different subunits, alpha and beta. The extracellular alpha-dystroglycan is a membrane-associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane beta-dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. alpha- and beta-Dystroglycan interact tightly but noncovalently. We have previously shown that the N-terminal region of beta-dystroglycan, beta-DG(654-750), binds to the C-terminal region of murine alpha-dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid-phase binding assays, the C-terminal sequence of alpha-dystroglycan containing the binding epitope for beta-dystroglycan has been defined more precisely. We found that a region of 36 amino acids, from position 550-585, is required for binding the extracellular region, amino acids 654-750 of beta-dystroglycan. Recently, a dystroglycan-like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550-585 sequence of vertebrate alpha-dystroglycan. We have expressed this Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) beta-dystroglycan (Kd = 6 +/- 1 microM). These data confirm the proper identification of the beta-dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan-specific binding drugs, that could have important biomedical applications.