Origin and Rapid Evolution of a Novel Murine Erythroleukemia Virus of the Spleen Focus-Forming Virus Family

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent M_r of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an M_r of 60,000. During further in vivo passaging, a virus that encodes an M_r-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.The independently isolated Friend and Rauscher erythroleukemia viruses (18, 48) are complexes of a replication competent murine leukemia virus (MuLV) and a replication-defective spleen focus-forming virus (SFFV) (39, 42, 47). The SFFVs encode Env glycoproteins (gp55) that are inefficiently processed to form larger cell surface derivatives (gp55^p) (19). The gp55 binds to erythropoietin receptors (EpoR) to cause erythroblast proliferation and splenomegaly in susceptible mice. Evidence has suggested that the critical mitogenic interaction occurs exclusively on cell surfaces (7, 16).SFFVs are structurally closely related to mink cell focus-inducing viruses (MCFs) (2, 5, 10, 50), a class of replication-competent murine retroviruses that has a broad host range termed polytropic (15, 21). Although MCFs are not inherited as replication-competent intact proviruses, the mouse genome contains numerous dispersed polytropic env gene sequences (8, 17, 27). MCFs apparently readily form de novo by recombination when ecotropic host range MuLVs replicate in mice (14, 15, 26, 43). MCF env genes typically are hybrid recombinants that contain a 5′ polytropic-specific region and a 3′ ecotropic-specific portion (26). They encode a gPr90 Env glycoprotein that is cleaved by partial proteolysis to form the products gp70 surface (SU) glycoprotein plus p15E transmembrane (TM) protein (32, 39, 47). In addition, MCFs often differ from ecotropic MuLVs in their long terminal repeat (LTR) sequences (8, 20, 26, 28, 29, 45).Based on their sequences, SFFVs could have derived from MCFs by a 585-base deletion and by a single-base addition in the ecotropic-specific portion of the env gene (10). Evidence suggests that both the 585-bp deletion and the frameshift mutation probably contribute to SFFV pathogenesis (3, 49). Several pathogenic differences among SFFV strains have also been ascribed to amino acid sequence differences in the ecotropic-specific portion of the Env glycoproteins (9, 41).This report describes the origin and rapid stepwise evolution of a new SFFV. This new pathogenic virus initially formed in a mouse that had been injected with an ecotropic strain of MuLV in the presence of a retroviral vector that does not encode any Env glycoprotein. The mouse developed erythroleukemia, splenomegaly, and polycythemia after a long lag phase. At that time the spleen contained viruses with env genes that were able to activate EpoR. Serial passage of this initial virus isolate resulted in selection of a novel SFFV that encodes a gp55 glycoprotein of 410 amino acids. This experimental system provides a method for isolating new SFFVs and for mapping the stages in their genesis.     

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu⁵⁴³-Asp⁵⁴⁴ in this linker or Leu⁶¹¹, Arg⁶⁸³, or Phe⁶⁹⁴ in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.The Janus family of tyrosine kinases (JAKs)² are key regulators of cytokine receptor signaling in hematopoiesis and immune responses (1). Of the four mammalian JAK kinases, JAK2 transmits signals for a variety of cytokine receptors, including the erythropoietin receptor (EpoR) that is essential for red blood cell production (2). Upon Epo stimulation, JAK2 activates downstream signaling, such as STAT5, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/AKT pathways (2). Mice deficient in Epo, EpoR, or JAK2 die embryonically due to the absence of definitive erythropoiesis (3–5).In addition to regulation by phosphatases and suppressors of cytokine signaling (6, 7), JAK2 kinase activity is critically controlled by an autoinhibitory mechanism. Like other JAK members, JAK2 contains an N-terminal segment followed by a pseudokinase domain and a C-terminal tyrosine kinase domain. The N-terminal segment, consisting of a FERM (protein 4.1, ezrin, moezin, radixin homologous) domain and an atypical SH2 domain (1), mediates association with the membrane-proximal region of the cytokine receptors (8). Binding of JAK2 through its N-terminal segment to the EpoR is essential for EpoR surface expression (9). The pseudokinase domain is predicted to adopt a kinase fold but lacks residues essential for catalysis (10). Deletion of the pseudokinase domain leads to a marked increase in JAK2 kinase activity and loss of response to cytokine stimulation (11–13). Therefore, this domain is essential for JAK2 autoinhibition and is essential for JAK2 activation upon cytokine stimulation. Consistent with this notion, a point mutation in the JAK2 pseudokinase domain was identified in the majority of myeloproliferative disorder patients, including 90% of polycythemia vera (PV) patients (14–18). This mutation, V617F, in the presence of a dimerized receptor scaffold, such as the EpoR, resulted in the constitutive activation of JAK2 and downstream signaling effectors (19, 20) and caused erythrocytosis in a murine bone marrow transplant model (14, 21–23). Recently, mutations immediately adjacent to the JAK2 pseudokinase domain in the SH2-pseudokinase domain linker were identified in PV patients and shown to cause constitutive activation of JAK2 and a PV-like phenotype in mice (24–26). The molecular mechanisms underlying the control of JAK2 activity (i.e. the swift augmentation of its activity upon receptor activation) are poorly understood. The residues involved in the autoinhibition in JAK2 are unknown.In this work, we sought to characterize the regulatory mechanisms controlling JAK2 kinase activity. Using a functional screen for activating JAK2 mutations that signal constitutively, we discovered 13 mutations in the pseudokinase domain and in the SH2-pseudokinase domain linker. These mutations identified specific residues that are important for the inhibition of basal JAK2 kinase activity and for cytokine-induced JAK2 activation. In addition, we showed that the SH2-pseudokinase domain linker is essential for interaction with the EpoR, autoinhibitory regulation, and Epo-inducible JAK2 activation and may act as a switch in relaying cytokine receptor engagement to JAK2 activation by flexing the pseudokinase domain hinge.     

Epstein-Barr Virus Lacking Glycoprotein gp42 Can Bind to B Cells but Is Not Able To Infect

The Epstein-Barr virus gH-gL complex includes a third glycoprotein, gp42, which is the product of the BZLF2 open reading frame (ORF). gp42 has been implicated as critical to infection of the B lymphocyte by virtue of its interaction with HLA class II on the B-cell surface. A neutralizing antibody that reacts with gp42 inhibits virus-cell fusion and blocks binding of gp42 to HLA class II; antibody to HLA class II can inhibit infection, and B cells that lack HLA class II can only be infected if HLA class II expression is restored. To confirm whether gp42 is an essential component of the virion, we derived a recombinant virus with a selectable marker inserted into the BZLF2 ORF to interrupt expression of the protein. A complex of gH and gL was expressed by the recombinant virus in the absence of gp42. Recombinant virus egressed from the cell normally and could bind to receptor-positive cells. It had, however, lost the ability to infect or transform B lymphocytes. Treatment with polyethylene glycol restored the infectivity of recombinant virus, confirming that gp42 is essential for penetration of the B-cell membrane.Entry of enveloped viruses into mammalian cells requires that the virion envelope fuse with the cell membrane after attachment to the cell surface. Herpesviruses require the functions of multiple protein species to mediate this event, and in keeping with the common origin and diverse habitats of these viruses, some of the proteins involved in penetration appear to be conserved throughout the family and some appear to be restricted to individual members or more closely related members with similar tropism. The two glycoproteins gH and gL fall into the first category of conserved proteins. Glycoprotein gH has been implicated as a major player in virus-cell fusion in many herpesviruses (8, 10, 11, 22, 28, 32, 34), and gL is an essential partner which is required for folding and transport of gH out of the endoplasmic reticulum (6, 19, 21, 27, 28, 35, 38, 45). The gH and gL homologs of Epstein-Barr virus (EBV) are gp85, the product of the BXLF2 open reading frame (ORF) (13, 31), and gp25, the product of the BKRF2 ORF (45), and these homologs appear to behave much as their counterparts in other herpesviruses do (45). However, a third glycoprotein, gp42, associates with the EBV gH-gL complex and falls into the second category of proteins, those with a more restricted distribution.Glycoprotein gp42 is the product of the BZLF2 ORF (26), and although there may be a functionally similar protein in cytomegalovirus (18, 24), it is not predicted to have a homolog in other human herpesviruses. It does, however, have a homolog in ORF51 of equine herpes virus 2 (43). Both EBV and equine herpes virus 2 infect B lymphocytes (1), and several lines of evidence suggest that, at least in the case of EBV, gp42 is critical to the infection of this cell type. A monoclonal antibody (MAb) called F-2-1 that reacts with gp42 has no affect on EBV attachment to its receptor, complement receptor type 2 (CR2) (CD21), but inhibits fusion of the virus with the B-cell membrane and neutralizes infection (29). Glycoprotein gp42 interacts with the β₁ domain of the HLA class II protein HLA-DR (39), and MAb F-2-1 interferes with this interaction (25). Like F-2-1, a MAb to HLA-DR or a soluble form of gp42 can block B-cell transformation, and B-cell lines which lack expression of HLA class II are not susceptible to superinfection with EBV unless expression of HLA class II is restored (25). Collectively these observations suggest that gp42, probably by virtue of its interaction with HLA class II, is essential to infection of the B lymphocyte. To answer directly the question of whether gp42 is an indispensable glycoprotein, we derived a virus that could be definitively shown to lack expression of the molecule and examined its ability to infect normal resting B lymphocytes. We report here that virus with expression of gp42 blocked can exit cells normally and can bind to receptor-positive target cells. However, it is unable to penetrate into cells and initiate infection.     

Toward Effective HIV Vaccination: INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY*

We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421–433 and 288–306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (V_H) domain framework (FR) residues. Substitution of the FR cavity V_H Lys-19 residue by an Ala residue resulted in attenuated binding of the 421–433 region peptide probe. The CDRs and V_H FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from V_H1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421–433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.Induction of neutralizing antibodies (Abs)² via adaptive immune processes is the cornerstone of vaccination against microbial antigens. The antigen-binding site is mostly formed by the complementarity determining regions (CDRs) of the light and heavy chain variable domains (V_L and V_H domains). Vaccine-induced adaptive Ab responses entail sequence diversification of Ab V domains expressed within the B cell receptor (BCR) complex, selective noncovalent antigen binding to the high affinity BCR mutants, and proliferation of the mutant B cell clones. No HIV vaccine is available. The surface of HIV is studded with noncovalently associated oligomers of gp120 complexed to gp41. HIV infection and experimental HIV vaccination attempts induce robust Ab responses to the immunodominant epitopes of gp120, which are structurally divergent in various HIV strains responsible for infection in different parts of the world. Abs to such epitopes express strain-specific neutralization (1, 2), i.e. they neutralize the HIV strain from which the immunogen was isolated but not strains genetically heterologous to the immunogen.The gp120 site responsible for binding host CD4 receptors (CD4BS) is structurally more conserved. Precise conformational details of the CD4BS expressed on the HIV surface are not available, but crystallography suggests a large, discontinuous determinant composed of regions distant from each other in the linear protein sequence (3, 4). The 421–433 peptide region is essential for CD4 binding by gp120, suggested by contacts in the crystallized complex and loss of CD4 binding function by site-directed mutagenesis in this region (5, 6). The 421–433 region is a member of a small group of microbial polypeptide sites recognized selectively by Abs produced by the immune system without prior infection by the microbe (preimmune Abs) (7–9). Such sites are designated B cell “superantigens” (SAgs) because of their selective and widespread recognition by the comparatively conserved framework regions (FRs) of Ab V domains (10, 11). Noncovalent SAg binding by preimmune Abs, however, is characterized by low-to-moderate binding strength (12). Most gp120-binding preimmune Abs from humans without infection display poor or no HIV neutralizing activity (13). Patients with the autoimmune disease lupus and no HIV infection produce increased amounts of Abs to the 421–433 CD4BS region (14). A single chain Fv (scFv; V_L and V_H domains linked by a flexible peptide) from the lupus Ab repertoire that binds the 421–433 region reversibly neutralizes genetically diverse strains of HIV (15). Following completion of the noncovalent binding step, certain Abs can hydrolyze polypeptides via nucleophilic attack on carbonyl groups (16–21). The proteolytic reaction imparts improved antigen inactivation potency to Abs (22). We reported the neutralization of HIV by secretory IgA from humans without infection, an Ab class distinguished by the ability to catalyze the hydrolysis of gp120 selectively because of initial noncovalent recognition of the 421–433 CD4BS region (13).The conserved character of the CD4BS in genetically diverse HIV strains renders it suitable as a vaccine target. The CD4BS, however, is poorly immunogenic. Traditional immunization methods do not stimulate the adaptive synthesis of neutralizing Abs to the 421–433 region or other CD4BS epitopes. Neutralizing Abs that bind the CD4BS are found in the blood of a subset of patients after years of HIV infection, but the target epitope is not identified, and Ab response is weak (23, 24). Certain monoclonal Abs (mAbs) that bind the CD4BS expressed by purified monomer gp120 do not neutralize HIV appreciably or display limited ability to neutralize genetically diverse HIV strains (25, 26). The CD4BS is a flexible structure expressed in differing conformational states by monomer gp120 and the native gp120 oligomers of the virus (27–30). Moreover, the process of binding CD4 may induce movements within the CD4BS (31). Reproducing the native CD4BS conformation in experimental vaccine candidates has been difficult. A CD4BS mimetic of the epitope recognized by a well known anti-CD4BS neutralizing mAb (clone b12) did not induce the synthesis of neutralizing Abs (32). Polyclonal Abs raised by immunization with synthetic peptides spanning the 421–433 CD4BS region neutralized laboratory-adapted, coreceptor CXCR4-dependent HIV strains inconsistently (33–35). Neutralization of coreceptor CCR5-dependent strains responsible for initiating most HIV infections was not studied. Importantly, small synthetic peptides are often more flexible than the corresponding native protein segments. Inducing a traditional adaptive immune response in which the Ab CDRs develop binding specificity for the peptide immunogen therefore does not ensure recognition of the native 421–433 CD4BS region (35, 36). From mutagenesis and sequence identity studies, the gp120-binding site of preimmune Abs, in contrast, is composed mainly of the V_H domain FR1 and FR3 (10, 11, 37). As certain preimmune Abs express HIV neutralizing activity attributable to recognition of the 421–433 region (13), the FR-dominated site must recognize the native state of this CD4BS epitope expressed on the viral surface.There is, however, substantial difficulty in amplifying and improving the subset of preimmune Abs with HIV neutralizing activity for vaccination against the virus; SAg binding to Ab FRs fails to stimulate adaptive B cell differentiation and synthesis of specific IgG class Abs (38, 39). Indeed, the binding at the FRs may even lead to premature death of the B cells (12, 40). The SAg character of the 421–433 CD4BS epitope is therefore predicted to render it hypoimmunogenic with respect to the adaptive synthesis of neutralizing Abs following infection or traditional vaccination procedures.We reported previously the induction of nucleophilic Abs by covalent immunization with full-length gp120 and a gp120 V3 peptide containing strongly electrophilic phosphonate groups (41–43). The electrophile reacts covalently with BCRs (44), resulting in adaptively strengthened nucleophilic reactivity coordinated with specific noncovalent recognition of gp120. The Abs obtained by covalent immunization formed very stable immune complexes with HIV resulting from pairing of Ab nucleophiles with the naturally occurring electrophilic groups of gp120 (e.g. the backbone and side chain carbonyls, see Refs. 42, 43). A minority of the Abs proceeded to catalyze the hydrolysis of gp120, aided by water attack on the covalent acyl-Ab complex (41). Here we report the neutralization of HIV strains heterologous to the full-length electrophilic gp120 immunogen (E-gp120) by mAbs with binary CD4BS and V3 loop recognition capability. We also present models that explain synthesis of the mAbs in response to immunization with E-gp120.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.Single-stranded DNA (ssDNA)³-binding proteins have been assigned the role of removing secondary structure in DNA and protecting ssDNA from hydrolysis by nucleases (1). However, in addition to these mundane roles, ssDNA-binding proteins are now recognized as a key component of the replisome where they physically and functionally interact with other replication proteins and with the primer-template (2–4). ssDNA-binding proteins are also engaged in DNA recombination and repair (5). In view of these multiple roles, it has been difficult to identify the specific defect in genetically altered ssDNA-binding proteins that leads to an observed phenotype.The crystal structures of several prokaryotic ssDNA-binding proteins have been determined (6–8). These proteins have a conserved oligosaccharide-oligonucleotide binding fold (OB-fold) that is thought to bind the ssDNA by means of stacking and electrostatic interactions (6). Prokaryotic ssDNA-binding proteins also have an acidic C-terminal tail that is essential for bacterial and phage growth (9–13).The ssDNA-binding protein of bacteriophage T7 is encoded by gene 2.5 (14). The gene 2.5 protein (gp2.5) is a homodimer in solution, a structure that is stabilized by its C-terminal tail (9, 15). The C-terminal tail of one monomer of gp2.5 binds in a trans mode to the ssDNA-binding cleft of the other subunit, thus stabilizing the dimer interface observed in the crystal structure (6). The current model proposes that the positively charged DNA-binding cleft is shielded by the electrostatic charges of the C-terminal tail in the absence of ssDNA, thus facilitating oligomerization of gp2.5. Upon binding ssDNA, the dimer dissociates to allow the C-terminal tail to interact with other replication proteins (16). The tail modulates the affinity for ssDNA and protein-protein interactions by functioning as a two-way switch (6, 17). This mode of function is applicable to other prokaryotic ssDNA-binding proteins, namely Escherichia coli SSB protein and T4 gp32 (10, 13, 15, 18–22).gp2.5 is one of four proteins that include the T7 replisome. The other three proteins are the T7 gene 5 DNA polymerase (gp5), its processivity factor, E. coli thioredoxin (trx), and the multifunctional gene 4 helicase-primase (gp4). gp5 and trx bind with high affinity (K_D of 5 nm), and the two proteins are normally found in complex (gp5/trx) at a stoichiometry of one to one (23). The acidic C-terminal tail of gp2.5 is critical for the interactions of the protein with gp5/trx and gp4 (9, 24). The C-terminal tail binds to a positively charged segment located in the thumb subdomain of the gp5 (25). This fragment, designated the trx binding domain (TBD), is also the site of binding of the processivity factor, E. coli trx, and the C terminus of gp4. The multiple interactions of the C terminus of gp2.5 could thus function to coordinate the dynamic reactions occurring at the replication fork. gp2.5 is known to be critical for establishing coordination during leading and lagging strand DNA synthesis (26, 27).This C-terminal tail of gp2.5 is an acidic 26-amino acid segment with an aromatic phenylalanine as the C-terminal residue. The C-terminal tail is not seen in the crystal structure because gp2.5Δ26, lacking the tail, was used for crystallization; the wild-type protein did not yield crystals that diffracted (6). gp2.5ΔF designates a genetically modified gp2.5 lacking the C-terminal phenylalanine. gp2.5ΔF does not support the growth of T7Δ2.5 phage lacking gene 2.5 (28). Interestingly, T7 gene 4 protein also has an acidic C-terminal tail with a C-terminal phenylalanine (29). Again, the phenylalanine is critical for the interaction of gp4 with gp5/trx (29). Further evidence for overlapping binding sites of the C termini of these two proteins comes from studies with chimeric proteins (28, 29). The C-terminal tails of gp2.5 and gp4 can be exchanged, and the chimeric proteins support the growth of T7 phage lacking the corresponding wild-type protein.We recently designed a screen for suppressors of dominant lethal mutations of gp2.5 (30). The screen identified mutations in gene 5, the structural gene for T7 DNA polymerase (Fig. 1), which suppresses the lethal phenotype of gp2.5 mutant in which the C-terminal phenylalanine was moved to the penultimate position (gp2.5ΔF232InsF231). One of the altered suppressor genes (gp5, gp5-sup1) encodes a gp5 in which where glycine at position 371 is replaced by lysine (G371K). Whereas the other (gp5-sup2) encodes a protein in which threonine 258 and alanine 411 are replaced by methionine and threonine, respectively (T258M and A411T). The suppressor mutations in gp5 are necessary and sufficient to suppress the lethal phenotype of gp2.5ΔF232InsF231. The affected residues map in proximity to aromatic residues and to residues in close proximity to DNA as seen in the crystal structure of gp5/trx in complex with DNA (31). Throughout this study, gp2.5ΔF232InsF231 mutant will be referred to as gp2.5-FD because it effectively switches the positions of the C-terminal phenylalanine and the adjacent aspartic acid. E. coli SSB protein also has a C-terminal phenylalanine, and recent studies have shown that this residue inserts into a hydrophobic region consisting of exonuclease I of E. coli (45, 46).Open in a separate windowFIGURE 1.Amino acid changes in gp5 suppressor mutant polymerase(s). The amino acid changes in gp5 arising from the suppressor mutations in gene 5 are identified in the crystal structure of gp5/trx in complex with a primer-template and a nucleoside triphosphate (31). gp5 (light gray), trx (dark gray), and primer/template (red) are depicted. The suppressor mutation G371K (gp5-sup1) is shown in yellow and T258M and A411T (gp5-sup2) in orange.In this study, we have purified the two suppressor DNA polymerases and characterized them individually and in interaction with the other T7 replication proteins. Whereas wild-type gp5 binds with low affinity to gp2.5-FD, the DNA polymerases harboring the suppressor mutations bind with a higher affinity. An interesting finding is that whereas wild-type gp2.5 enables gp5/trx to catalyze strand displacement synthesis at a nick in DNA, gp2.5-FD does not support this reaction. Strand displacement synthesis is necessary for the initiation of leading strand DNA synthesis at a nick because it creates a 5′-single-stranded DNA tail for loading of the T7 helicase (32).     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Syncytium-Inhibiting Monoclonal Antibodies Produced against Human T-Cell Lymphotropic Virus Type 1-Infected Cells Recognize Class II Major Histocompatibility Complex Molecules and Block by Protein Crowding

Four new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).Human T-cell lymphotropic virus type 1 (HTLV-1) is a type C retrovirus and the etiologic agent of adult T-cell leukemia (43, 56, 59) and HTLV-1-associated myelopathy or tropical spastic paraparesis (15, 17, 49, 61). Although HTLV-1 shows tropism primarily for T cells, it can infect a variety of cell types including cells from some nonhuman species (6, 9, 27, 46, 48, 60, 62). Infection by free HTLV-1 tends to be highly inefficient, and the virus appears to be transmitted primarily by the cell-to-cell route (37). The HTLV-1 envelope glycoprotein is synthesized as a 61-kDa precursor which is cleaved into surface (gp46) and transmembrane (gp21) proteins (40, 57). gp46 is thought to serve as the virus attachment protein, as does gp120 for human immunodeficiency virus (HIV) (40, 57). Although previous reports have identified host cell molecules which might potentially mediate virus binding (9, 14), the cellular receptor for HTLV-1 has not been definitively identified. A recent study in which affinity chromatography was carried out with a gp46 peptide has provided evidence that the heat shock protein HSC70 binds directly to gp46 and may serve as a virus receptor (47).gp21 contains an N-terminal hydrophobic fusion domain and likely serves as a fusion protein similar to HIV gp41 (12, 61). Like many other retroviruses, HTLV-1 can induce syncytium formation between infected cells and certain uninfected cell types (28, 39). However, there are no data to indicate that virus transmission or virus persistence in vivo depends on syncytium formation. It is thought that cell-cell fusion involves the same receptors and occurs in a manner similar to virus-cell fusion. For this reason, HTLV-1 syncytium assays have been used to screen for cell surface molecules that may serve as virus receptors (13, 14, 25, 29). Monoclonal antibodies (MAbs) against a number of membrane proteins including members of the tetraspanner family (30, 31) have been found to block syncytium formation. My colleagues and I recently reported that expression of the cell adhesion molecule vascular cell adhesion molecule 1 (VCAM-1) on uninfected cells can confer sensitivity to HTLV-1-mediated syncytium formation (25). In this previous study, we were not able to block HTLV-1 cell fusion with MAbs against the major VCAM-1 counterreceptor VLA-4 (25). Others have reported that MAbs to other adhesion molecules including intercellular adhesion molecule 3 (ICAM-3) also block HTLV-1 syncytium formation (29). We have demonstrated that adhesion molecules also facilitate HIV type 1 (HIV-1) infection and syncytium formation (16, 24). Thus, adhesion molecules may be important accessory molecules for retroviruses generally.Earlier studies on accessory molecules involved in HTLV-1 biology have been extended by immunizing mice with HTLV-1-infected cells and screening for MAbs that block VCAM-1-supported HTLV-1 syncytium formation. Four new MAbs that completely block HTLV-1-mediated cell fusion have been generated. The MAbs were all determined to be specific for class II major histocompatibility complex (MHC) molecules. These MAbs had no effect on syncytium formation induced by HIV-1. Studies on the mechanism by which the MAbs mediate this effect have revealed a novel mode of antibody blockade of virus-induced cell fusion: protein crowding at the infected cell surface resulting in steric blockade of critical receptor-ligand interactions.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Evidence that Antibody-Mediated Neutralization of Human Immunodeficiency Virus Type 1 by Sera from Infected Individuals Is Independent of Coreceptor Usage

Human immunodeficiency virus type 1 (HIV-1) uses a variety of chemokine receptors as coreceptors for virus entry, and the ability of the virus to be neutralized by antibody may depend on which coreceptors are used. In particular, laboratory-adapted variants of the virus that use CXCR4 as a coreceptor are highly sensitive to neutralization by sera from HIV-1-infected individuals, whereas primary isolates that use CCR5 instead of, or in addition to, CXCR4 are neutralized poorly. To determine whether this dichotomy in neutralization sensitivity could be explained by differential coreceptor usage, virus neutralization by serum samples from HIV-1-infected individuals was assessed in MT-2 cells, which express CXCR4 but not CCR5, and in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), where multiple coreceptors including CXCR4 and CCR5 are available for use. Our results showed that three of four primary isolates with a syncytium-inducing (SI) phenotype and that use CXCR4 and CCR5 were neutralized poorly in both MT-2 cells and PBMC. The fourth isolate, designated 89.6, was more sensitive to neutralization in MT-2 cells than in PBMC. We showed that the neutralization of 89.6 in PBMC was not improved when CCR5 was blocked by having RANTES, MIP-1α, and MIP-1β in the culture medium, indicating that CCR5 usage was not responsible for the decreased sensitivity to neutralization in PBMC. Consistent with this finding, a laboratory-adapted strain of virus (IIIB) was significantly more sensitive to neutralization in CCR5-deficient PBMC (homozygous Δ32-CCR5 allele) than were two of two SI primary isolates tested. The results indicate that the ability of HIV-1 to be neutralized by sera from infected individuals depends on factors other than coreceptor usage.Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS, utilizes the HLA class II receptor, CD4, as its primary receptor to gain entry into cells (17, 30). Entry is initiated by a high-affinity interaction between CD4 and the surface gp120 of the virus (32). Subsequent to this interaction, conformational changes that permit fusion of the viral membrane with cellular membranes occur within the viral transmembrane gp41 (9, 58, 59). In addition to CD4, one or more recently described viral coreceptors are needed for fusion to take place. These coreceptors belong to a family of seven-transmembrane G-protein-coupled proteins and include the CXC chemokine receptor CXCR4 (3, 4, 24, 44), the CC chemokine receptors CCR5 (1, 12, 13, 18, 21, 23, 45) and, less commonly, CCR3 and CCR2b (12, 21), and two related orphan receptors termed BONZO/STRL33 and BOB (19, 34). Coreceptor usage by HIV-1 can be blocked by naturally occurring ligands, including SDF-1 for CXCR4 (4, 44), RANTES, MIP-1α, and MIP-1β in the case of CCR5 (13, 45), and eotaxin for CCR3 (12).The selective cellular tropisms of different strains of HIV-1 may be determined in part by coreceptor usage. For example, all culturable HIV-1 variants replicate initially in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), but only a minor fraction are able to infect established CD4⁺ T-cell lines (43). This differential tropism is explained by the expression of CXCR4 together with CCR5 and other CC chemokine coreceptors on PBMC and the lack of expression of CCR5 on most T-cell lines (5, 10, 19, 35, 39, 50, 53). Indeed, low-passage field strains (i.e., primary isolates) of HIV-1 that fail to replicate in T-cell lines use CCR5 as their major coreceptor and are unable to use CXCR4 (1, 12, 18, 21, 23, 28). Because these isolates rarely produce syncytia in PBMC and fail to infect MT-2 cells, they are often classified as having a non-syncytium-inducing (NSI) phenotype. Primary isolates with a syncytium-inducing (SI) phenotype are able to use CXCR4 alone or, more usually, in addition to CCR5 (16, 20, 51). HIV-1 variants that have been passaged multiple times in CD4⁺ T-cell lines, and therefore considered to be laboratory adapted, exhibit a pattern of coreceptor usage that resembles that of SI primary isolates. Most studies have shown that the laboratory-adapted strain IIIB uses CXCR4 alone (3, 13, 20, 24, 51) and that MN and SF-2 use CXCR4 primarily and CCR5 to a lesser degree (11, 13). Sequences within the V3 loop of gp120 have been shown to be important, either directly or indirectly, for the interaction of HIV-1 with both CXCR4 (52) and CCR5 (12, 14, 54, 60). This region of gp120 contains multiple determinants of cellular tropism (43) and is a major target for neutralizing antibodies to laboratory-adapted HIV-1 but not to primary isolates (29, 46, 57).It has been known for some time that the ability of sera from HIV-1-infected individuals to neutralize laboratory-adapted strains of HIV-1 does not predict their ability to neutralize primary isolates in vitro (7). In general, the former viruses are highly sensitive to neutralization whereas the latter viruses are neutralized poorly by antibodies induced in response to HIV-1 infection (7, 43). Importantly, neutralizing antibodies generated by candidate HIV-1 subunit vaccines have been highly specific for laboratory-adapted viruses (26, 37, 38). In principle, the dichotomy in neutralization sensitivity between these two categories of virus could be related to coreceptor usage. To test this, we investigated whether the use of CXCR4 in the absence of CCR5 would render SI primary isolates highly sensitive to neutralization in vitro by sera from HIV-1-infected individuals. Two similar studies using human monoclonal antibodies and soluble CD4 have been reported (31a, 55).     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]