A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.     

Interaction with decay-accelerating factor facilitates coxsackievirus B infection of polarized epithelial cells

All coxsackie B (CB) viruses can initiate infection by attaching to the coxsackievirus and adenovirus receptor (CAR). Although some CB isolates also bind to decay-accelerating factor (DAF), the role of DAF interaction during infection remains uncertain. We recently observed that CAR in polarized epithelial cells is concentrated at tight junctions, where it is relatively inaccessible to virus. In the experiments reported here we found that, unlike CAR, DAF was present on the apical surface of polarized cells and that DAF-binding isolates of CB3 and CB5 infected polarized epithelial cells more efficiently than did isolates incapable of attaching to DAF. Virus attachment and subsequent infection of polarized cells by DAF-binding isolates were prevented in the presence of anti-DAF antibody. Serial passage on polarized cell monolayers selected for DAF-binding virus variants. Taken together, these results indicate that interaction with DAF on the apical surface of polarized epithelial cells facilitates infection by a subset of CB virus isolates. The results suggest a possible role for DAF in infection of epithelial cells at mucosal surfaces.     

Interaction of decay-accelerating factor with echovirus 7

Echovirus 7 (EV7) belongs to the Enterovirus genus within the family Picornaviridae. Many picornaviruses use IgG-like receptors that bind in the viral canyon and are required to initiate viral uncoating during infection. However, in addition, some of the enteroviruses use an alternative or additional receptor that binds outside the canyon. Decay-accelerating factor (DAF) has been identified as a cellular receptor for EV7. The crystal structure of EV7 has been determined to 3.1-Å resolution and used to interpret the 7.2-Å-resolution cryo-electron microscopy reconstruction of EV7 complexed with DAF. Each DAF binding site on EV7 is near a 2-fold icosahedral symmetry axis, which differs from the binding site of DAF on the surface of coxsackievirus B3, indicating that there are independent evolutionary processes by which DAF was selected as a picornavirus accessory receptor. This suggests that there is an advantage for these viruses to recognize DAF during the initial process of infection.Echoviruses (EVs) belong to the family Picornaviridae, which contains some of the most common viral pathogens of vertebrates (43, 50, 51, 55, 58, 63). Picornaviruses are small, icosahedral, nonenveloped animal viruses. Their capsids have 60 copies each of four viral proteins, VP1, VP2, VP3, and VP4, that form an ∼300-Å-diameter icosahedral shell filled with a positive-sense, single-stranded RNA genome. A distinctive feature of the capsid surface is a depression around the 5-fold axes of symmetry, called the “canyon” (47). The results of both genetic and structural studies have shown that the canyon is the site of receptor binding for many of these viruses (4, 11, 23, 25, 36, 47, 68), including echoviruses, which utilize β-integrins (6, 33, 66). Receptor molecules that bind in the canyon have been found to belong to the immunoglobulin superfamily (49). When these receptor molecules bind within the canyon, they dislodge a “pocket factor” within a pocket immediately below the surface of the canyon. The shape and environment of the pocket factor suggest that it might be a lipid (13, 32, 45, 54). When a receptor binds within the canyon, it depresses the floor of the canyon, corresponding to the roof of the pocket. Similarly, when a lipid or antiviral compound binds to the pocket, it expands the roof of the pocket, corresponding to the floor of the canyon (39, 45). Thus, receptors that bind to the canyon and the pocket factor compete with each other for binding to the virus. An absence of the hydrophobic pocket factor destabilizes the virus and initiates transition to altered “A” particles, a likely prelude to uncoating of the virion, possibly during passage through an endosomal vesicle (45).Not all receptors of picornaviruses bind in the canyon. A minor group of human rhinoviruses (HRV) bind to the low-density-lipoprotein receptor family (17, 34, 61, 62), and some other picornaviruses, including certain coxsackie- and echoviruses, utilize decay-accelerating factor (DAF; also called CD55) as a cellular receptor (9, 28, 40, 52).DAF is a member of a family of proteins that regulate complement activation by binding to and accelerating the decay of both classical and alternative pathway C3 and C5 convertases (7, 18, 26), the central amplification enzymes of the complement cascade. DAF is expressed on virtually all cell surfaces, protecting self cells from the immune system by rapidly dissociating any convertases that assemble, thereby halting the progression of a complement attack directed at the cell. Recent work (15, 27, 29, 56) has shown that DAF also participates in T-cell antiviral immunity (56) and protects against T-cell autoimmunity (29) by regulating complement that is produced locally by immune cells. The functional region of DAF consists of four short consensus repeats (SCR1, -2, -3, and -4). The structures have been determined for the SCR2-SCR3 fragment, the SCR3-SCR4 fragment, and the full four-domain region (30, 60, 65). Each of the SCR domains contains about 60 residues and is folded into a β structure stabilized by disulfide bridges. The four SCR domains form a relatively rigid extended rod with dimensions of 160 by 50 by 30 Å (30). The four domains rise about 180 Å above the plasma membrane, on a serine- and threonine-rich stalk of 94 amino acids, 11 of which are O-glycosylated, and is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor.Structural and genetic studies have shown that closely related picornaviruses have adapted to bind to DAF at different sites on the receptor surface (9, 31, 38, 42, 52, 64). Although DAF binding is likely to facilitate viral adsorption, the availability of DAF receptor molecules on the host is normally not sufficient for echovirus 7 (EV7) to enter cells. Presumably, viral adaptation to bind DAF offers some advantage to the virus, such as increasing the efficiency of infection.In an earlier publication (14), a 16-Å-resolution cryo-electron microscopy (cryo-EM) density map of the EV7-DAF complex was interpreted with the homologous structures of coxsackievirus B3 (CVB3) for EV7 (74% sequence identity) and virus complement protein for DAF (25% sequence identity). Because of the limited resolution of the earlier cryo-EM reconstruction, it was concluded that DAF bound to EV7 by laying across the icosahedral 2-fold axes. This implied that there were two alternative DAF binding modes occupying the same site, but with DAF oriented in opposite directions, and that only one of these alternative sites could be occupied at a time. Here we describe an improved, 7.2-Å-resolution cryo-EM reconstruction of DAF bound to EV7 and 3.1-Å-resolution X-ray crystal structures of EV7. Together with previously determined structures of DAF (30), we now show that 2-fold axis-related DAF molecules bind close to the icosahedral 2-fold axes on the viral surface but (in contradiction to the earlier results and consistent with predictions made by Pettigrew et al. [38]) do not cross these axes. This is consistent with the results of DAF binding to EV12, which binds DAF similarly to the manner reported here and also predicted for EV7 (38). Thus, the binding modes of DAF to EV12 and EV7 are now shown to be similar, but not the same, and are completely different from the binding mode of DAF to CVB3.     

Single amino acid changes in the virus capsid permit coxsackievirus B3 to bind decay-accelerating factor

Many coxsackievirus B isolates bind to human decay-accelerating factor (DAF) as well as to the coxsackievirus and adenovirus receptor (CAR). The first-described DAF-binding isolate, coxsackievirus B3 (CB3)-RD, was obtained during passage of the prototype strain CB3-Nancy on RD cells, which express DAF but very little CAR. CB3-RD binds to human DAF, whereas CB3-Nancy does not. To determine the molecular basis for the specific interaction of CB3-RD with DAF, we produced cDNA clones encoding both CB3-RD and CB3-Nancy and mutated each of the sites at which the RD and Nancy sequences diverged. We found that a single amino acid change, the replacement of a glutamate within VP3 (VP3-234E) with a glutamine residue (Q), conferred upon CB3-Nancy the capacity to bind DAF and to infect RD cells. Readaptation of molecularly cloned CB3-Nancy to RD cells selected for a new virus with the same VP3-234Q residue. In experiments with CB3-H3, another virus isolate that does not bind measurably to DAF, adaptation to RD cells resulted in a DAF-binding isolate with a single amino acid change within VP2 (VP2-138 N to D). Both VP3-234Q and VP2-138D were required for binding of CB3-RD to DAF. In the structure of the CB3-RD-DAF complex determined by cryo-electron microscopy, both VP3-234Q and VP2-138D are located at the contact site between the virus and DAF.     

Binding to decay-accelerating factor is not required for infection of human leukocyte cell lines by enterovirus 70

Enterovirus 70 (EV70) is one of several human enteroviruses that exhibit a propensity for infecting the central nervous system (CNS). The mechanisms by which neurotropic enteroviruses gain access to and invade the CNS are poorly understood. One possibility is that circulating leukocytes become infected and carry neurotropic enteroviruses to the CNS. We examined the ability of EV70 to infect cell lines derived from lymphoid, myeloid, and monocytic lineages. Most leukocyte cell lines tested bound radiolabeled EV70 and were permissive for EV70 replication, suggesting that EV70, in contrast to other enteroviruses, has an in vitro tropism that includes lymphoid, monocytic, and myeloid cell lines. For some of the cell lines, virus binding and infection correlated with surface expression of decay-accelerating factor (DAF), an attachment protein for EV70 on HeLa cells. However, EV70 also adsorbed to and infected cell lines that expressed little or no DAF. In contrast to what was observed for HeLa cells, neither DAF-specific monoclonal antibodies nor phosphatidylinositol-specific phospholipase C treatment inhibited EV70 binding to permissive leukocyte cell lines, and antibody blockade of DAF had little or no effect on EV70 replication. We also found that neither the human coxsackievirus-adenovirus receptor nor intercellular cell adhesion molecule 1, which mediate the entry of coxsackie B viruses and coxsackievirus A21, respectively, functions as a receptor for EV70. EV70 binding to all cell lines was sensitive to sialidase treatment and to inhibition of O glycosylation by benzyl N-acetyl-alpha-D-galactosaminide. Taken together, these results suggest that a sialylated molecule(s) other than DAF serves as a receptor for EV70 on permissive human leukocyte cell lines.     

Interaction of decay-accelerating factor with coxsackievirus B3

Many entero-, parecho-, and rhinoviruses use immunoglobulin (Ig)-like receptors that bind into the viral canyon and are required to initiate viral uncoating during infection. However, some of these viruses use an alternative or additional receptor that binds outside the canyon. Both the coxsackievirus-adenovirus receptor (CAR), an Ig-like molecule that binds into the viral canyon, and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). A cryoelectron microscopy reconstruction of a variant of CVB3 complexed with DAF shows full occupancy of the DAF receptor in each of 60 binding sites. The DAF molecule bridges the canyon, blocking the CAR binding site and causing the two receptors to compete with one another. The binding site of DAF on CVB3 differs from the binding site of DAF on the surface of echoviruses, suggesting independent evolutionary processes.     

Novel role for decay-accelerating factor in coxsackievirus A21-mediated cell infectivity

Decay-accelerating factor (DAF) is involved in the cell membrane attachment of many human enteroviruses. Presently, further specific active roles of DAF in mediating productive cell infection and in the pathogenesis of natural enterovirus infection are poorly understood. In an attempt to more fully understand the role of DAF in lytic cell infection we examined the specific interactions of the prototype strain of coxsackievirus A21 (CVA21) with surface-expressed DAF. Investigations into discrete DAF-CVA21 interactions focused on viral binding; viral particle elution with respect to the parameters of time, temperature, and pH; and subsequent cell infection. Radiolabeled-virus binding assays revealed that peak elution of CVA21 from DAF occurred within 15 min of initial attachment and that the DAF-eluted virus increased in a linear fashion with respect to temperature and pH. CVA21 eluted from endogenous surface-expressed DAF was highly infectious, in contrast to CVA21 eluted from intercellular adhesion molecule 1 (ICAM-1), which retained little to no infectivity. Using an adenovirus transduction system, we demonstrate that CVA21 can remain infectious for up to 24 h after DAF binding and is capable of initiating a multicycle lytic infection upon delayed ICAM-1 surface expression. Taken together, the data suggest that a major role of DAF in cell infection by the prototype strain of CVA21 is to provide membrane concentration of infectious virions, effectively increasing viral interactions with endogenous or induced ICAM-1.     

Caspases mediate processing of the capsid precursor and cell release of human astroviruses

In this work we have shown that astrovirus infection induces apoptosis of Caco-2 cells, since fragmentation of cellular DNA, cleavage of cellular proteins which are substrate of activated caspases, and a change in the mitochondrial transmembrane potential occur upon virus infection. The human astrovirus Yuc8 polyprotein capsid precursor VP90 is initially processed to yield VP70, and we have shown that this processing is trypsin independent and occurs intracellularly through four cleavages at its carboxy-terminal region. We further showed that VP90-VP70 processing is mediated by caspases, since it was blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk), and it was promoted by the apoptosis inducer TNF-related apoptosis-inducing ligand (TRAIL). Although the cell-associated virus produced in the presence of these compounds was not affected, the release of infectious virus to the cell supernatant was drastically reduced in the presence of z-VAD-fmk and increased by TRAIL, indicating that VP90-VP70 cleavage is important for the virus particles to be released from the cell. This is the first report that describes the induction and utilization of caspase activity by a virus to promote processing of the capsid precursor and dissemination of the viral particles.     

Epstein-Barr virus lytic infection is required for efficient production of the angiogenesis factor vascular endothelial growth factor in lymphoblastoid cell lines

Although Epstein-Barr virus (EBV)-associated malignancies are primarily composed of cells with one of the latent forms of EBV infection, a small subset of tumor cells containing the lytic form of infection is often observed. Whether the rare lytically infected tumor cells contribute to the growth of the latently infected tumor cells is unclear. Here we have investigated whether the lytically infected subset of early-passage lymphoblastoid cell lines (LCLs) could potentially contribute to tumor growth through the production of angiogenesis factors. We demonstrate that supernatants from early-passage LCLs infected with BZLF1-deleted virus (Z-KO LCLs) are highly impaired in promoting endothelial cell tube formation in vitro compared to wild-type (WT) LCL supernatants. Furthermore, expression of the BZLF1 gene product in trans in Z-KO LCLs restored angiogenic capacity. The supernatants of Z-KO LCLs, as well as supernatants from LCLs derived with a BRLF1-deleted virus (R-KO LCLs), contained much less vascular endothelial growth factor (VEGF) in comparison to WT LCLs. BZLF1 gene expression in Z-KO LCLs restored the VEGF level in the supernatant. However, the cellular level of VEGF mRNA was similar in Z-KO, R-KO, and WT LCLs, suggesting that lytic infection may enhance VEGF translation or secretion. Interestingly, a portion of the vasculature in LCL tumors in SCID mice was derived from the human LCLs. These results suggest that lytically infected cells may contribute to the growth of EBV-associated malignancies by enhancing angiogenesis. In addition, as VEGF is a pleiotropic factor with effects other than angiogenesis, lytically induced VEGF secretion may potentially contribute to viral pathogenesis.     

Simian virus 40 prevents activation of M-phase-promoting factor during lytic infection.

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.     

The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55).

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.     

The CCAAT binding factor can mediate interactions between CONSTANS-like proteins and DNA

CONSTANS-Like (COL) proteins are plant-specific nuclear regulators of gene expression but do not contain a known DNA-binding motif. We tested whether a common DNA-binding protein can deliver these proteins to specific cis-acting elements. We screened for proteins that interact with two members of a subgroup of COL proteins. These COL proteins were Tomato COL1 (TCOL1), which does not seem to be involved in the control of flowering time, and the Arabidopsis thaliana CONSTANS (AtCO) protein which mediates photoperiodic induction of flowering. We show that the C-terminal plant-specific CCT (CO, CO-like, TIMING OF CAB EXPRESSION 1) domain of both proteins binds the trimeric CCAAT binding factor (CBF) via its HAP5/NF-YC component. Chromatin immunoprecipitation demonstrated that TCOL is recruited to the CCAAT motifs of the yeast CYC1 and HEM1 promoters by HAP5. In Arabidopsis, each of the three CBF components is encoded by several different genes that are highly transcribed. Under warm long days, high levels of expression of a tomato HAP5 (THAP5a) gene can reduce the flowering time of Arabidopsis. A mutation in the CCT domain of TCOL1 disrupts the interaction with THAP5 and the analogous mutation in AtCO impairs its function and delays flowering. CBFs are therefore likely to recruit COL proteins to their DNA target motifs in planta.     

Noncovalent interactions of the Apple 4 domain that mediate coagulation factor XI homodimerization

The Apple 4 (A4) domain of human plasma factor XI (FXI) was used to investigate the process of FXI noncovalent dimer formation. Recombinant 6-histidine-tagged A4 domain proteins were prepared utilizing a bacterial expression system. Purification was accomplished under denaturing conditions, followed by a refolding protocol to facilitate correct disulfide bond formation. Analysis of the A4 domain (C321S mutant) by size exclusion chromatography indicated the presence of a slowly equilibrating reversible monomer-dimer equilibrium. The elution profiles reveal highly symmetrical peaks for both dimeric and monomeric species with elution times that were highly reproducible for varying amounts of both the dimeric and monomeric species. The monomer-dimer equilibrium was found to be dependent upon changes in both pH and salt concentration. Under conditions approximating physiologic salt concentration and pH (20 mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH 7.4), it was determined that the monomer-dimer equilibrium was characterized by a dissociation constant (K(D)) value of 229 +/- 26 nm with a calculated Delta G value of 9.1 kcal/mol. This report identifies electrostatic contributions and the presence of a hydrophobic component that mediate interactions at the A4 domain interface. The rate of dissociation for the recombinant A4 domain C321S mutant was examined by monitoring the increase in 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt fluorescence under dissociating conditions, giving a value for a dissociation rate constant (k(off)) of 4.3 x 10(-3) s(-1).     

Endogenous association of decay-accelerating factor (DAF) with C4b and C3b on cell membranes

Decay-accelerating factor (DAF) is a membrane glycoprotein found on various cells that are in contact with complement. It inhibits the formation of the C3 convertases of the complement system, both the classic (C4b2a) and alternative (C3bBb) pathways. In this investigation, we used a homobifunctional cross-linking reagent to search for a DAF ligand on the surface of cells subjected to complement attack. We found that DAF forms complexes with C4b and C3b deposited on the same erythrocytes, but not with the physiologic degradation products of these complement fragments, that is, C4d or C3dg. Taken together with prior observations that DAF action is reversible, and DAF does not affect the structure of C4b or C3b, these findings suggest that DAF functions by competitively inhibiting the uptake of C2 or factor B, and preventing the assembly of the C3 convertases.     

Idiotype-specific T suppressor factor alternatively interacts with a nonspecific T-acceptor-like cell to mediate immune suppression

The ability of the idiotype (Id)-specific second-order T suppressor factor (TsF2) to interact with a final effector Ts cell type other than the previously reported third-order Ts (Ts3) subset was studied in the phenyltrimethylamino (TMA) hapten system. Hence, mice were primed with unrelated heterologous haptens to induce the nonspecific T acceptor (Tacc) cells following published procedures. When enriched T cell populations containing these nonspecific Ts were briefly incubated in vitro with TMA-TsF2, they produced suppression upon adoptive transfer into cyclophosphamide-treated mice which had been previously immunized for TMA-specific delayed-type hypersensitivity. Despite the fact that the effector population studied in this report also required Id-binding TsF2 for its function, it differs markedly from the Ts3 subset studied previously in the TMA system. First, the cell type studied herein could be easily generated with noncrossreacting heterologous chemically reactive haptens when applied directly to the skin of mice. Furthermore, these Ts effector cells had no detectable intrinsic receptors for homologous haptens and most importantly, unlike Ts3, this population had no affinity for the TMA hapten. Nevertheless, the nonspecifically induced Ts once activated by TsF2 suppresses TMA-directed, but not similar immune responses specific for heterologous haptens. Thus the results indicate that TsF2 can functionally interact with a final effector Ts subset (very similar to the Tacc) other than the well described Ts3 population. The ramifications of these findings are discussed with reference to a generalized view of the cellular basis of terminal phases of immune suppression.     

Structure stability of lytic peptides during their interactions with lipid bilayers.

In this work, molecular dynamics simulations were used to examine the consequences of a variety of analogs of cecropin A on lipid bilayers. Analog sequences were constructed by replacing either the N- or C-terminal helix with the other helix in native or reverse sequence order, by making palindromic peptides based on both the N- and C-terminal helices, and by deleting the hinge region. The structure of the peptides was monitored throughout the simulation. The hinge region appeared not to assist in maintaining helical structure but help in motion flexibility. In general, the N-terminal helix of peptides was less stable than the C-terminal one during the interaction with anionic lipid bilayers. Sequences with hydrophobic helices tended to regain helical structure after an initial loss while sequences with amphipathic helices were less able to do this. The results suggests that hydrophobic design peptides have a high structural stability in an anionic membrane and are the candidates for experimental investigation.     

De novo infection with rhesus monkey rhadinovirus leads to the accumulation of multiple intranuclear capsid species during lytic replication but favors the release of genome-containing virions

Rhesus monkey rhadinovirus (RRV) is one of the closest phylogenetic relatives to the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), yet it has the distinct experimental advantage of entering efficiently into lytic replication and growing to high titers in culture. RRV therefore holds promise as a potentially attractive model with which to study gammaherpesvirus structure and assembly. We have isolated RRV capsids, determined their molecular composition, and identified the genes encoding five of the main capsid structural proteins. Our data indicate that, as with other herpesviruses, lytic infection with RRV leads to the synthesis of three distinct intranuclear capsid species. However, in contrast to the inefficiency of KSHV maturation following reactivation from latently infected B-cell lines (K. Nealon, W. W. Newcomb, T. R. Pray, C. S. Craik, J. C. Brown, and D. H. Kedes, J. Virol. 75:2866-2878, 2001), de novo infection of immortalized rhesus fibroblasts with RRV results in the release of high levels of infectious virions with genome-containing C capsids at their center. Together, our findings argue for the use of RRV as a powerful model with which to study the structure and assembly of gammaherpesviruses and, specifically, the human rhadinovirus,KSHV.     

Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry.

It is becoming increasingly apparent that many viruses employ multiple receptor molecules in their cell entry mechanisms. The human enterovirus coxsackievirus A21 (CAV21) has been reported to bind to the N-terminal domain of intercellular adhesion molecule 1 (ICAM-1) and undergo limited replication in ICAM-1-expressing murine L cells. In this study, we show that in addition to binding to ICAM-1, CAV21 binds to the first short consensus repeat (SCR) of decay-accelerating factor (DAF). Dual antibody blockade using both anti-ICAM-1 (domain 1) and anti-DAF (SCR1) monoclonal antibodies (MAbs) is required to completely abolish binding and replication of high-titered CAV21. However, the binding of CAV21 to DAF, unlike that to ICAM-1, does not initiate a productive cell infection. The capacity of an anti-DAF (SCR3) MAb to block CAV21 infection but not binding, coupled with immunoprecipitation data from chemical cross-linking studies, indicates that DAF and ICAM-1 are closely associated on the cell surface. It is therefore suggested that DAF may function as a low-affinity attachment receptor either enhancing viral presentation or providing a viral sequestration site for subsequent high-affinity binding to ICAM-1.     

Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.