Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference

Crowell, Richard L. (Hahnemann Medical College, Philadelphia, Pa.). Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference. J. Bacteriol. 91:198-204. 1966.-Exposure of HeLa cells to high levels of coxsackievirus B3 produced cells which were refractory to attachment of coxsackievirus B1, whereas poliovirus T2 attached normally. Under similar conditions, poliovirus T2 was found to interfere with the attachment of poliovirus T1 to HeLa cells without affecting the attachment rate of coxsackievirus B3. The data confirm earlier findings that the receptor sites on HeLa cells, which bind members of group B coxsackieviruses, are distinct from those for polioviruses. Quantitatively, coxsackieviruses B1 and B3 were found to be mutually exclusive in the attachment interference assay to suggest that they compete for the same receptors on the HeLa cell surface. The finding that input multiplicities of B3 virus which exceeded 500 saturated the homologous viral receptors of HeLa cells was unexpected, but was consistent with the results of interference assays. Excessive amounts of input virus did not, however, inhibit eclipse of homologous cell-associated virus. Attachment interference between enteroviruses occurred even though the interfering virus was eclipsed prior to addition of challenge virus. The finding that enterovirus attachment interference was reversible with acid pH suggested that attachment and eclipse of enterovirus does not result in a permanent alteration of the cell membrane and that these events occur at the cell surface.     

Comparative Studies of the Regeneration of HeLa Cell Receptors for Poliovirus T1 and Coxsackievirus B3

Enterovirus receptors of live HeLa cells have been shown to reappear after inactivation by proteolytic enzymes, provided the cells are incubated at 37 C in a nutritionally adequate medium. Regeneration of receptor activity for poliovirus T1 occurred at a significantly faster rate than for coxsackievirus B3. The regenerative process for both types of receptors studied evidently required an active process of protein synthesis, since it was found that reappearance of receptor activity was inhibited by streptovitacin A, puromycin, and actinomycin D. Substitution of p-fluorophenylalanine for the naturally occurring amino acid, at concentrations which inhibited virus synthesis, was without effect on regeneration of receptor activity. It is anticipated that these findings will aid in the study of the biosynthesis and subsequent chemical characterization of viral receptors of living host cells.     

Flexibility of the adenovirus fiber is required for efficient receptor interaction

The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.     

Elution and uncoating of Coxsackievirus B3 by isolated HeLa cell plasma membranes.

Plasma membranes isolated from HeLa cells on discontinuous sucrose gradients were assayed for their capacity to elute and uncoat coxsackievirus B3 at 37 C. Because the viral receptors are limited to the surface of HeLa cells, the addition of radioactively labeled virus to the cells prior to cell homogenization provided a useful marker for locating the plasma membranes during the fractionation procedure. Four bands were formed on the discontinuous sucrose gradients with approximately 70% or more of the membrane-associated viral label being recovered in the most dense bands, designated as bands 3 and 4. Bands 3 and 4 also possessed the plasma membrane marker enzymes, Na+, K+ adenosine triphosphatase and 5'-nucleotidase and revealed typical structures characteristic of plasma membranes as revealed by electron microscopy. Pelleted and washed membranes from band 3 both eluted and uncoated B3 32P-labeled virus, whereas membranes from band 4 eluted virus but failed to uncoat it. The membranes from band 4 were shown to inhibit the viral uncoating activity when mixed with membranes of band 3. Characteristically, unfractionated homogenates of cell membranes eluted but did not uncoat virus. The finding of a naturally occurring inhibitor of virus uncoating provides for the first time a way to distinguish between the membrane activities of virus elution and virus uncoating. The inhibitor remains to be characterized.     

Human Astrocytic Cells Support Persistent Coxsackievirus B3 Infection

Enteroviruses can frequently target the human central nervous system to induce a variety of neurological diseases. Although enteroviruses are highly cytolytic, emerging evidence has shown that these viruses can establish persistent infections both in vivo and in vitro. Here, we investigated the susceptibility of three human brain cell lines, CCF-STTG1, T98G, and SK-N-SH, to infection with three enterovirus serotypes: coxsackievirus B3 (CVB3), enterovirus 71, and coxsackievirus A9. Persistent infection was observed in CVB3-infected CCF-STTG1 cells, as evidenced by prolonged detection of infectious virions, viral RNA, and viral antigens. Of note, infected CCF-STTG1 cells expressed the nonfunctional canonical viral receptors coxsackievirus-adenovirus receptor and decay-accelerating factor, while removal of cell surface chondroitin sulfate from CCF-STTG1 cells inhibited the replication of CVB3, suggesting that receptor usage was one of the major limiting factors in CVB3 persistence. In addition, CVB3 curtailed the induction of beta interferon in infected CCF-STTG1 cells, which likely contributed to the initiation of persistence. Furthermore, proinflammatory chemokines and cytokines, such as vascular cell adhesion molecule 1, interleukin-8 (IL-8), and IL-6, were upregulated in CVB3-infected CCF-STTG1 cells and human progenitor-derived astrocytes. Our data together demonstrate the potential of CCF-STTG1 cells to be a novel cell model for studying CVB3-central nervous system interactions, providing the basis toward a better understanding of CVB3-induced chronic neuropathogenesis.     

Monoclonal antibodies that inhibit attachment of group B coxsackieviruses.

Hybridoma cell lines that secrete monoclonal antibodies which react with HeLa cell surface antigens were produced. The monoclonal antibodies prevented cytopathic effects caused by coxsackievirus B1 and significantly reduced the amounts of coxsackieviruses B1, B5, and B6 that absorb to HeLa cells. These antibodies did not protect the cells from poliovirus cytopathic effects, and they had no effect on the attachment of other picornaviruses to HeLa cells.     

Human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and B7-H1 expression

Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.     

Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples

The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 degrees C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h.     

GP96 Interacts with HHV-6 during Viral Entry and Directs It for Cellular Degradation

CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus.     

Specific Alterations of Coxsackievirus B3 Eluted from Hela Cells

After the attachment of radioactive coxsackievirus B3 to HeLa cells at 0 C and subsequent incubation at 37 C, 50 to 80% of attached virus radioactivity was eluted from the cells within 1 hr. Eluted virus had a buoyant density of 1.21 in a potassium tartrate gradient, sedimented more slowly than native virus in sucrose gradients, was resistant to ribonuclease, was unstable in CsCl centrifugation, and did not reattach to uninfected cells. Electrophoretic studies of sodium dodecyl sulfate-disrupted B3 virus in sodium dodecyl sulfate-polyacrylamide gels revealed four radioactive virus polypeptides (VP 1 to 4), of which the three largest migrated slightly faster than their poliovirus T1 counterparts. In contrast, electrophoretic analysis of eluted virus, after banding in a tartrate gradient or pelleting by centrifugation, showed the absence of the fastest migrating polypeptide, VP 4. VP 4 was recovered in the supernatant fluid when the eluted virions were removed by high-speed centrifugation. The results indicate that VP 4 is located at the surface of the native virion, and its dissociation from the capsid may represent the first specific alteration of the virion after virus-receptor interaction at the cell surface.     

Soluble V domain of Nectin-1/HveC enables entry of herpes simplex virus type 1 (HSV-1) into HSV-resistant cells by binding to viral glycoprotein D

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1(123)), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1(123), approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1(123) did not associate with the cell surface. sNec1(123)-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.     

Coxsackievirus B3 infection alters plasma membrane of neonatal skin fibroblasts.

Replication of coxsackievirus B3 occurred for days in cultures of murine neonatal skin fibroblasts in the absence of cytopathology and resulted in alteration of the plasma membrane. Dual immunofluorescence studies showed that the lectin Ulex europaeus agglutinin I bound only to cells producing viral capsid antigens. Cultures of coxsackievirus B3-inoculated murine neonatal skin fibroblasts showed maximum binding of this lectin at 72 h postinoculation. These data show that in a nonlytic infection a picornavirus can alter the surface of an infected cell.     

Interaction with coxsackievirus and adenovirus receptor, but not with decay-accelerating factor (DAF), induces A-particle formation in a DAF-binding coxsackievirus B3 isolate

Although many coxsackie B viruses interact with decay accelerating factor (DAF), attachment to DAF by itself is not sufficient to initiate infection. We examined the early events in infection that follow virus interaction with DAF, and with the coxsackievirus and adenovirus receptor (CAR). Interaction with soluble CAR in a cell-free system, or with CAR on the surfaces of transfected cells, induced the formation of A particles; interaction with soluble or cell surface DAF did not. The results suggest that CAR, but not DAF, is capable of initiating the conformational changes in the viral capsid that lead to release of viral nucleic acid.     

Permeability to alpha sarcin in virus-infected cells

Summary Incubation of HeLa cells with Encephalomyocarditis virus (EMC) induces permeability of the cell membrane to protein toxins, such as alpha sarcin. To induce permeability to this toxin only 5 min incubation of cells with virus is needed. On the other hand, less than 1 min exposure of the susceptible cells to alpha sarcin produces maximal inhibition of protein synthesis. EMC virus treated with UV-light, although unable to replicate, can still induce the entrance of alpha sarcin into HeLa cells, but the virion loses this capacity after heating at 60 °C for 10 min. These findings suggest that an integral viral genome is not necessary to make the cells permeable to alpha sarcin, and that a virion protein might be involved in this phenomenon. Although human interferon prevents productive EMC infection, it does not affect the virus-induced entrance of alpha sarcin into the cells. The plasma membrane of cells that have been treated with virion particles can recover its initial lack of permeability to alpha sarcin after 2 h at 37 °C. Poliovirus modifies membrane permeability in human HeLa cells, but it has no effect on mouse L cells. This fact suggests that viral attachment to specific cell surface receptors is necessary to induce permeability, since receptors to poliovirus are only present in primate cells.     

The gp49B1 inhibitory receptor regulates the IFN-gamma responses of T cells and NK cells

The magnitude and diversity of Ag-specific T cell effector activity have been proposed to be controlled by an integration of positive signals transduced by the TCR and negative signals originating from inhibitory cell surface molecules. Although the lectin family of NK cell-associated inhibitory receptors has been reported to regulate the function of murine CTLs, gp49B1, the Ig superfamily member is not known to be expressed on T cells. Moreover, the consequences of the lack of an endogenously expressed NK cell-associated inhibitory receptor on T cell functions are not known. We report that gp49B1 is expressed by nearly all activated CD8 and CD4 T cells in addition to NK cells during an immune response to viral, bacterial, or tumor challenge. Kinetics of gp49B1 expression parallel functional capability and subside in the memory phase. Following vaccinia viral infection, IFN-gamma production by both subsets of T cells and NK cells is enhanced in gp49B1-deficient mice compared with gp49B1(+/+) mice. The stimulation threshold for IFN-gamma production is also lower in gp49B1-deficient T cells. In contrast, no significant differences were observed in the cytotoxic responses. We conclude that gp49B1 is a unique inhibitory receptor that is induced in multiple lineages of innate and adaptive immune cells during an infection and controls their IFN-gamma, but not cytotoxic responses.     

Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes

Adenovirus (Ad) fiber proteins are responsible for the initial attachment of the virion to the cell membrane. Most Ad vectors currently in use are based on the Ad type 5 (Ad5), which belong to subgroup C, and use the coxsackievirus and adenovirus receptors (CAR) as the initial receptor. Ad35, which belongs to subgroup B, recognizes unknown receptor(s) other than CAR. In this study, the feasibility of the Ad vector containing Ad5/35 chimeric fiber protein was examined in a wide variety of cell types, such as CAR-positive or -negative human tumor cells, rodent cells, and blood cells (a total of 20 cell types), and in mice in vivo. Transduction data suggested that the Ad vectors containing the Ad5/35 chimeric fiber protein exhibited altered and expanded tropism when compared with the Ad5-based vector. The chimeric vector also allows the packaging of larger foreign DNAs than the conventional Ad5-based vector, which can package approximately 8.1-8.2 kb of foreign DNA. The chimeric vector containing approximately 8.8 kb of foreign DNA was generated without affecting the viral growth rate and titer. These results suggested that inclusion of the Ad35 fiber protein into the Ad5-based vector could lead to an improved efficiency in gene therapy and in gene transfer experiments, especially for the cells lacking in sufficient CAR expression.     

The multiple facets of HIV attachment to dendritic cell lectins

Entry of enveloped viruses into host cells depends on the interactions of viral surface proteins with cell surface receptors. Many enveloped viruses maximize the efficiency of receptor engagement by first binding to attachment‐promoting factors, which concentrate virions on target cells and thus increase the likelihood of subsequent receptor engagement. Cellular lectins can recognize glycans on viral surface proteins and mediate viral uptake into immune cells for subsequent antigen presentation. Paradoxically, many viral and non‐viral pathogens target lectins to attach to immune cells and to subvert cellular functions to promote their spread. Thus, it has been proposed that attachment of HIV to the dendritic cell lectin DC‐SIGN enables the virus to hijack cellular transport processes to ensure its transmission to adjacent T cells. However, recent studies show that the consequences of viral capture by immune cell lectins can be diverse, and can entail negative and positive regulation of viral spread. Here, we will describe key concepts proposed for the role of lectins in HIV attachment to host cells, and we will discuss recent findings in this rapidly evolving area of research.     

Differences in the susceptibility of herpes simplex virus types 1 and 2 to modified heparin compounds suggest serotype differences in viral entry.

Although heparan sulfate (HS) serves as an initial receptor for the binding of both herpes simplex virus type 1 (HSV-1) and HSV-2 to cell surfaces, the two serotypes differ in epidemiology, cell tropism, and ability to compete for viral receptors in vitro. These observations are not necessarily contradictory and can be explained if the two serotypes recognize different structural features of HS. To compare the specific features of HS important for the binding and infection of HSV-1 and HSV-2, we took advantage of structural similarities between heparin and cell surface HS and compared the abilities of chemically modified heparin compounds to inhibit plaque formation. We found that the antiviral activity of heparin for both serotypes was independent of anticoagulant activity. Moreover, specific negatively charged regions of the polysaccharide, including N sulfations and the carboxyl groups, are key structural features for interactions of both HSV-1 and HSV-2 with cell surfaces since N desulfation or carboxyl reduction abolished heparin's antiviral activity. In contrast, 6-O sulfations and 2-,3-O sulfations are important determinants primarily for HSV- 1 infection. The O-desulfated heparins had little or no inhibitory effect on HSV-1 infection but inhibited HSV-2 infection. Using a series of intertypic recombinant mutant viruses, we found that susceptibility to O-desulfated heparins can be transferred to HSV-1 by the gene for glycoprotein C of HSV-2 (gC-2). This supports the notion that the envelope glycoproteins of HSV-1 and HSV-2 interact with different affinities for different structural features of heparin. To determine if the modified heparin compounds inhibited plaque formation by competing with cell surface HS for viral attachment, binding studies were also performed. As anticipated, most compounds inhibited binding and plaque formation in parallel. However, several compounds inhibited the binding of HSV-1 to cells during the initial attachment period at 4 degrees C; this inhibitory effect was reversed when the cells and inoculum were shifted to 37 degrees C. This temperature-dependent differential response to modified heparin compounds was evident primarily when glycoprotein C of HSV-1 (gC-1) was present in the virion envelope. Minimal temperature-dependent differences were seen for HSV-1 with gC-1 deleted and for HSV-2. These results suggest differences in the interactions of HSV-1 and HSV-2 with cell surface HS that may influence cell tropism.     

The class of surface immunoglobulin on virgin and memory B lymphocytes.

The class of surface immunoglobulin receptors for antigen on B cell precursors of different classes of antibody-forming cells was determined by utilizing a technique of class-specific antigen suicide. Spleen cells are first treated with a class-specific antiserum under conditions that result in the stripping of that class from the cell surface. The cells are then permitted to bind a highly radioactive trinitrophenyl (TNP)-conjugated protein, which leads to lethal irradiation of all TNP-specific B cells except those whose TNP receptors had been removed by the class-specific stripping of surface immunoglobulin. In this way, the class of antibody-forming cells resulting from TNP stimulation of B cells with different classes of surface immunoglobulin can be examined. It was found that the virgin B cell precursors of IgM-producing cells are two types: cells bearing IgM receptors only and those bearing both IgM and IgD receptors. All virgin B cells that gave rise to IgG1 antibody-forming cells had both IgM and IgD on their surfaces, demonstrating that an antigen-dependent switch from IgM and IgD to IgG1 production is a common feature of B cell maturation. In contrast, memory B cell precursors of IgG1 antibody-forming cells had predominantly IgG1 as their surface antigen receptor. The implications of these findings on current models of B cell maturation are analyzed.     

A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.