Vaccinia virus morphogenesis and dissemination

Vaccinia virus is the smallpox vaccine. It is the most intensively studied poxvirus, and its study has provided important insights about virus replication in general and the interactions of viruses with the host cell and immune system. Here, the entry, morphogenesis and dissemination of vaccinia virus are considered. These processes are complicated by the existence of two infectious vaccinia virus particles, called intracellular mature virus (IMV) and extracellular enveloped virus (EEV). The IMV particle is surrounded by one membrane, and the EEV particle comprises an IMV particle enclosed within a second lipid membrane containing several viral antigens. Consequently, these virions have different biological properties and play different roles in the virus life cycle.     

Activity of vaccinia virus-neutralizing antibody in the sera of smallpox vaccinees

Individuals vaccinated against smallpox maintain substantial antiviral antibody responses for many years after vaccination. In this study, we examined the ability of antiviral antibodies from 104 unique serum samples to neutralize the two infectious forms of vaccinia virus, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). While we found direct correlations between antiviral antibody titers and the ability to neutralize IMV and EEV, correlation with EEV neutralization was weaker. To determine factors that may influence more varied EEV neutralization within a vaccinated population, we asked the following questions. (1) Does vaccinia virus-neutralizing ability remain constant over time? (2) Do multiple vaccinations boost IMV and EEV neutralization activity? We found that serum from vaccinated individuals retained ability to neutralize EEV for a relatively long time, but there was a significant drop in EEV neutralization ability in the third decade after vaccination. While all vaccinees maintained some ability to neutralize IMV, a number of individuals lost the capacity to neutralize EEV. Interestingly, the ability to neutralize either virus form was not altered by the number of vaccinations received. Since it is likely that neutralizing antibodies against both IMV and EEV are required for maximal protective immunity, a loss of anti-EEV-neutralizing ability may warrant the revaccination of individuals who have been vaccinated >20 years ago, should widespread pre-event smallpox vaccination be instituted.     

Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.     

Glycosaminoglycans and sialylated glycans sequentially facilitate Merkel cell polyomavirus infectious entry

Merkel cell polyomavirus (MCV or MCPyV) appears to be a causal factor in the development of Merkel cell carcinoma, a rare but highly lethal form of skin cancer. Although recent reports indicate that MCV virions are commonly shed from apparently healthy human skin, the precise cellular tropism of the virus in healthy subjects remains unclear. To begin to explore this question, we set out to identify the cellular receptors or co-receptors required for the infectious entry of MCV. Although several previously studied polyomavirus species have been shown to bind to cell surface sialic acid residues associated with glycolipids or glycoproteins, we found that sialylated glycans are not required for initial attachment of MCV virions to cultured human cell lines. Instead, glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS), serve as initial attachment receptors during the MCV infectious entry process. Using cell lines deficient in GAG biosynthesis, we found that N-sulfated and/or 6-O-sulfated forms of HS mediate infectious entry of MCV reporter vectors, while CS appears to be dispensable. Intriguingly, although cell lines deficient in sialylated glycans readily bind MCV capsids, the cells are highly resistant to MCV reporter vector-mediated gene transduction. This suggests that sialylated glycans play a post-attachment role in the infectious entry process. Results observed using MCV reporter vectors were confirmed using a novel system for infectious propagation of native MCV virions. Taken together, the findings suggest a model in which MCV infectious entry occurs via initial cell binding mediated primarily by HS, followed by secondary interactions with a sialylated entry co-factor. The study should facilitate the development of inhibitors of MCV infection and help shed light on the infectious entry pathways and cellular tropism of the virus.     

Identification of Functional Domains in the 14-Kilodalton Envelope Protein (A27L) of Vaccinia Virus

The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vázquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126-10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacI operator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread.     

Incomplete but Infectious Vaccinia Virions Are Produced in the Absence of Oncolysis in Feline SCCF1 Cells

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.     

Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein.

There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV). To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation. The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene. Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation. By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions. Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface. The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion. These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane. By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.     

Vaccinia Virus Protein F12 Associates with Intracellular Enveloped Virions through an Interaction with A36

Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.     

Dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic HIV-1 infection

Understanding the interactions between human immunodeficiency virus type 1 (HIV-1) virions and antibodies (Ab) produced during acute HIV-1 infection (AHI) is critical for defining antibody antiviral capabilities. Antibodies that bind virions may prevent transmission by neutralization of virus or mechanically prevent HIV-1 migration through mucosal layers. In this study, we quantified circulating HIV-1 virion-immune complexes (ICs), present in approximately 90% of AHI subjects, and compared the levels and antibody specificity to those in chronic infection. Circulating HIV-1 virions coated with IgG (immune complexes) were in significantly lower levels relative to the viral load in acute infection than in chronic HIV-1 infection. The specificities of the antibodies in the immune complexes differed between acute and chronic infection (anti-gp41 Ab in acute infection and anti-gp120 in chronic infection), potentially suggesting different roles in immunopathogenesis for complexes arising at different stages of infection. We also determined the ability of circulating IgG from AHI to bind infectious versus noninfectious virions. Similar to a nonneutralizing anti-gp41 monoclonal antibody (MAb), purified plasma IgG from acute HIV-1 subjects bound both infectious and noninfectious virions. This was in contrast to the neutralizing antibody 2G12 MAb that bound predominantly infectious virions. Moreover, the initial antibody response captured acute HIV-1 virions without selection for different HIV-1 envelope sequences. In total, this study demonstrates that the composition of immune complexes are dynamic over the course of HIV-1 infection and are comprised initially of antibodies that nonselectively opsonize both infectious and noninfectious virions, likely contributing to the lack of efficacy of the antibody response during acute infection.     

Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo

An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L(-)) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L(-) mutant virus. IMV from the H3L(-) mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L(-) mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.     

Deletion of the vaccinia virus B5R gene encoding a 42-kilodalton membrane glycoprotein inhibits extracellular virus envelope formation and dissemination.

The structure, formation, and function of the virion membranes are among the least well understood aspects of vaccinia virus replication. In this study, we investigated the role of gp42, a glycoprotein component of the extracellular enveloped form of vaccinia virus (EEV) encoded by the B5R gene. The B5R gene was deleted by homologous recombination from vaccinia virus strains IHD-J and WR, which produce high and low levels of EEV, respectively. Isolation of recombinant viruses was facilitated by the insertion into the genome of a cassette containing the Escherichia coli gpt and lacZ genes flanked by the ends of the B5R gene to provide simultaneous antibiotic selection and color screening. Deletion mutant viruses of both strains formed tiny plaques, and those of the IHD-J mutant lacked the characteristic comet shape caused by release of EEV. Nevertheless, similar yields of intracellular infectious virus were obtained whether cells were infected with the B5R deletion mutants or their parental strains. In the case of IHD-J, however, this deletion severely reduced the amount of infectious extracellular virus. Metabolic labeling studies demonstrated that the low extracellular infectivity corresponded with a decrease in EEV particles in the medium. Electron microscopic examination revealed that mature intracellular naked virions (INV) were present in cells infected with mutant virus, but neither membrane-wrapped INV nor significant amounts of plasma membrane-associated virus were observed. Syncytium formation, which occurs in cells infected with wild-type WR and IHD-J virus after brief low-pH treatment, did not occur in cells infected with the B5R deletion mutants. By contrast, syncytium formation induced by antibody to the viral hemagglutinin occurred, suggesting that different mechanisms are involved. When assayed by intracranial injection into weanling mice, both IHD-J and WR mutant viruses were found to be significantly attenuated. These findings demonstrate that the 42-kDa glycoprotein of the EEV is required for efficient membrane enwrapment of INV, externalization of the virus, and transmission and that gp42 contributes to viral virulence in strains producing both low and high levels of EEV.     

Susceptibility to duck hepatitis B virus infection is associated with the presence of cell surface receptor sites that efficiently bind viral particles.

To test the hypothesis that susceptibility of hepatocytes to duck hepatitis B virus (DHBV) infection requires cell surface receptors that bind the virus in a specific manner, we developed an assay for the binding of DHBV particles to monolayers of intact cells, using radiolabeled immunoglobulin G specific for DHBV envelope protein. Both noninfectious DHBV surface antigen particles and infectious virions bound to a susceptible fraction (approximately 60%) of Pekin duck hepatocytes. In contrast, binding did not occur to cells that were not susceptible to DHBV infection, including Pekin duck fibroblasts and chicken hepatocytes, and binding to Muscovy duck hepatocytes, which are only weakly susceptible (approximately 1% of cells) to DHBV infection, was virtually undetectable. Within a monolayer, individual Pekin duck hepatocytes appeared to differ markedly in the capacity to bind DHBV, which may explain difficulties that have been encountered in infecting 100% of cells in culture. We have also found that the loss of susceptibility to infection with DHBV that occurs when Pekin duck hepatocytes are maintained for more than a few days in culture correlates with a decline in the number of cells that bind virus particles efficiently. All of these results support the interpretation that the binding event detected by our assay is associated with the interaction between DHBV and specific cell surface receptors that are required for initiation of infection. Our assay may facilitate isolation and identification of hepatocyte receptors for this virus.     

Vaccinia Virus Intracellular Mature Virions Contain only One Lipid Membrane

Vaccinia virus (VV) morphogenesis commences with the formation of lipid crescents that grow into spherical immature virus (IV) and then infectious intracellular mature virus (IMV) particles. Early studies proposed that the lipid crescents were synthesized de novo and matured into IMV particles that contained a single lipid bilayer (S. Dales and E. H. Mosbach, Virology 35:564–583, 1968), but a more recent study reported that the lipid crescent was derived from membranes of the intermediate compartment (IC) and contained a double lipid bilayer (B. Sodiek et al., J. Cell Biol. 121:521–541, 1993). In the present study, we used high-resolution electron microscopy to reinvestigate the structures of the lipid crescents, IV, and IMV particles in order to determine if they contain one or two membranes. Examination of thin sections of Epon-embedded, VV-infected cells by use of a high-angular-tilt series of single sections, serial-section analysis, and high-resolution digital-image analysis detected only a single, 5-nm-thick lipid bilayer in virus crescents, IV, and IMV particles that is covered by a 8-nm-thick protein coat. In contrast, it was possible to discern tightly apposed cellular membranes, each 5 nm thick, in junctions between cells and in the myelin sheath of Schwann cells around neurons. Serial-section analysis and angular tilt analysis of sections detected no continuity between virus lipid crescents or IV particles and cellular membrane cisternae. Moreover, crescents were found to form at sites remote from IC membranes—namely, within the center of virus factories and within the nucleus—demonstrating that crescent formation can occur independently of IC membranes. These data leave unexplained the mechanism of single-membrane formation, but they have important implications with regard to the mechanism of entry of IMV and extracellular enveloped virus into cells; topologically, a one-to-one membrane fusion suffices for delivery of the IMV core into the cytoplasm. Consistent with this, we have demonstrated previously by confocal microscopy that uncoated virus cores within the cytoplasm lack the IMV surface protein D8L, and we show here that intracellular cores lack the surface protein coat and lipid membrane.     

Quantification of antibody responses against multiple antigens of the two infectious forms of Vaccinia virus provides a benchmark for smallpox vaccination

Smallpox was eradicated without an adequate understanding of how vaccination induced protection. In response to possible bioterrorism with smallpox, the UK government vaccinated approximately 300 health care workers with vaccinia virus (VACV) strain Lister. Antibody responses were analyzed using ELISA for multiple surface antigens of the extracellular enveloped virus (EEV) and the intracellular mature virus (IMV), plaque reduction neutralization and a fluorescence-based flow cytometric neutralization assay. Antibody depletion experiments showed that the EEV surface protein B5 is the only target responsible for EEV neutralization in vaccinated humans, whereas multiple IMV surface proteins, including A27 and H3, are targets for IMV-neutralizing antibodies. These data suggest that it would be unwise to exclude the B5 protein from a future smallpox vaccine. Repeated vaccination provided significantly higher B5-specific and thus EEV-neutralizing antibody responses. These data provide a benchmark against which new, safer smallpox vaccines and residual immunity can be compared.     

Role of cell-associated enveloped vaccinia virus in cell-to-cell spread.

The roles of intracellular naked (INV), cell-associated enveloped (CEV), and extracellular enveloped (EEV) forms of vaccinia virus in cell-to-cell and longer-range spread were investigated by using two closely related strains of vaccinia virus, WR and IHD-J. We confirmed previous results that WR and IHD-J produced similar amounts of INV and formed similar-size primary plaques but that IHD-J produced 10 to 40 times more EEV and spread to distant cells much more efficiently than did WR. Nevertheless, cells infected with WR and IHD-J had similar amounts of CEV, indicating that wrapping and transport of WR virions were unimpaired. A WR mutant with a deletion in VP37, the major outer envelope protein, formed normal amounts of INV; however, the generation of CEV was blocked and plaque formation was inhibited. These results suggested that CEV is the form of virus that mediates cell-to-cell spread. Marker rescue experiments indicated that the differences in EEV production by WR and IHD-J were not due to sequence differences in VP37. The low amount of WR EEV could be attributed to retention of CEV on the cell membrane. In support of this hypothesis, mild treatment with trypsin released as much or more infectious virus from cells infected with WR as it did with cells infected with IHD-J. Most of the virus released by trypsin sedimented with the buoyant density of EEV. Also, addition of trypsin to cells following inoculation with WR led to a comet-shaped distribution of secondary plaques characteristic of IHD-J. These results demonstrated that the release of CEV from the cell surface was limiting for extracellular virus formation and affirmed the role of EEV in long-range spread.     

Vaccinia virus entry into cells is dependent on a virion surface protein encoded by the A28L gene

The A28L gene of vaccinia virus is conserved in all poxviruses and encodes a protein that is anchored to the surface of infectious intracellular mature virions (IMV) and consequently lies beneath the additional envelope of extracellular virions. A conditional lethal recombinant vaccinia virus, vA28-HAi, with an inducible A28L gene, undergoes a single round of replication in the absence of inducer, producing IMV, as well as extracellular virions with actin tails, but fails to infect neighboring cells. We show here that purified A28-deficient IMV appeared to be indistinguishable from wild-type IMV and were competent to synthesize RNA in vitro. Nevertheless, A28-deficient virions did not induce cytopathic effects, express early genes, or initiate a productive infection. Although A28-deficient IMV bound to the surface of cells, their cores did not penetrate into the cytoplasm. An associated defect in membrane fusion was demonstrated by the failure of low pH to trigger syncytium formation when cells were infected with vA28-HAi in the absence of inducer (fusion from within) or when cells were incubated with a high multiplicity of A28-deficient virions (fusion from without). The correlation between the entry block and the inability of A28-deficient virions to mediate fusion provided compelling evidence for a relationship between these events. Because repression of A28 inhibited cell-to-cell spread, which is mediated by extracellular virions, all forms of vaccinia virus regardless of their outer coat must use a common A28-dependent mechanism of cell penetration. Furthermore, since A28 is conserved, all poxviruses are likely to penetrate cells in a similar way.     

Initial interaction of herpes simplex virus with cells is binding to heparan sulfate.

We have shown that cell surface heparan sulfate serves as the initial receptor for both serotypes of herpes simplex virus (HSV). We found that virions could bind to heparin, a related glycosaminoglycan, and that heparin blocked virus adsorption. Agents known to bind to cell surface heparan sulfate blocked viral adsorption and infection. Enzymatic digestion of cell surface heparan sulfate but not of dermatan sulfate or chondroitin sulfate concomitantly reduced the binding of virus to the cells and rendered the cells resistant to infection. Although cell surface heparan sulfate was required for infection by HSV types 1 and 2, the two serotypes may bind to heparan sulfate with different affinities or may recognize different structural features of heparan sulfate. Consistent with their broad host ranges, the two HSV serotypes use as primary receptors ubiquitous cell surface components known to participate in interactions with the extracellular matrix and with other cell surfaces.     

Herpes simplex virus type 1 and pseudorabies virus bind to a common saturable receptor on Vero cells that is not heparan sulfate.

Herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) infect different natural hosts but are very similar in structure, replicative cycle, and entry into cultured cells. We determined whether HSV-1 and PRV use the same cellular components during entry into Vero cells, which are highly susceptible to each virus but are not from native hosts for either. UV-inactivated virions of either HSV-1 or PRV could saturate cell surfaces to block infection of challenge HSV-1 or PRV. In the presence of saturating levels for infection of either virus, radiolabeled virus bound well and in a heparin-sensitive manner. This result shows that heparan sulfate proteoglycans on Vero cells are not the limiting cellular component. To identify the virus component required for blocking, we used an HSV-1 null mutant virus lacking gB, gD, or gH as blocking virus. Virions lacking gB were able to block infection of challenge virus to the same level as did virus containing gB. In contrast, virions lacking gD lost all and most of the ability to block infection of HSV-1 and PRV, respectively. HSV-1 lacking gH and PRV lacking gp50 also were less competent in blocking infection of challenge virus. We conclude that HSV-1 and PRV bind to a common receptor for infection of Vero cells. Although both viruses bind a heparin-like cell component on many cells, including Vero cells, they also attach to a different and limited cell surface component that is bound at least by HSV-1 gD and possibly gH and to some degree by PRV gp50 but not gB. These results clearly demonstrate binding of both HSV-1 and PRV to a common cell receptor that is not heparan sulfate and demonstrate that several types of attachment occur for both viruses during infectious entry.