Hemadsorption expressed by cloned H genes from subacute sclerosing panencephalitis (SSPE) viruses and their possible progenitor measles viruses isolated in Osaka, Japan

Most subacute sclerosing panencephalitis (SSPE) viruses, including our Osaka-1, -2, and -3 strains isolated in Osaka, have shown negative hemadsorption (HAD) by African green monkey red blood cells. This property has been thought to be characteristic of SSPE virus as compared to the positive reaction of the standard Edmonston strain of measles virus (MV). However, this assumption has become quite obscure because MV mutates frequently at the genetic level during its multiplication and also because recent field strains isolated by lymphoblastoid cell lines have shown negative HAD. To investigate the above issue, the nucleotide sequences of the hemagglutinin (H) genes from SSPE virus Osaka-1, -2, or -3 strains were compared to those of various MV field strains isolated in Osaka by Vero cells. The H gene sequences of three SSPE strains were relatively conserved without such biased hypermutation as had been observed in the matrix (M) gene of three SSPE strains. However, this analysis of the H gene sequence of the SSPE viruses enabled us to deduce possible progenitor MVs, which are in agreement with the deduction from the M gene analysis we reported previously. The HAD of Vero cells transfected with the cloned H cDNAs from the SSPE strains and their progenitors suggested that negative HAD of the SSPE viruses has been maintained as one of original properties of the progenitor MVs rather than having been acquired as an altered one during long-term persistent infection in the brains of patients with SSPE.     

A Recombinant Measles Vaccine Virus Expressing Wild-Type Glycoproteins: Consequences for Viral Spread and Cell Tropism

Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism.     

Moesin Is Not a Receptor for Measles Virus Entry into Mouse Embryonic Stem Cells

The involvement of moesin in measles virus (MV) entry was investigated with moesin-positive and -negative mouse embryonic stem (ES) cells. MV infection of these cells was very ineffective and was independent of moesin expression. Furthermore, when these cells were transfected to express human CD46, a 100-fold increase in syncytium formation was observed with these cells and was independent of the expression of moesin. The only obvious difference between moesin-positive and -negative ES cells was the shape of the syncytia formed. Moesin-negative ES cells expressing or not expressing human CD46 formed separate pieces of fragmented syncytia which were torn apart during spreading, whereas ES cells expressing moesin exhibited typical syncytia. In addition, moesin was not detected on the surface of any murine cells or cell lines that we have tested by a flow cytometric assay with moesin-specific antibodies. These findings indicate that murine moesin is neither a receptor nor a CD46 coreceptor for MV entry into mouse ES cells. Moesin is involved in actin filament-plasma membrane interactions as a cross-linker, and it affects only the spreading and shape of MV-mediated syncytia.     

Efficiency of measles virus entry and dissemination through different receptors

The efficiency with which different measles virus (MV) strains enter cells through the immune cell-specific protein SLAM (CD150) or other receptors, including the ubiquitous protein CD46, may influence their pathogenicity. We compared the cell entry efficiency of recombinant MV differing only in their attachment protein hemagglutinin (H). We constructed these viruses with an additional gene expressing an autofluorescent reporter protein to allow direct detection of every infected cell. A virus with a wild-type H protein entered cells through SLAM two to three times more efficiently than a virus with the H protein of the attenuated strain Edmonston, whereas cell entry efficiency through CD46 was lower. However, these subtle differences were amplified at the cell fusion stage because the wild-type H protein failed to fuse CD46-expressing cells. We also proved formally that a mutation in H protein residue 481 (asparagine to tyrosine) results in improved CD46-specific entry. To define the selective pressure exerted on that codon, we monitored its evolution in different H protein backgrounds and found that several passages in CD46-expressing Vero cells were necessary to shift it in the majority of the MV RNA. To verify the importance of these observations for human infections, we examined MV entry into peripheral blood mononuclear cells and observed that viruses with asparagine 481 H proteins infect these cells more efficiently.     

Measles virus infects both polarized epithelial and immune cells by using distinctive receptor-binding sites on its hemagglutinin

Measles is one of the most contagious human infectious diseases and remains a major cause of childhood morbidity and mortality worldwide. The signaling lymphocyte activation molecule (SLAM), also called CD150, is a cellular receptor for measles virus (MV), presumably accounting for its tropism for immune cells and its immunosuppressive properties. On the other hand, pathological studies have shown that MV also infects epithelial cells at a later stage of infection, although its mechanism has so far been unknown. In this study, we show that wild-type MV can infect and produce syncytia in human polarized epithelial cell lines independently of SLAM and CD46 (a receptor for the vaccine strains of MV). Progeny viral particles are released exclusively from the apical surface of these polarized epithelial cell lines. We have also identified amino acid residues on the MV attachment protein that are likely to interact with a putative receptor on epithelial cells. All of these residues have aromatic side chains and may form a receptor-binding pocket located in a different position from the putative SLAM- and CD46-binding sites on the MV attachment protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells by using distinctive receptor-binding sites on the attachment protein corresponding to each of their respective receptors. The ability of MV to infect polarized epithelial cells and its exclusive release from the apical surface may facilitate its efficient transmission via aerosol droplets, resulting in its highly contagious nature.     

A human lung carcinoma cell line supports efficient measles virus growth and syncytium formation via a SLAM- and CD46-independent mechanism

Measles virus (MV) propagates mainly in lymphoid organs throughout the body and produces syncytia by using signaling lymphocyte activation molecule (SLAM) as a receptor. MV also spreads in SLAM-negative epithelial tissues by unknown mechanisms. Ubiquitously expressed CD46 functions as another receptor for vaccine strains of MV but not for wild-type strains. We here show that MV grows and produces syncytia efficiently in a human lung adenocarcinoma cell line via a SLAM- and CD46-independent mechanism using a novel receptor-binding site on the hemagglutinin protein. This infection model could advance our understanding of MV infection of SLAM-negative epithelial cells and tissues.     

Ablation of Nectin4 Binding Compromises CD46 Usage by a Hybrid Vesicular Stomatitis Virus/Measles Virus

Measles virus (MV) immunosuppression is due to infection of SLAM-positive immune cells, whereas respiratory shedding and virus transmission are due to infection of nectin4-positive airway epithelial cells. The vaccine lineage MV strain Edmonston (MV-Edm) acquired an additional tropism for CD46 which is the basis of its oncolytic specificity. VSVFH is a vesicular stomatitis virus (VSV) encoding the MV-Edm F and H entry proteins in place of G. The virus spreads faster than MV-Edm and is highly fusogenic and a potent oncolytic. To determine whether ablating nectin4 tropism from VSVFH might prevent shedding, increasing its safety profile as an oncolytic, or might have any effect on CD46 binding, we generated VSVFH viruses with H mutations that disrupt attachment to SLAM and/or nectin4. Disruption of nectin4 binding reduced release of VSVFH from the basolateral side of differentiated airway epithelia composed of Calu-3 cells. However, because nectin4 and CD46 have substantially overlapping receptor binding surfaces on H, disruption of nectin4 binding compromised CD46 binding and greatly diminished the oncolytic potency of these viruses on human cancer cells. Thus, our results support continued preclinical development of VSVFH without ablation of nectin4 binding.     

Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 envelope protein.

The CC chemokine receptors CCR5, CCR2, and CCR3 and the CXC chemokine receptor CXCR4 have been implicated as CD4-associated cofactors in the entry of primary and cell line-adapted human immunodeficiency virus type 1 (HIV-1) strains. CXCR4 is also a receptor for T-cell-line-adapted, CD4-independent strains of HIV-2. With the exception of this latter example, little has been reported on the entry cofactors used by HIV-2 strains. Here we show that a CD4-dependent, T-cell-line-adapted HIV-2 strain uses CXCR4 and, to a lesser extent, CCR3 for fusion with and infectious entry into cells. In a cell-to-cell fusion assay, the envelope protein of this virus can utilize a wider repertoire of chemokine receptors to induce fusion. These include CCR1, CCR2, CCR3, CCR4, CCR5, CXCR2, and CXCR4. Kinetic analysis indicated that cell lines expressing the receptors that support infection, CXCR4 and CCR3, form syncytia more rapidly than do cell lines expressing the other receptors. Nevertheless, although less efficient, fusion with CXCR2 expressing cells was specific, since it was inhibited by antibodies against CXCR2. The extensive use of chemokine receptors in cell-to-cell fusion has implications for understanding the molecular basis of CD4-chemokine receptor-induced lentivirus fusion and may have relevance for syncytium formation and the direct cell-to-cell transfer of virus in vivo.     

SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein

Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.     

Single-chain antibody displayed on a recombinant measles virus confers entry through the tumor-associated carcinoembryonic antigen

To redirect the tropism of the vaccine strain of measles virus (MV), Edmonston B, to a targeted cell population, we displayed on the viral hemagglutinin (H) a single-chain antibody (scAb) specific for the tumor-associated carcinoembryonic antigen (CEA). We generated H fusion proteins with three forms of the scAb appended, differing in the lengths of the linkers separating the VH and VL domains and thus in the oligomerization states of the scAbs. All proteins were stable, appeared properly folded, and were transported to the cell surface, but only H displaying the long-linker form of scAb was functional in supporting cell-cell fusion. This protein induced extensive syncytia in cells expressing the normal virus receptor CD46 and also in CD46-negative cells expressing the targeted receptor, human CEA. Replication-competent MV with H replaced by H displaying the long-linker form of scAb was recovered and replicated efficiently in both CD46-positive and CD46-negative, CEA-positive cells. Thus, MV not only tolerates the addition of a scAb on its H protein but also infects cells via a novel interaction between the scAb and its targeted receptor.     

cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor

DC-SIGN is a C-type lectin expressed on dendritic cells and restricted macrophage populations in vivo that binds gp120 and acts in trans to enable efficient infection of T cells by human immunodeficiency virus type 1 (HIV-1). We report here that DC-SIGN, when expressed in cis with CD4 and coreceptors, allowed more efficient infection by both HIV and simian immunodeficiency virus (SIV) strains, although the extent varied from 2- to 40-fold, depending on the virus strain. Expression of DC-SIGN on target cells did not alleviate the requirement for CD4 or coreceptor for viral entry. Stable expression of DC-SIGN on multiple lymphoid lines enabled more efficient entry and replication of R5X4 and X4 viruses. Thus, 10- and 100-fold less 89.6 (R5/X4) and NL4-3 (X4), respectively, were required to achieve productive replication in DC-SIGN-transduced Jurkat cells when compared to the parental cell line. In addition, DC-SIGN expression on T-cell lines that express very low levels of CCR5 enabled entry and replication of R5 viruses in a CCR5-dependent manner, a property not exhibited by the parental cell lines. Therefore, DC-SIGN expression can boost virus infection in cis and can expand viral tropism without affecting coreceptor preference. In addition, coexpression of DC-SIGN enabled some viruses to use alternate coreceptors like STRL33 to infect cells, whereas in its absence, infection was not observed. Immunohistochemical and confocal microscopy data indicated that DC-SIGN was coexpressed and colocalized with CD4 and CCR5 on alveolar macrophages, underscoring the physiological significance of these cis enhancement effects.     

Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins

The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVDeltaG*). MV glycoproteins were efficiently incorporated into VSVDeltaG*, producing the VSV pseudotypes. VSVDeltaG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVDeltaG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVDeltaG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVDeltaG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role.     

Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence

Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.     

Coexistence of fusion receptors for human T-cell leukemia virus type-I (HTLV-I) and human immunodeficiency virus type-1 (HIV-1) on MOLT-4 cells.

Human immunodeficiency virus type-1 (HIV-1) and human T-cell leukemia virus type-I (HTLV-I) have a similar tropism for target cell types, especially for CD4+ T cells. In this study, we provide evidence that receptors of these two viruses exist independently on the target cell. We established an HTLV-I-producing CD8+ T cell line (ILT-8M2) with a remarkable cell fusion capacity. When cocultured with MOLT-4 cells, ILT-8M2 cells induced giant syncytia more efficiently than any other tested HTLV-I-producer cell lines. In contrast to other HTLV-I-producers, ILT-8M2 cells were minimally susceptible to cytopathic effects of HIV-1 due to very low expression of CD4, although they were able to be persistently infected by HIV-1. The indicator MOLT-4 cells are known to respond well to HIV-1-induced cell fusion, but they lose this ability if they become persistently infected with HIV-1 because of the reduction of CD4 receptor expression. ILT-8M2 was, however, still capable of inducing syncytia with the MOLT-4 cells persistently infected by HIV-1 (MOLT-4/IIIB). This syncytium formation was dependent on the HTLV-I-envelope, as it was inhibited by HTLV-I-positive human sera or a monoclonal antibody to HTLV-I gp46 but not by monoclonal antibodies to HIV-1 gp120 or CD4. Moreover, ILT-8M2 cells persistently infected by HIV-1 (ILT-8M2/IIIB) induced both HTLV-I- and HIV-1-mediated syncytia with uninfected MOLT-4 cells. These results suggest that HTLV-I induces cell fusion utilizing receptors on the target cells independent of HIV-1-receptors.     

Recent topics related to human herpesvirus 6 cell tropism

Human herpesvirus 6 (HHV-6) is a T lymphotropic herpes virus that is categorized into two variants, A (HHV-6A) and B (HHV-6B), on the basis of distinct genetic, immunological and biological characteristics. HHV-6 uses human CD46 as a cellular receptor. Without viral replication, HHV-6A induces cell–cell fusion between cells expressing human CD46. Some HHV-6B strains can also induce CD46-mediated cell–cell fusion. A multiple glycoprotein complex composed of glycoprotein (g) H-gL complexed with gQ1 and gQ2 has been identified, and found to be a viral ligand for the human CD46 receptor. Moreover, a novel complex consisting of gH/gL/gO, which does not associate with CD46, has also been identified. The evidence suggests that an additional receptor for HHV-6B or both variants may play a role in determining the cell tropism of this virus. Finally, cholesterol in the HHV-6 envelope and plasma membrane of the host cells plays an important role in HHV-6 entry, although how this function relates to cell–envelope fusion remains to be elucidated.     

Failure of human immunodeficiency virus entry and infection in CD4-positive human brain and skin cells.

CD4 molecules on human cells function as a major receptor for human immunodeficiency virus (HIV); however, certain CD4-negative cell types may also be susceptible to infection. Therefore, we attempted to quantitate the relationship between HIV infection and CD4 expression on human cell lines before and after introduction of the CD4 gene by using a retrovirus vector. Prior to introduction of the CD4 expression vector, low levels of HIV infection were detected by a sensitive focal immunoassay on all three cell types studied. With several HIV strains in clones of human cervical carcinoma (HeLa) cells expressing different levels of CD4, HIV titer increased with increasing CD4 expression. In contrast, in squamous cell carcinoma cells (SCL1) and astroglial cells (U87MG), even high levels of CD4 expression failed to augment HIV infection. The CD4 protein expressed in these two cell lines had the expected molecular weight and was capable of binding HIV virions. However, in contrast to CD4-positive HeLa cells, CD4-positive U87MG and SCL1 cells were unable to form syncytia when cultured with cells expressing HIV envelope protein. Thus, the inability of HIV to infect these cells appeared to be due to lack of fusion between HIV virion envelope proteins and CD4-positive cell membranes. This block is infectivity was overcome when cells were infected with HIV which was pseudotyped with the envelope protein of amphotropic murine leukemia virus. Thus, in addition to CD4, other cell surface molecules appear to be required for successful HIV entry into and infection of these two human cell lines.     

Inhibition of human immunodeficiency virus fusion by a monoclonal antibody to a coreceptor (CXCR4) is both cell type and virus strain dependent.

CXCR4 (also termed fusin, LESTR, or HUMSTR) is a member of the G-protein-coupled chemokine receptor family with seven membrane-spanning domains. CXCR4 acts as a coreceptor for syncytium-inducing human immunodeficiency virus type 1 (HIV-1) strains, conferring entry into CD4+ cells. We show here that a novel mouse monoclonal antibody (12G5) that recognizes CXCR4 blocked cell-to-cell fusion and cell free-virus infection of CXCR4+ CD4+ RD rhabdomyosarcoma cells by seven HIV-1 and HIV-2 strains that had various cell tropisms for different CD4+ human cell types. Yet the majority of the members of the same virus panel resisted 12G5 inhibition on T-cell lines. When inhibition was observed on these cell types, it was both cell type and virus strain dependent. In at least one situation, 12G5 failed to block LAI infection of cells expressing CXCR4 as the only available coreceptor. Our observations suggest that CXCR4 could be processed or presented differently depending on the cell type, allowing some strains to evade 12G5 inhibition. Alternatively, since several of the viruses could infect certain CXCR4- CD4+ cell lines, it is conceivable that alternative coreceptors are active, enabling individual HIV strains to choose between compatible coreceptors during entry into cells. Moreover, the strain dependency of 12G5 inhibition implies that the interaction of different HIVs with CXCR4 varies.     

Human immunodeficiency virus type 1 membrane fusion mediated by a laboratory-adapted strain and a primary isolate analyzed by resonance energy transfer.

Previous studies of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein-mediated membrane fusion have focused on laboratory-adapted T-lymphotropic strains of the virus. The goal of this study was to characterize membrane fusion mediated by a primary HIV-1 isolate in comparison with a laboratory-adapted strain. To this end, a new fusion assay was developed on the basis of the principle of resonance energy transfer, using HeLa cells stably transfected with gp120/gp41 from the T-lymphotropic isolate HIV-1LA1 or the macrophage-tropic primary isolate HIV-1JR-FL. These cells fused with CD4+ target cell lines with a tropism mirroring that of infection by the two viruses. Of particular note, HeLa cells expressing HIV-1JR-FL gp120/gp41 fused only with PM1 cells, a clonal derivative of HUT 78, and not with other T-cell or macrophage cell lines. These results demonstrate that the envelope glycoproteins of these strains play a major role in mediating viral tropism. Despite significant differences exhibited by HIV-1JR-FL and HIV-1LAI in terms of tropism and sensitivity to neutralization by CD4-based proteins, the present study found that membrane fusion mediated by the envelope glycoproteins of these viruses had remarkably similar properties. In particular, the degree and kinetics of membrane fusion were similar, fusion occurred at neutral pH and was dependent on the presence of divalent cations. Inhibition of HIV-1JR-FL envelope glycoprotein-mediated membrane fusion by soluble CD4 and CD4-IgG2 occurred at concentrations similar to those required to neutralize this virus. Interestingly, higher concentrations of these agents were required to inhibit HIV-1LAI envelope glycoprotein-mediated membrane fusion, in contrast to the greater sensitivity of HIV-1LAI virions to neutralization by soluble CD4 and CD4-IgG2. This finding suggests that the mechanisms of fusion inhibition and neutralization of HIV-1 are distinct.