Enhancement Of Virus Infection Research Articles

Utilization of Sialic Acid as a Coreceptor Enhances Reovirus Attachment by Multistep Adhesion Strengthening

Many serotype 3 reoviruses bind to two different host cell molecules, sialic acid and an unidentified protein, using discrete receptor-binding domains in viral attachment protein, final sigma1. To determine mechanisms by which these receptor-binding events cooperate to mediate cell attachment, we generated isogenic reovirus strains that differ in the capacity to bind sialic acid. Strain SA+, but not SA-, bound specifically to sialic acid on a biosensor chip with nanomolar avidity. SA+ displayed 5-fold higher avidity for HeLa cells when compared with SA-, although both strains recognized the same proteinaceous receptor. Increased avidity of SA+ binding was mediated by increased k(on). Neuraminidase treatment to remove cell-surface sialic acid decreased the k(on) of SA+ to that of SA-. Increased k(on) of SA+ enhanced an infectious attachment process, since SA+ was 50-100-fold more efficient than SA- at infecting HeLa cells in a kinetic fluorescent focus assay. Sialic acid binding was operant early during SA+ attachment, since the capacity of soluble sialyllactose to inhibit infection decreased rapidly during the first 20 min of adsorption. These results indicate that reovirus binding to sialic acid enhances virus infection through adhesion of virus to the cell surface where access to a proteinaceous receptor is thermodynamically favored.

Pretreatment with protease is a useful experimental strategy for enhancing adenovirus-mediated cancer gene therapy.

A key impediment to the development of effective virus-mediated gene therapy for cancer is the low level of gene transfer that occurs after the administration of recombinant viral vectors. Improving in vivo infection and transduction efficiency is an important goal for gene therapy. The limited distribution of gene delivery is particularly problematic when large vectors such as recombinant adenoviruses and retroviruses are used to mediate transgene delivery to solid tumors. To facilitate the spread of virus, we have investigated the potential of administering proteases prior to the intratumoral inoculation of recombinant replication deficient adenovirus. For these studies, we chose proteases that are active against collagen and the other extracellular matrix proteins found in primary brain tumor tissue, but are not widely expressed in normal brain. Various concentrations of a mixture of collagenase/dispase or trypsin were inoculated into xenografts of human glioblastoma multiforme-derived brain tumor cell lines U87, U251, and SF767. Subsequently, recombinant adenovirus encoding the beta-galactosidase gene was administered and tumor tissue was examined for evidence of virus infection. Both collagenase/dispase and trypsin enhanced virus infection, indicating that protease pretreatment may be a useful strategy for enhancing virus-mediated gene transduction for many in vivo applications.

Two Amino Acid Substitutions in the SIV Nef Protein Mediate Associations with Distinct Cellular Kinases

A functional Nef protein is crucial in vivo for viral replication leading to pathogenesis in SIV-infected macaques. Moreover, a full-length Nef protein is required for optimal virus replication in primary cells, and both HIV and SIV Nef proteins enhance virion infectivity. Enhanced infectivity may result in part from the ability of Nef to incorporate cellular kinases into virions. In two previous reports, we compared in vitro kinase profiles of SIV recombinant clones that express nef genes derived either from the prototypic lymphocyte-tropic SIVmac239, clone SIV/Fr-2, or from our neurovirulent clone SIV/17E-Fr. While the SIV/Fr-2 Nef protein associated with the previously described PAK-related kinase and an unidentified serine kinase present in a Nef-associated kinase complex (NAKC), SIV/17E-Fr Nef was found to associate with a novel serine kinase activity that was biochemically distinct from both PAK and NAKC. Interestingly, while both Nef proteins were incorporated into virus particles, Nef-associated kinase activity was detected only in virions containing the SIV/17E-Fr Nef protein. Because sequence analysis identified only five amino acids that differed between the Nef proteins of SIV/Fr-2 and SIV/17E-Fr, we were able to evaluate the contribution of each amino acid to Nef-associated kinase activity as well as virus infectivity by constructing a panel of SIV clones containing individual reversions of each differing amino acid in SIV/17E-Fr Nef to the corresponding amino acid in SIV/Fr-2 Nef. In this report, we identify previously uncharacterized amino acids in the N terminus and the conserved core domain of Nef that are essential for the detection of Nef/kinase interactions as well as Nef phosphorylation during SIV infection. Further, via a novel infectivity assay recently developed in our laboratory that utilizes CEMX174 reporter cells stably expressing an SIV/LTR-luciferase construct, we find no direct correlation between specific Nef kinase associations and enhanced virion infectivity.

Simian immunodeficiency virus containing mutations in N-terminal tyrosine residues and in the PxxP motif in Nef replicates efficiently in rhesus macaques.

SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a putative SH3-ligand domain (P(104)xxP(107)). In the present study, we investigated the effects of combined mutations in these tyrosine and proline residues on simian immunodeficiency virus (SIV) Nef interactions with the cellular signal transduction and endocytic machinery. We found that mutation of Y(28)F, Y(39)F, P(104)A, and P(107)A (FFAA-Nef) had little effect on Nef functions such as the association with the cellular tyrosine kinase Src, downregulation of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef variant showed high viral loads during the acute phase of infection. Reversions in the mutated prolines were observed between 12 and 20 weeks postinfection. Importantly, reversion of A(107)-->P, which restored the ability of Nef to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did not precede the development of a high viral load. The Y(28)/Y(39)-->F(28)/F(39) substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt major aspects of SIV Nef function in vivo.

Enhancement of adenovirus-mediated gene transfer into dermal fibroblasts in vitro and in vivo by polyethylene glycol 6000.

Gene therapy directed to the skin requires efficient transfer of the desired gene into cutaneous cells. In this study, we examined several chemical substances present in various ointments by enhancement of virus infectivity. The recombinant adenovirus vector, AxCALacZ, was used to infect dermal fibroblasts with some chemicals both in vitro and in vivo, and the expression of the LacZ gene was determined by X-Gal reaction. As the result, it was shown that PEG 6000 had the highest ability to enhance the exogenous gene expression in cultured fibroblasts with little toxicity. In vivo, it was also demonstrated that fibroblasts in mouse skin were efficiently gene transfer by adenovirus vector and 20% PEG 6000-treatment. These results suggest that this chemical treatment appears to be a simple, safe, convenient, and effective method for facilitating virus-mediated gene therapy in the skin.

Cell-dependent functional roles of HIV-1 Nef for virus replication (review).

The stage in the human immunodeficiency virus type 1 (HIV-1) replication cycle that is affected by Nef has been investigated. Nef is dispensable during the transcription to virion production step. In contrast, Nef is critical in the early phase of the virus replication cycle, from virion adsorption to integration. Furthermore, virions produced in the absence of Nef have been demonstrated to be inefficient to complete the virus DNA synthesis. When virions are produced in particular cells without Nef, they exhibit a severe defect in the virus entry process into cells. Taken together, it is concluded that HIV-1 Nef acts via modulation of viral particles to enhance virus infectivity in a cell-dependent manner.

Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway.

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.

Enhancement of human immunodeficiency virus type 1 infectivity by Nef is producer cell-dependent.

The growth kinetics of wild-type and nef mutant viruses of human immunodeficiency virus type 1 were comparatively analysed in several human CD4+ cell lines. Delayed replication of nef mutant virus was observed in all cell lines examined. To determine the stage in the virus replication cycle that is affected by Nef, a single-round replication assay was performed. Initially, the expression of marker genes in transfected cells was examined in order to study the role of Nef in the late phase of infection. The results obtained indicated that Nef is dispensable during the transcription to virion production stage. Next, the effect of Nef on the early phase was investigated with a single-round infection. It was demonstrated that Nef is required in the early phase of the virus replication cycle, from virion adsorption to integration. Finally, the infectivity of virus stocks prepared from four cell lines was determined. The relative infectivity of the nef mutant from the four cell lines differed. Taken together, we conclude that Nef acts via modulation of viral particles to enhance virus infectivity in a cell-dependent manner.

Enhanced in vitro and in vivo gene delivery using cationic agent complexed retrovirus vectors.

Retroviruses are, at present, the most efficient integrative vectors available for gene delivery. However, these viruses are still limited by relatively low titres. Although several protocols exist to improve virus titre most of them are time-consuming and unable to provide sufficient virus for in vivo applications. Virus titre can be enhanced by polybrene and other cationic agents. By investigating a broad range of cationic agents for their ability to enhance virus infectivity we found that both ecotropic and amphotropic retrovirus infection could be increased. In particular, the lipopolyamine dioctadecylamidoglycylspermine (DOGS) gave up to one order of magnitude enhancement above polybrene-mediated infection without cytotoxicity. To increase virus infectivity further we combined the enhancing effect of DOGS on virus infectivity with concentration of virus particles by ultrafiltration to reach titres of 1 x 10(9) IU/ml. The in vivo transduction of regenerating rat liver, by an amphotropic retrovirus was increased approximately five-fold by the addition of DOGS compared with virus alone. There was no animal toxicity observed following the administration of DOGS. The improved transduction efficiency seen both in vitro and in vivo following the co-administration of DOGS/virus complexes may be useful for future gene therapy applications.

The proteolytic cleavage of human immunodeficiency virus type 1 Nef does not correlate with its ability to stimulate virion infectivity.

The Nef protein of human immunodeficiency virus type 1 (HIV-1) promotes virion infectivity through mechanisms that are yet ill defined. Some Nef is incorporated into particles, where it is cleaved by the viral protease between amino acids 57 and 58. The functional significance of this event, which liberates the C-terminal core domain of the protein from its membrane-associated N terminus, is unknown. To address this question, we examined the modalities of Nef virion association and processing. We found that although significant levels of Nef were detected in HIV-1 virions partly in a cleaved form, cell-specific variations existed in the efficiency of Nef proteolytic processing. The virion association of Nef was strongly enhanced by myristoylation but did not require other HIV-1-specific proteins, since Nef was efficiently incorporated into and cleaved inside murine leukemia virus particles. Substituting alanine for tryptophan57 decreased the efficiency of Nef processing, while mutating leucine58 had little effect. In contrast, replacing both of these residues simultaneously almost completely prevented this process. However, when the resulting mutants were compared with a wild-type control in viral infectivity assays, no correlation was found between the levels of cleavage and the ability to stimulate virion infectivity. Furthermore, simian immunodeficiency virus Nef, which lacks the sequence recognized by the protease and as a consequence is not cleaved despite its incorporation into virions, could stimulate the infectivity of a nef-defective HIV-1 variant as efficiently as HIV-1 Nef. On these bases, we conclude that the proteolytic processing of Nef is not required for the ability of this protein to enhance virion infectivity.

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.

Human Immunodeficiency Virus Type I Nef Independently Affects Virion Incorporation of Major Histocompatibility Complex Class I Molecules and Virus Infectivity

We have recently reported that HIV-1 Nef down-regulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules. MHC-I molecules are one of the predominant cellular proteins associated with HIV-1 virions. Wild-type or nef-mutated HIV-1 virions were analyzed by immunoelectronic microscopy and Western blot for particle-associated MHC-I molecules. The number of MHC-I molecules was significantly higher in HIV-1 virions produced in the absence of Nef than in wild-type virions, indicating that Nef affects the incorporation of MHC-I molecules into virions. Wild-type HIV particles have been shown to be more infectious than Nef−viruses. This difference was maintained when Nef+and Nef−virions devoid of MHC-I molecules were produced in Daudi-CD4 cells. Therefore, the enhancement of virion infectivity and the down-regulation of MHC-I represent independent biological properties of Nef.

Induction of phosphorylation of human immunodeficiency virus type 1 Nef and enhancement of CD4 downregulation by phorbol myristate acetate.

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) encodes a 27 to 34 kDa myristoylated protein that induces downregulation of CD4 from the cell surface and enhances virus infectivity. As shown by experiments on SIV-infected adult macaques, Nef is important in pathogenesis and disease progression. In vitro, protein kinase C (PKC) phosphorylates Nef, but the role of phosphorylation in the function and expression of this protein has not yet been determined. Here we show that in HIV type 1-infected cells, phosphorylation of Nef increased 8- to 12-fold after treatment with phorbol myristate acetate and phytohemagglutinin (PMA/PHA). Basal and PMA/PHA-induced phosphorylation occurred on serine residues of Nef and was independent of other HIV proteins. The PMA/PHA-induced phosphorylation of Nef was inhibited by bisindolylmaleimide I, a potent and specific inhibitor of PKC, but was unaffected by H89, an inhibitor of protein kinase A. In contrast, treatment with bisindolylmaleimide I did not affect the basal level of Nef phosphorylation, suggesting two different phosphorylation pathways. A PMA-insensitive CD4 mutant in which three serine residues in the cytoplasmic domain have been replaced by alanines was used to determine whether PMA-induced phosphorylation affects Nef-induced CD4 downregulation. In Nef-expressing cells, treatment with PMA enhanced downregulation of the CD4 serine triple mutant from the cell surface, suggesting that phosphorylation is important for Nef function.

Regulated expression vectors demonstrate cell-type-specific sensitivity to human immunodeficiency virus type 1 Nef-induced cytostasis.

The nef gene product of both human and simian immunodeficiency viruses is critically important for virus replication and disease progression in vivo. However, the precise biological function of Nef remains poorly characterized in vitro, with previous reports suggesting that Nef might be either cytotoxic or cytostatic. As a result of difficulties encountered by several groups in establishing cell lines constitutively expressing Nef, we have developed two inducible systems resulting in stable Nef expression in various mammalian cell lines. Tetracycline-regulated Nef expression was achieved in HeLa cells but could not be established in human T cell lines. Jurkat E6-1 T cell and RAW264.7 murine macrophage cell lines expressing a regulated nef gene were generated using a system in which Nef expression was controlled by a mutated version of the heavy metal-inducible human metallothionein IIA promoter. Induction of high levels of Nef expression in HeLa-Nef and Jurkat-Nef cells resulted in a moderate (2-fold) and a dramatic (10-fold) retardation of cell growth respectively, supporting the contention that Nef may be a cytotoxic or cytostatic factor. This property was also observed at low basal levels of Nef expression in RAW264.7-Nef macrophage clones (5-fold reduction in growth) and was associated with an altered morphological phenotype suggesting that different cell types may be more susceptible to the cytostatic activity of Nef. The regulated Nef-expression systems provide tools for investigating the molecular basis of Nef function, including Nef-mediated cytopathogenicity, CD4 down-regulation and enhancement of virus infectivity.

5475090 Gene coded for a polypeptide which enhances virus infection of host insects : Granados Robert R; Hashimoto Yoshifumi Ithaca, NY, United States assigned to Boyce Thompson Institute for Plant Research

5475090 Gene coded for a polypeptide which enhances virus infection of host insects : Granados Robert R; Hashimoto Yoshifumi Ithaca, NY, United States assigned to Boyce Thompson Institute for Plant Research

HIV-1 Nef Association with Cellular Serine Kinase Correlates with Enhanced Virion Infectivity and Efficient Proviral DNA Synthesis

We previously reported on the association of Nef with a cellular serine kinase (E. T. Sawaiet al., Proc. Natl. Acad. Sci. USA91, 1539–1543, 1994). In the present study, we further define the Nef sequence requirements for this kinase association and investigate the effect of this kinase association on functions of HIV-1 Nef. We observe that, in addition to the membrane targeting signal and the conserved arg–arg residues within the core region, mutations in the proline-rich domain of Nef also affect its ability to associate with the serine kinase activity. The region encompassing the arg–arg residues of Nef is shown to be important for Nef-mediated cell-surface CD4 down-modulation as well as enhancement of viral growth properties. This is similar to what has previously been observed for the membrane targeting site at the N-terminus of Nef. In contrast, the proline-rich region of Nef is found to be involved in mediating efficient proviral DNA synthesis and the enhanced virion-infectivity function, but is not necessary for CD4 down-modulation by Nef. Thus, it appears that serine kinase association of Nef is necessary for efficient proviral DNA synthesis and for promotion of virion infectivity of Nef+viruses, but is dispensable for down-regulation of the CD4 receptor by Nef. These findings define three functional domains of Nef that are required for its interaction with the serine kinase activity and suggest that the cellular interaction events via the myristoylation and arg–arg regions of Nef lie upstream of the interaction event via the proline-rich domain.

Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell.

We have analyzed the replication of Nef+ and Nef- isogenic human immunodeficiency virus in CEM, HUT78, MT4 lymphoid, and U937 monocytic cell lines. At each passage of infected cells, we have assessed the relative infectivity of the virus particles released in culture media by measuring the number of infections units per nanogram of p24 protein. Values appeared to be 3- to 10-fold higher for the Nef+ virus than for the Nef- number The positive effect of Nef was observed regardless of the cell line, the multiplicity of infection, and the number of virus replication cycles achieved. We showed, by using cells expressing glycosylphosphatidylinositol-linked CD4, that the enhancement of virion infectivity could be dissociated from the down-regulation of cell surface CD4 also induced by Nef. The gp120-to-p24 ratio and the RNA content of virus particles produced in the presence or in the absence of Nef were equivalent. Virions bound to cell surface CD4 receptors with equal efficiencies. Equivalent reverse transcriptase activities were measured both on exogenous substrate and on particle genomic RNAs. In contrast, reverse transcription in infected cells generated 5- to 10-fold less DNA when the virions were produced in the absence of Nef, indicating that these particles performed reverse transcription in a suboptimal environment. These data suggest that the expression of Nef in virus-producing cells is required for efficient processing of the early stages of virus replication in target cells, including the internalization in an appropriate cell compartment and the uncoating of the particle.

Enhancement of infectivity of hantavirus in cell culture by centrifugation

Centrifugation was introduced during virus adsorption to Vero E6 cells to improve the infectivity of hantavirus. Centrifugal adsorption of a stock solution of Hantaan virus strain 76–118 to a monolayer of Vero E6 cells enhanced virus infectivity depending on the centrifugation time and the centrifugal force. The maximum level of infectivity (3.1 × 10 6 FFU/ml) was enhanced after a 2 h centrifugation at 671 × g, which was almost 9-times higher than that of conventional adsorption of the virus at 37°C for 1 h. Vero E6 cells were inoculated with a new hantavirus strain, KI-91-40, isolated with a low infectious titer (400 FFU/ml) from an urban rat and adsorbed by centrifugation. A higher virus titer was detected sooner compared to when using conventional adsorption. To analyze the mechanism of the enhancement, the centrifugation was carried out before and after virus adsorption. The infectivity was reduced when Vero E6 monolayers were centrifuged before virus inoculation. When the centrifugation proceeded after inoculation, the infectivity was almost equal to that without centrifugation. The infectivity was only enhanced when centrifugation was carried out during inoculation. These results indicate that centrifugation promotes a very early event of infection, probably attachment of the virus to cells.

Failure of Neutralizing Antibody to Regulate CAE Lentivirus Expression in Vivo

Sequential isolates of caprine arthritis-encephalitis lentivirus (CAEV) were obtained from clinically arthritic carpal joints of three Saanen goats between 36.4 and 44.9 months after infection with biologically cloned CAEV isolate 63. These virus isolates and the CAEV-63 inoculum were evaluated for neutralization by autologous sera. Neutralizing antibody to CAEV-63 persisted throughout the period of virus isolations. Five CAEV isolates from cell-free synovial fluid and six isolates from synovial fluid cells were neutralized by autologous sera at the time of virus isolation and up to 38.7 months prior to virus isolation. Comparison of serum neutralizing antibody titers for CAEV-63 and selected CAEV isolates demonstrated that neutralizing antibody responses were predominantly type-specific. Six virus isolates were resistant to neutralization by coincident autologous sera; however, neutralization assays using postisolation sera indicated that four of these isolates did not elicit detectable neutralizing antibody responses. Results indicate that (i) CAEV replication persists in the presence of neutralizing antibody and (ii) the neutralization phenotype of CAEV variants depends on the relative immunodominance of antigenically variable neutralization epitopes. We suggest that neutralizing antibody responses are a consequence of CAEV antigenic diversity and that variants are not selected based on resistance to neutralization. Enhancement of virus infectivity by nonneutralizing antibody is a possible mechanism of positive selection of antigenic variants.

Neutralization and infection-enhancement epitopes of influenza A virus hemagglutinin.

We studied 18 mAb specific for the H3 hemagglutinin (HA) to analyze the relationships between neutralizing and infection-enhancing epitopes on the influenza HA. The mAb could be separated into three groups based on their neutralization (N) and enhancement (E) activity in assays with the prototype virus; group I (N+E+), group II (N+E-) and group III (N +/- E+). A representative mAb from each group was analyzed for its effect on the infectivity of a group of escape mutants, selected with mAb to three sites on the H3 HA, and wild-type H3 viruses to define the relationship between neutralizing epitopes and infection-enhancing epitopes. A group I mAb (N+E+), which recognized site A on the HA, neutralized virus infection at high concentrations of antibody and enhanced virus infection at low concentrations. A group II mAb (N+E-), which recognized site B, had high neutralizing but no enhancing activity. The failure of this mAb to enhance virus uptake was a result of the inability of the Fc portion of virus-mAb complexes to bind to Fc receptor. The addition of anti-murine IgG as a second antibody to these virus-mAb complexes augmented virus uptake. A group III mAb (N +/- E+), which recognized site C, had enhancing but little neutralizing activity. This is the first definition of distinct epitopes that induce neutralizing and/or enhancing mAb.