Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly

Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites.     

The cytoplasmic domain of alphavirus E2 glycoprotein contains a short linear recognition signal required for viral budding.

Intracellular alphavirus nucleocapsids express a binding site for the cytoplasmic domain of the viral E2 spike glycoprotein. This binding site is recognized by the anti-idiotype monoclonal antibody, F13. The monoclonal anti-anti-idiotype antibody, raised against F13 and designated 3G10, recognizes the carboxy-terminal eight residues of the E2 cytoplasmic domain in Semliki Forest virus (SFV), identifying this as the signal for nucleocapsid interaction. F13 binding to cells infected with SFV or a second alphavirus, Sindbis virus, is inhibited by a synthetic peptide corresponding to the entire 31 residue cytoplasmic domain (E2c), and also by a synthetic peptide corresponding to the eight residue epitope recognized by 3G10. Both E2c and the eight residue peptide inhibited viral budding in microinjection experiments and when conjugated to colloidal gold are bound specifically to nucleocapsids in infected cells. These results identify a short linear signal in the E2 cytoplasmic domain required for the interaction with nucleocapsids which leads to budding of at least two alphaviruses from infected cells.     

Epitope-specific antibody response to murine hepatitis virus-4 (strain JHM)

Monoclonal hybridoma antibodies to the structural proteins of murine hepatitis virus-4, strain JHM (MHV-4) were used in a competition binding enzyme immunoassay to analyze at the epitope level the antibody response of mice after infection with MHV-4. Colonized mice often had pre-existing MHV antibodies directed against epitopes on the E2 glycoprotein, the E1 glycoprotein, and the nucleocapsid protein. These mice generated a secondary antibody response after virus inoculation, reaching peak levels 7 days after infection. In contrast, Nude/+ mice raised in a pathogen-free colony had no detectable circulating MHV antibodies and generated a primary antibody response which gradually increased to peak levels 14 to 28 days after infection. Kinetics of antibody responses against specific epitopes usually correlated well with measured total virus-specific antibody responses, but variation was observed. Mice injected with three antigenically distinct strains of MHV made antibody responses to conserved epitopes but not to an antigenic determinant absent in these strains. Measurement of epitope-specific responses in a polyclonal population of viral specific antibodies is feasible and a valuable adjunct in understanding viral immunity.     

Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants.

Baculovirus and vaccinia virus vectors were used to express the small (S) and medium (M) genome segments of Hantaan virus. Expression of the complete S or M segments yielded proteins electrophoretically indistinguishable from Hantaan virus nucleocapsid protein or envelope glycoproteins (G1 and G2), and expression of portions of the M segment, encoding either G1 or G2 alone, similarly yielded proteins which closely resembled authentic Hantaan virus proteins. The expressed envelope proteins retained all antigenic sites defined by a panel of monoclonal antibodies to Hantaan virus G1 and G2 and elicited antibodies in animals which reacted with authentic viral proteins. A Hantaan virus infectivity challenge model in hamsters was used to assay induction of protective immunity by the recombinant-expressed proteins. Recombinants expressing both G1 and G2 induced higher titer antibody responses than those expressing only G1 or G2 and protected most animals from infection with Hantaan virus. Baculovirus recombinants expressing only nucleocapsid protein also appeared to protect some animals from challenge. Passively transferred neutralizing monoclonal antibodies similarly prevented infection, suggesting that an antibody response alone is sufficient for immunity to Hantaan virus.     

人源中和性抗汉滩病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面表达技术,获得人源和中性抗滩滩病毒汉滩型Ｇ１基因工程单克隆抗体Ｆａｂ段基因及其表达,并同时获得抗汉滩病毒核蛋白的Ｆａｂ抗体。从能综合征出血热疫区恢复期病人抗凝血中分离到的外周淋巴细胞中,提取了部细胞ＲＮＡ。通过ＲＴ－ＰＣＲ方法,用一组人ＩｇＧ Ｆａｂ基因特异性引物,从合成了ｃＤＮＡ中经ＰＣＲ扩增了一组轻链和重链Ｆａｂ段基因,将轻链和重链先后插入噬菌体载体ｐＣｏｍｂ３,ｄｎａｌｆ ｖｆ     

Requirements for budding of paramyxovirus simian virus 5 virus-like particles

Enveloped viruses are released from infected cells after coalescence of viral components at cellular membranes and budding of membranes to release particles. For some negative-strand RNA viruses (e.g., vesicular stomatitis virus and Ebola virus), the viral matrix (M) protein contains all of the information needed for budding, since virus-like particles (VLPs) are efficiently released from cells when the M protein is expressed from cDNA. To investigate the requirements for budding of the paramyxovirus simian virus 5 (SV5), its M protein was expressed in mammalian cells, and it was found that SV5 M protein alone could not induce vesicle budding and was not secreted from cells. Coexpression of M protein with the viral hemagglutinin-neuraminidase (HN) or fusion (F) glycoproteins also failed to result in significant VLP release. It was found that M protein in the form of VLPs was only secreted from cells, with an efficiency comparable to authentic virus budding, when M protein was coexpressed with one of the two glycoproteins, HN or F, together with the nucleocapsid (NP) protein. The VLPs appeared similar morphologically to authentic virions by electron microscopy. CsCl density gradient centrifugation indicated that almost all of the NP protein in the cells had assembled into nucleocapsid-like structures. Deletion of the F and HN cytoplasmic tails indicated an important role of these cytoplasmic tails in VLP budding. Furthermore, truncation of the HN cytoplasmic tail was found to be inhibitory toward budding, since it prevented coexpressed wild-type (wt) F protein from directing VLP budding. Conversely, truncation of the F protein cytoplasmic tail was not inhibitory and did not affect the ability of coexpressed wt HN protein to direct the budding of particles. Taken together, these data suggest that multiple viral components, including assembled nucleocapsids, have important roles in the paramyxovirus budding process.     

Monoclonal antibodies against baboon endogenous virus and against host cell antigens.

Monoclonal antibodies were produced by murine hybridomas after immunization with semipurified baboon endogenous virus. In a solid-phase radioimmunoassay, two antibodies (F12-9 and B9-18) reacted with viral antigen only. The antibodies A6-8 and C9-12 also reacted with virus-producing cells but not with control cells, whereas antibodies E4-6 and D12-2 bound to virus-free cells as well. The cytofluorometry technique confirmed these results and showed a competition between antibodies A6-8 and C9-12 for binding to virus-producing cells as well as a competition between antibodies D12-2 and E4-6 for binding to virus-free human cells. An immune precipitation assay with disrupted virions indicated that antibodies A6-8, B9-18, and C9-12 were directed against the gp70 glycoprotein, and that antibody F12-9 reacted with a viral antigen with a molecular weight of 18,000. The syncytia induced in RSa cells by baboon molecular weight of 18,000. The syncytia induced in RSa cells by baboon endogenous virus could be inhibited either when antibody A6-8 or C9-12 was combined to the virus or when the RSa cells were treated with the anticellular antibody D12-2 or E4-6. These two effects were not observed with Mason-Pfizer virus. Thus, of three antibodies with specificities for viral gp70, two (A6-8 and C9-12) were directed at viral sites responsible for syncytium formation. Another antiviral antibody (F12-9) reacted with a protein of unknown function with a molecular weight of 18,000. The two anticellular antibodies were directed at similar or neighboring epitopes, which may be situated within the receptor to the virus.     

Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene.

A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody. The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay. These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.     

Control of central nervous system viral persistence by neutralizing antibody

Replication of the neurotropic JHM strain of mouse hepatitis virus within the central nervous system is controlled by cellular immunity. However, following initial clearance, virus reactivates in the absence of humoral immunity. Viral recrudescence is prevented by the transfer of antiviral antibody (Ab). To characterize the specificity and biological functions of Ab critical for maintaining viral persistence, monoclonal Abs specific for the viral spike, matrix, and nucleocapsid proteins were transferred into infected B-cell-deficient mice following initial virus clearance. Neutralizing immunoglobulin G (IgG) but not IgA anti-spike Ab suppressed virus recrudescence, reduced viral antigen in most cell types except oligodendroglia, and was associated with reduced demyelination. Nonneutralizing monoclonal Abs specific for the spike, matrix, and nucleocapsid proteins did not prevent recrudescence, demonstrating that neutralization is critical for maintaining JHM mouse hepatitis virus persistence within the central nervous system. Ab-mediated protection was not associated with alterations in virus-specific T-cell function or inflammation. Furthermore, neutralizing Ab delayed but did not prevent virus recrudescence. These data indicate that following acute viral clearance cellular immunity is ineffective in controlling virus recrudescence and suggest that the continued presence of neutralizing Ab is the essential effector in maintaining viral persistence within the central nervous system.     

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.     

Mapping a region of hepatitis C virus E2 that is responsible for escape from neutralizing antibodies and a core CD81-binding region that does not tolerate neutralization escape mutations

Understanding the interaction between broadly neutralizing antibodies and their epitopes provides a basis for the rational design of a preventive hepatitis C virus (HCV) vaccine. CBH-2, HC-11, and HC-1 are representatives of antibodies to overlapping epitopes on E2 that mediate neutralization by blocking virus binding to CD81. To obtain insights into escape mechanisms, infectious cell culture virus, 2a HCVcc, was propagated under increasing concentrations of a neutralizing antibody to isolate escape mutants. Three escape patterns were observed with these antibodies. First, CBH-2 escape mutants that contained mutations at D431G or A439E, which did not compromise viral fitness, were isolated. Second, under the selective pressure of HC-11, escape mutations progressed from a single L438F substitution at a low antibody concentration to double substitutions, L438F and N434D or L438F and T435A, at higher antibody concentrations. Escape from HC-11 was associated with a loss of viral fitness. An HCV pseudoparticle (HCVpp) containing the L438F mutation bound to CD81 half as efficiently as did wild-type (wt) HCVpp. Third, for HC-1, the antibody at a critical concentration completely suppressed viral replication and generated no escape mutants. Epitope mapping revealed contact residues for CBH-2 and HC-11 in two regions of the E2 glycoprotein, amino acids (aa) 425 to 443 and aa 529 to 535. Interestingly, contact residues for HC-1 were identified only in the region encompassing aa 529 to 535 and not in aa 425 to 443. Taken together, these findings point to a region of variability, aa 425 to 443, that is responsible primarily for viral escape from neutralization, with or without compromising viral fitness. Moreover, the region aa 529 to 535 is a core CD81 binding region that does not tolerate neutralization escape mutations.     

Peptide-mediated interference with baculovirus transduction

Baculovirus represents a multifunctional platform with potential for biomedical applications including disease therapies. The importance of F3, a tumor-homing peptide, in baculovirus transduction was previously recognized by the ability of F3 to augment viral binding and gene delivery to human cancer cells following display on the viral envelope. Here, F3 was utilized as a molecular tool to expand understanding of the poorly characterized baculovirus-mammalian cell interactions. Baculovirus-mediated transduction of HepG2 hepatocarcinoma cells was strongly inhibited by coincubating the virus with synthetic F3 or following incorporation of F3 into viral nucleocapsid by genetic engineering, the former suggesting direct interaction of the soluble peptide with the virus particles. Since internalization and nuclear accumulation of the virus were significantly inhibited or delayed, but the kinetics of viral binding, initial uptake, and endosomal release were unaffected, F3 likely interferes with cytoplasmic trafficking and subsequent nuclear transport of the virus. A polyclonal antibody raised against nucleolin, the internalizing receptor of F3, failed to inhibit cellular binding, but considerably reduced viral transduction efficiency, proposing the involvement of nucleolin in baculovirus entry. Together, these results render the F3 peptide a tool for elucidating the mechanism and molecular details conferring to baculovirus-mediated gene transduction in mammalian cells.     

4E10-resistant variants in a human immunodeficiency virus type 1 subtype C-infected individual with an anti-membrane-proximal external region-neutralizing antibody response

The broadly neutralizing monoclonal antibody (MAb) 4E10 recognizes a linear epitope in the C terminus of the membrane-proximal external region (MPER) of gp41. This epitope is particularly attractive for vaccine design because it is highly conserved among human immunodeficiency virus type 1 (HIV-1) strains and neutralization escape in vivo has not been observed. Multiple env genes were cloned from an HIV-1 subtype C virus isolated from a 7-year-old perinatally infected child who had anti-MPER neutralizing antibodies. One clone (TM20.13) was resistant to 4E10 neutralization as a result of an F673L substitution in the MPER. Frequency analysis showed that F673L was present in 33% of the viral variants and in all cases was linked to the presence of an intact 2F5 epitope. Two other envelope clones were sensitive to 4E10 neutralization, but TM20.5 was 10-fold less sensitive than TM20.6. Substitutions at positions 674 and 677 within the MPER rendered TM20.5 more sensitive to 4E10 but had no effect on TM20.6. Using chimeric and mutant constructs of these two variants, we further demonstrated that the lentivirus lytic peptide-2 domain in the cytoplasmic tail affected the accessibility of the 4E10 epitope, as well as virus infectivity. Collectively, these genetic changes in the face of a neutralizing antibody response to the MPER strongly suggested immune escape from antibody responses targeting this region.     

Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines

A number of antibodies generated during human respiratory syncytial virus (RSV) infection have been cloned by the phage library approach. Antibodies reactive with an immunodominant epitope on the F glycoprotein of this virus have a high affinity for affinity-purified F antigen. These antibodies, however, have a much lower affinity for mature F glycoprotein on the surface of infected cells and are nonneutralizing. In contrast, a potent neutralizing antibody has a high affinity for mature F protein but a much lower affinity for purified F protein or F protein in viral lysates. The data indicate that at least two F protein immunogens are produced during natural RSV infection: immature F, found in viral lysates, and mature F, found on infected cells or virions. Binding studies with polyclonal human immunoglobulin G suggest that the antibody responses to the two immunogens are of similar magnitudes. Competitive binding studies suggest that overlap between the responses is relatively limited. A mature envelope with an antigenic configuration different from that of the immature envelope has an evolutionary advantage in that the infecting virus is less subject to neutralization by the humoral response to the immature envelope that inevitably arises following lysis of infected cells. Subunit vaccines may be at a disadvantage because they most often resemble immature envelope molecules and ignore this aspect of viral evasion.     

Stoichiometry of murine leukemia virus envelope protein-mediated fusion and its neutralization

Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.     

Characterization of the structural proteins of the murine coronavirus strain A59 using monoclonal antibodies

Monoclonal antibodies reacting with the A59 strain of mouse hepatitis virus (MHV-A59) were characterized and those specific to the E2 major envelope glycoprotein were studied in detail. Antibodies were tested for their ability to neutralize viral infectivity (N+ characteristic) and prevent viral-induced cell-to-cell fusion (F+ characteristic). All four possible combinations of activities reflecting E2 functions were found, i.e., N+F+, N-F-, N+F-, and N-F+. In addition, competitive binding studies with these monoclonal antibodies revealed two nonoverlapping antigenic regions. The first region, designated A, was recognized by antibodies which included each of the four functional types. Region B was identified by a single monoclonal antibody with N-F- activities. The existence of antibodies which only neutralize virus or only block viral-induced fusion implies that the structures on E2 which serve as targets for neutralization and which induce fusion are not identical. The critical determinants for neutralization and fusion must be closely related topographically on E2 since both N+F- and N-F+ antibodies recognize the same antigenic region.     

Antigenic modulation induced by monoclonal antibodies: antibodies to measles virus hemagglutinin alters expression of other viral polypeptides in infected cells

Polyclonal antibody to measles virus can have profound effects on external (outer plasma membrane) as well as internal (cytoplasmic) viral polypeptides expressed in infected cells. The process, termed "antibody-induced antigenic modulation," was further investigated by using monoclonal antibody to several viral polypeptides. Four monoclonal antibodies against the viral hemagglutinin had the ability to decrease the expression of the phosphoprotein, fusion, and membrane protein. A monoclonal antibody to the nucleocapsid protein did not cause these changes. The observed decreases were not due to preferential degradation of viral polypeptides as determined by pulse-chase experiments. Our results indicate that a specific signal to an epitope on the plasma membrane (monoclonal antibody measles virus hemagglutinin) can alter the expression of measles virus phosphoprotein and membrane protein, both polypeptides present in the cytoplasm of infected cells.     

Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.