Enterovirus capsid interactions with decay-accelerating factor mediate lytic cell infection

The cellular receptor usage of numerous human enteroviruses can differ significantly between low-cell-culture-passaged clinical isolates and highly laboratory-passaged prototype strains. The prototype strain of coxsackievirus A21 (CVA21) displays a dual-receptor specificity as determined with a receptor complex consisting of decay-accelerating factor (DAF) and intercellular adhesion molecule 1 (ICAM-1). In this study, the cellular receptor interactions of low-cell-passage CVA21 clinical isolates with respect to their interactions with cell surface-expressed DAF and ICAM-1 were compared to those of the CVA21 prototype (Kuykendall) strain. Dual-receptor usage of DAF and ICAM-1 by CVA21 clinical isolates was confirmed by cell transfection and radiolabeled binding assays. The cellular attachment of clinical and prototype CVA21 strains to cells that coexpressed DAF and ICAM-1 was not additive compared to the viral binding to cells expressing one or other receptor. In fact, the binding data suggest there is an inhibition of CVA21 cellular attachment in environments where high-level coexpression of both DAF and ICAM-1 occurs. Antibody cross-linking of DAF rendered cells susceptible to lytic infection by the CVA21 clinical isolates. In a novel finding, three clinical isolates could, to various degrees, infect and lyse DAF-expressing cells in the absence of DAF-antibody cross-linking and ICAM-1 expression. Sequence analysis of the P1 region of clinical and prototype virus genomes identified a number of coding changes that may contribute to the observed enhanced DAF usage phenotype of the clinical CVA21 isolates. None of the amino acid changes was located in the previously postulated ICAM-1 footprint, a receptor-binding environment that was conserved on the capsid surface of all CVA21 clinical isolates. Taken together, the data suggest that community-circulating strains of CVA21 can infect target cells expressing either ICAM-1 or DAF alone and that such interactions extend tissue tropism and impact directly on viral pathogenesis.     

Novel role for decay-accelerating factor in coxsackievirus A21-mediated cell infectivity

Decay-accelerating factor (DAF) is involved in the cell membrane attachment of many human enteroviruses. Presently, further specific active roles of DAF in mediating productive cell infection and in the pathogenesis of natural enterovirus infection are poorly understood. In an attempt to more fully understand the role of DAF in lytic cell infection we examined the specific interactions of the prototype strain of coxsackievirus A21 (CVA21) with surface-expressed DAF. Investigations into discrete DAF-CVA21 interactions focused on viral binding; viral particle elution with respect to the parameters of time, temperature, and pH; and subsequent cell infection. Radiolabeled-virus binding assays revealed that peak elution of CVA21 from DAF occurred within 15 min of initial attachment and that the DAF-eluted virus increased in a linear fashion with respect to temperature and pH. CVA21 eluted from endogenous surface-expressed DAF was highly infectious, in contrast to CVA21 eluted from intercellular adhesion molecule 1 (ICAM-1), which retained little to no infectivity. Using an adenovirus transduction system, we demonstrate that CVA21 can remain infectious for up to 24 h after DAF binding and is capable of initiating a multicycle lytic infection upon delayed ICAM-1 surface expression. Taken together, the data suggest that a major role of DAF in cell infection by the prototype strain of CVA21 is to provide membrane concentration of infectious virions, effectively increasing viral interactions with endogenous or induced ICAM-1.     

Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry.

It is becoming increasingly apparent that many viruses employ multiple receptor molecules in their cell entry mechanisms. The human enterovirus coxsackievirus A21 (CAV21) has been reported to bind to the N-terminal domain of intercellular adhesion molecule 1 (ICAM-1) and undergo limited replication in ICAM-1-expressing murine L cells. In this study, we show that in addition to binding to ICAM-1, CAV21 binds to the first short consensus repeat (SCR) of decay-accelerating factor (DAF). Dual antibody blockade using both anti-ICAM-1 (domain 1) and anti-DAF (SCR1) monoclonal antibodies (MAbs) is required to completely abolish binding and replication of high-titered CAV21. However, the binding of CAV21 to DAF, unlike that to ICAM-1, does not initiate a productive cell infection. The capacity of an anti-DAF (SCR3) MAb to block CAV21 infection but not binding, coupled with immunoprecipitation data from chemical cross-linking studies, indicates that DAF and ICAM-1 are closely associated on the cell surface. It is therefore suggested that DAF may function as a low-affinity attachment receptor either enhancing viral presentation or providing a viral sequestration site for subsequent high-affinity binding to ICAM-1.     

A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.     

Variations of coxsackievirus B3 capsid primary structure, ligands, and stability are selected for in a coxsackievirus and adenovirus receptor-limited environment

While group B coxsackieviruses (CVB) use the coxsackievirus and adenovirus receptor (CAR) as the receptor through which they infect susceptible cells, some CVB strains are known for their acquired capacity to bind other molecules. The CVB3/RD strain that emerged from a CVB3/Nancy population sequentially passaged in the CAR-poor RD cell line binds decay-accelerating factor (DAF) (CD55) and CAR. A new strain, CVB3/RDVa, has been isolated from RD cells chronically infected with CVB3/RD and binds multiple molecules in addition to DAF and CAR. The capsid proteins of CVB3/RD differ from those of CVB3/28, a cloned strain that binds only CAR, by only four amino acids, including a glutamate/glutamine dimorphism in the DAF-binding region of the capsid. The capsid proteins of CVB3/RD and CVB3/RDVa differ by seven amino acids. The ability of CVB3/RDVa to bind ligands in addition to CAR and DAF may be attributed to lysine residues near the icosahedral 5-fold axes of symmetry. Considered with differences in the stability of the CVB3 strains, these traits suggest that in vitro selection in a CAR-limited environment selects for virus populations that can associate with molecules on the cell surface and survive until CAR becomes available to support infection.     

Single amino acid changes in the virus capsid permit coxsackievirus B3 to bind decay-accelerating factor

Many coxsackievirus B isolates bind to human decay-accelerating factor (DAF) as well as to the coxsackievirus and adenovirus receptor (CAR). The first-described DAF-binding isolate, coxsackievirus B3 (CB3)-RD, was obtained during passage of the prototype strain CB3-Nancy on RD cells, which express DAF but very little CAR. CB3-RD binds to human DAF, whereas CB3-Nancy does not. To determine the molecular basis for the specific interaction of CB3-RD with DAF, we produced cDNA clones encoding both CB3-RD and CB3-Nancy and mutated each of the sites at which the RD and Nancy sequences diverged. We found that a single amino acid change, the replacement of a glutamate within VP3 (VP3-234E) with a glutamine residue (Q), conferred upon CB3-Nancy the capacity to bind DAF and to infect RD cells. Readaptation of molecularly cloned CB3-Nancy to RD cells selected for a new virus with the same VP3-234Q residue. In experiments with CB3-H3, another virus isolate that does not bind measurably to DAF, adaptation to RD cells resulted in a DAF-binding isolate with a single amino acid change within VP2 (VP2-138 N to D). Both VP3-234Q and VP2-138D were required for binding of CB3-RD to DAF. In the structure of the CB3-RD-DAF complex determined by cryo-electron microscopy, both VP3-234Q and VP2-138D are located at the contact site between the virus and DAF.     

Viral Cell Entry Induced by Cross-Linked Decay-Accelerating Factor

Decay-accelerating factor (DAF) mediates cellular attachment for many human picornaviruses. In most cases, viral binding to DAF is itself insufficient to permit cell infectivity, with a second, functional internalization receptor being required to facilitate this process. Previously, we postulated that the role of DAF in enterovirus cell infection is as a sequestration receptor, maintaining a reservoir of bound virus in an infectious state, awaiting interaction with functional internalization receptors. Many of these functional receptors possess the capacity to induce relatively rapid changes in capsid conformations, resulting in the formation of altered particles (A-type particles). In this report, we show that antibody-cross-linked DAF, in contrast to endogenous surface-expressed forms, can act as a functional virus receptor to mediate coxsackie A21 virus (CAV21) lytic cell infection. In contrast to the situation with ICAM-1-mediated CAV21 infection, in which high levels of A-type particles are formed, cross-linked DAF-induced CAV21 replication occurs in the absence of detectable A-particle formation.     

Heparan sulfates and coxsackievirus-adenovirus receptor: each one mediates coxsackievirus B3 PD infection

Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-O-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR.     

The capsid determinant of fibrotropism for the MVMp strain of minute virus of mice functions via VP2 and not VP1.

The minute virus of mice, prototype strain MVMp, productively infects cultured murine fibroblasts but not T cells. The immunosuppressive strain, MVMi, shows the converse tropism. These reciprocal tropisms are mediated by the viral capsids, in which their determinants have been mapped to a few specific amino acids in the primary sequence shared by VP1 and VP2. Which of these proteins is relevant in presenting these determinants during infection is not known. We have approached this question using a recombinant parvovirus system in which a LuIII-derived transducing genome, containing the luciferase reporter in place of viral coding sequences, can be packaged by capsid proteins from separate helper sources. We generated transducing virions by using helper constructs expressing either VP1 or VP2, containing the MVMp or MVMi tropic determinant region, in various combinations. The virions were used to infect human NB324K cells and murine A9 fibroblasts. Transduction of the human cells (permissive for both MVMp and MVMi) required both VP1 and VP2 and was successful with all combinations of these proteins. In contrast, significant transducing activity for A9 cells was detected only with recombinant virions containing VP2 of MVMp, while the use of either source of VP1 had little effect. We conclude that VP2 from MVMp is necessary to enable infection of murine A9 fibroblasts.     

Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide.

We established a human cell line which was persistently infected (PI) by the normally cytolytic echovirus 6. All of the cultured PI cells contained genome-size viral RNA which was synthesized continuously and incorporated into virus particles. This steady-state infection has been maintained for more than 6 years. In contrast to RNA of wild-type echovirus 6, the viral RNA from PI cells was not lytic when transfected into uninfected, susceptible cells. The capsid polypeptides of the virus particles produced during lytic infections were compared with those of virus particles from PI cells. Wild-type virions contained five polypeptides with molecular masses of 31.5, 27, 25.8, 21.2, and 9.5 kilodaltons. Comparison of polypeptide profiles of virions and empty immature capsids along with peptide analyses by immunoblotting and partial proteolysis of isolated viral proteins identified the cleavage products of the 31.5-kilodalton polypeptide (VP0) as the two smaller polypeptides (VP2 and VP4). The virus particles produced by PI cells as well as cellular extracts of PI cells contained only the three largest proteins (VP0, VP1, and VP3), indicating that VP0 was not processed during persistent infection. The lack of VP2 and VP4 in the defective virus particles coincided with their inability to attach to uninfected, susceptible cells. The maintenance of the steady-state infection of echovirus 6 was not dependent upon the release of virus particles from PI cells.     

The Crystal Structure of a Coxsackievirus B3-RD Variant and a Refined 9-Angstrom Cryo-Electron Microscopy Reconstruction of the Virus Complexed with Decay-Accelerating Factor (DAF) Provide a New Footprint of DAF on the Virus Surface

The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses.     

Host range determinants located on the interior of the poliovirus capsid.

The inability of certain poliovirus strains to infect mice can be overcome by the expression of human poliovirus receptors in mice or by the presence of a particular amino acid sequence of the B-C loop of the viral capsid protein VP1. We have identified changes in an additional capsid structure that permit host-restricted poliovirus strains to infect mice. Variants of the mouse-virulent P2/Lansing strain were constructed containing amino acid changes, deletions and insertions in the B-C loop of VP1. These variants were attenuated in mice, demonstrating the importance of the B-C loop sequence in host range. Passage of two of the B-C loop variants in mice led to the selection of viruses that were substantially more virulent. The increased neurovirulence of these strains was mapped to two different suppressor mutations in the N-terminus of VP1. Whereas the B-C loop of VP1 is highly exposed on the surface of the capsid, near the five-fold axis of symmetry, the suppressor mutations are in the interior of the virion, near the three-fold axis. Introduction of the suppressor mutations into the genome of the mouse-avirulent P1/Mahoney strain resulted in neurovirulent viruses, demonstrating that the P2/Lansing B-C loop sequence is not required to infect mice. Because the internal host range determinants are in a structure known to be important in conformational transitions of the virion, the host range of poliovirus may be determined by the ability of virions to undergo transitions catalyzed by cell receptors.     

A simian virus 40-encoded protein of Mr 74,000 daltons is structurally related to the capsid proteins of the virus

We have demonstrated the synthesis of a 74,000-dalton protein (74K protein) in African green monkey kidney cells infected with simian virus (SV)40. The 74K protein was detected late during the lytic cycle. Its synthesis was inhibited by arabinosyl cytosine as was the synthesis of the capsid proteins. Monospecific antibodies raised against VP1 and VP3 precipitated the structural proteins and the 74K protein. The 74K protein was not found in purified virions. Tryptic peptide analysis demonstrated that the 74K protein shares methionine- and serine-containing peptides with VP1 and VP3 and thus is structurally related to the capsid proteins.     

The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55).

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.     

Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55).

A coxsackievirus B3 (CB3) isolate adapted to growth in RD cells shows an alteration in cell tropism as a result of its capacity to bind a 70-kDa cell surface molecule expressed on these cells. We now show that this molecule is the complement regulatory protein, decay-accelerating factor (DAF) (CD55). Anti-DAF antibodies prevented CB3 attachment to the cell surface. Radiolabeled CB3 adapted to growth in RD cells bound to CHO cells transfected with human DAF, whereas CB3 (strain Nancy), the parental strain, did not bind to DAF transfectants. These results indicate that growth of CB3 in RD cells selected for a virus strain that uses DAF for cell surface attachment.     

The minor receptor group of human rhinovirus (HRV) includes HRV23 and HRV25, but the presence of a lysine in the VP1 HI loop is not sufficient for receptor binding

Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore "true" major group viruses.     

Level of ICAM-1 Surface Expression on Virus Producer Cells Influences both the Amount of Virion-Bound Host ICAM-1 and Human Immunodeficiency Virus Type 1 Infectivity

Using virions harvested from 293T cells stably expressing either low or high levels of surface ICAM-1, we determined that the number of virus-embedded host ICAM-1 proteins is positively influenced by the expression level of ICAM-1 on virus producer cells. Moreover, the increase in virion-bound host cell membrane ICAM-1 led to a concomitant enhancement of virus infectivity when a T-cell-tropic strain of human immunodeficiency virus type 1 (HIV-1) was used. The phenomenon was also seen when primary human cells were infected with virions pseudotyped with the envelope protein from a macrophage-tropic HIV-1 isolate, thus ruling out any envelope-specific effect. We also observed that target cells treated with NKI-L16, an anti-LFA-1 antibody known to increase the affinity of LFA-1 for ICAM-1, were markedly more susceptible to infection with HIV-1 particles bearing on their surfaces large numbers of host-derived ICAM-1 proteins. Given that cellular activation of leukocytes is known to modify the conformational state of LFA-1 and induce ICAM-1 surface expression, it is tempting to speculate that activation of virus-infected cells will lead to the production of HIV-1 particles bearing more host ICAM-1 on their surfaces and that such progeny virions will preferentially infect and replicate more efficiently in activated cells which are prevalent in lymphoid organs.     

A single-amino-acid substitution in the P2 domain of VP1 of murine norovirus is sufficient for escape from antibody neutralization

Noroviruses cause epidemic outbreaks of acute viral gastroenteritis worldwide, and the number of reported outbreaks is increasing. Human norovirus strains do not grow in cell culture. However, murine norovirus (MNV) replicates in the RAW 264.7 macrophage cell line and thus provides a tractable model to investigate norovirus interactions with host cells. Epitopes recognized by monoclonal antibodies (MAbs) against the human norovirus strains Norwalk virus and Snow Mountain virus (SMV) identified regions in the P domain of major capsid protein VP1 important for interactions with putative cellular receptors. To determine if there was a relationship between domains of MNV VP1 and VP1 of human norovirus strains involved in cell binding, epitope mapping by phage display was performed with an MNV-1-neutralizing MAb, A6.2.1. A consensus peptide, GWWEDHGQL, was derived from 20 third-round phage clones. A synthetic peptide containing this sequence and constrained through a disulfide linkage reacted strongly with the A6.2.1 MAb, whereas the linear sequence did not. Four residues in the A6.2.1-selected peptide, G327, G333, Q334, and L335, aligned with amino acid residues in the P2 domain of MNV-1 VP1. This sequence is immediately adjacent to the epitope recognized by anti-SMV MAb 61.21. Neutralization escape mutants selected with MAb A6.2.1 contained a leucine-to-phenylalanine substitution at position 386 in the P2 domain. The predicted location of these residues on VP1 suggests that the phage peptide and the mutation in the neutralization-resistant viruses may be in close proximity to each other and to residues reported to be important for carbohydrate binding to VP1 of human norovirus strains.     

The abundant nuclear enzyme PARP participates in the life cycle of simian virus 40 and is stimulated by minor capsid protein VP3

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.     

柯萨奇病毒A组16型衣壳蛋白VP1的原核表达及初步活性测定

目的:原核表达柯萨奇病毒A组16型(CVA16)衣壳蛋白VP1,以便于研制血清学检测试剂。方法:在基因库中钓取CVA16-VP1的全长序列,采用PCR逐步合成法合成其全长基因,测序正确后克隆到表达载体pET28a(+)中,构建重组表达质粒pET28a(+)/VP1,转化大肠杆菌BL21,IPTG诱导表达,利用Ni2+亲和层析柱对重组蛋白进行纯化;建立捕获免疫酶联法检测IgM抗体,检测20份手足口病阳性血清和30份阴性血清,评价重组抗原的灵敏度和特异性;采用CVA16全病毒免疫的抗小鼠血清进行Western印迹。结果:重组CVA16-VP1蛋白在大肠杆菌中获得高效表达;用重组蛋白抗原检测,20份手足口病患儿阳性血清中有4份阳性,其中1份同时为肠道病毒71型(EV71)VP1阳性,30份阴性血清无反应。结论:实现了CVA16-VP1的高效表达,初步结果显示重组蛋白具有较好的抗原性,为柯萨奇病毒A组16型诊断试剂的研究奠定了基础。