MDV-1 VP22: a transporter that can selectively deliver proteins into cells

The tegument protein VP22 of Marek’s disease virus (MDV) was previously shown to be able to travel between cells. To further characterize the transport property of VP22 and assess whether it can be used for protein delivery, we investigated the subcellular localization of VP22 fused to five heterologous proteins, including green fluorescent protein, nucleoprotein of avian influenza virus, bovine IFN-γ, F protein of Newcastle disease virus and VP2 protein of infectious bursa disease virus. The transport of these fusion proteins in monolayer cells was assayed by immunofluorescence assay. The results showed that all except VP2 could be delivered by VP22 of MDV serotype 1 (MDV-1), at different efficiencies. After being transported to surrounding cells, VP22 fused to avian influenza nucleoprotein, bovine IFN-γ, or to F was localized in the nucleus. Our data suggest that VP22 of MDV-1 can be used as transport tool in protein delivery and that the original localization of cargo proteins may be changed after transport by MDV-1 VP22. Finally, infectious bursa disease VP2 protein could not be transported by MDV-1 VP22.     

Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: Inclusion of the largest protein VP1 in the core and virus-like particles

Bluetongue virus (BTV) VP1 protein, a component of the viral RNA-directed RNA polymerase, but not the VP4 or VP6 proteins, was specifically incorporated into baculovirus expressed BTV core-like particles (composed of VP3 and VP7) and BTV virus-like particles (composed of VP2, VP3, VP5, and VP7). The VP1 protein has been shown to be associated with subcore particles composed of VP3. The data suggest that the VP1 protein of BTV has both enzymatic and structural roles in the virus life cycle.     

Presentation of hepatitis B virus preS2 epitope on bluetongue virus core-like particles.

A chimeric protein containing most of the hepatitis B virus preS2 region (amino acid residues 1-48) upstream to, and colinear with the amino-terminus of bluetongue virus VP7 protein (preS2-VP7) was expressed by a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). The chimeric protein formed BTV core-like particles (CLPs) in Spodoptera frugiperda cells only when the cells were coinfected with this recombinant virus and a recombinant baculovirus that expresses unmodified VP7 and VP3 of BTV. The ratio of preS2-VP7 incorporated into CLPs was influenced by the relative multiplicities of infection of the two viruses. Immunoelectron microscopy of the chimeric particles indicated that the preS2 epitope was exposed on the surface of the CLPs. When insect cells were coinfected with the preS2-VP7 recombinant virus and a baculovirus vector that synthesized only the VP3 protein, no CLPs were identified.     

Presentation of hepatitis B virus preS2 epitope on bluetongue virus core-like particles

A chimeric protein containing most of the hepatitis B virus preS₂ region (amino acid residues 1–48) upstream to, and colinear with the amino-terminus of bluetongue virus VP7 protein (preS₂VP7) was expressed by a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). The chimeric protein formed BTV core-like particles (CLPs) in Spodoptera frugiperda cells only when the cells were coinfected with this recombinant virus and a recombinant baculovirus that expresses unmodified VP7 and VP3 of BTV. The ratio of preS₂VP7 incorporated into CLPs was influenced by the relative multiplicities of infection of the two viruses. Immunoelectron microscopy of the chimeric particles indicated that the preS₂ epitope was exposed on the surface of the CLPs. When insect cells were coinfected with the preS₂ VP7 recombinant virus and a baculovirus vector that synthesized only the VP3 protein, no CLPs were identified.     

Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype

Analyses of reassortant and parental strains of BTV serotypes 3 and 10, in serum neutralization tests, confirmed the major role of outer capsid protein VP2 in determination of virus serotype and its involvement in serum neutralization. However, a reassortant BTV strain (R70), containing protein VP5 derived from BTV 3 and VP2 derived from BTV 10, cross-neutralized with both parental virus strains (BTV 3 and BTV 10). It is concluded that VP5 also plays some part in serotype determination of these virus isolates, as analyzed by serum-neutralization, but its role may be less significant than that of VP2.     

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.     

Cell-free translation of foot-and-mouth disease virus RNA into identifiable non-capsid and capsid proteins.

Foot-and-mouth disease virus (a member of the picornavirus group) RNA could be translated effectively in an S-30 extract from Ehrlich ascites tumour cells. This translation was inhibited by aurintricarboxylic acid, cycloheximide, puromycin and RNase. Cell-free products of translation were identified by disc gel electrophoresis and immunoprecipitation with specific antisera. Gel electrophoresis of the products without prior immunoprecipitation suggested the synthesis of some of the non-capsid proteins and capsid proteins VP1, VP2 and VP3 of the virus. Immunoprecipitations with antisera against whole virus and VP3 indicated the synthesis of VP3 and of at least two additional peptides of 100 000 and 56 000 daltons containing antigenic sites of VP3. Gel electrophoresis after immunoprecipitation with antiserum against virus infection-associated antigen indicated the synthesis of a different 56 000-dalton protein appearing to resemble non-capsid protein NCVP5. The amount of foot-and-mouth disease virus and VP3-specific peptides in the virus RNA-directed products were measured by immunoprecipitation.     

Morphogenesis of a bluetongue virus variant with an amino acid alteration at a neutralization site in the outer coat protein, VP2

Neutralization-resistant variants of bluetongue virus, selected with a monoclonal antibody to the outer coat protein VP2, have been used to delineate a neutralization epitope on the VP2 protein. Comparison of the RNA 2 sequence of four variants with that of the wild-type virus indicated that each variant contained a single nucleotide substitution which in turn resulted in a single amino acid alteration in VP2. The changes were clustered within a span of eight amino acids at positions 328 to 335 in the VP2 protein. In addition, analyses of cells infected with wild-type and a variant virus V35B2 have provided information on the site of VP2 addition to virus particles during morphogenesis. Electron microscopic examination revealed few virus-like particles around virus inclusion bodies (VIB) in wild-type virus-infected cells and cytoskeletons. In contrast, VIB in cells infected with the neutralization-resistant variant V35B2 were surrounded by particles identified as virus cores on the basis of their size and morphology. Probing of cytoskeletons with gold-labeled anti-VP2 monoclonal antibody revealed that in wild-type virus-infected cells the antibodies reacted weakly with VIB and only at locations where virus particles appeared to be leaving. The core-like particles surrounding VIB in V35B2-infected cells labeled very weakly with the anti-VP2 antibody. In contrast, wild-type and V35B2 virus particles which bound to the cytoskeleton at locations distal to VIB and those outside the infected cell bound significant amounts of antibody. These results suggest that although some VP2 may be added to developing virus particles at the periphery of VIB, the remainder of the VP2 protein is added outside the VIB either in the cytosol or following attachment of the particles to the cytoskeleton.     

Characterization of structural proteins of Solenopsis invicta virus 1

Purification of Solenopsis invicta virus 1 (SINV-1) from its host, S. invicta, and subsequent examination by electron microscopy revealed a homogeneous fraction of spherical particles with a diameter of 30-35 nm. Quantitative PCR with SINV-1-specific oligonucleotide primers verified that this fraction contained high copy numbers of the SINV-1 genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SINV-1 purified fraction revealed three major and one minor protein bands. The protein bands were labeled VP1 (40.8+/-1.4 kDa), VP2 (35.7+/-2.8 kDa), VP3 (25.2+/-1.8 kDa), and VP4 (22.2+/-2.5 kDa) based on mass. N-terminal sequence was acquired successfully for VP1, VP2, and VP3, but not VP4, and delineated each capsid protein within the 3'-proximal open reading frame of SINV-1. Positional organization of the viral proteins within the SINV-1 structural polyprotein was consistent with dicistroviruses (when based on sequence similarity). Blastp analysis of SINV-1 VP1, VP2, and VP3 revealed significant identity with corresponding structural capsid proteins of positive-strand RNA viruses, particularly acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV). Amino acid residues about the scissile bonds for VP1 and VP3 were consistent with dicistroviruses and insect-infecting picorna-like viruses. N-terminal sequencing of VP2 also established that translation initiation of the SINV-1 structural polyprotein was mediated by an internal ribosomal entry site and is AUG-independent.     

Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion.

We identified by immunochemical methods 13 polypeptides associated with the infectious respiratory syncytial virus virion. Eight of these polypeptides (VP200, VP84, VP66, VP43, VP40, VP37, VP28, and VP19) were identified as virus specific. Two other polypeptides, (VP) 22 and (VP) 12, are provisionally considered to be of viral origin. Three nonviral proteins are also intimately associated with the infectious virion. These nonviral proteins were identified as cellular actin and two proteins with bovine serum albumin immunospecificity. VP40 was identified as the major ribonucleoprotein. Based on biochemical and biophysical similarities with paramyxovirus proteins, other respiratory syncytial virus proteins are believed to have these specific viral functions: VP84, "hemagglutinin"; VP66, undissociated fusion protein, F1,2; VP43, F1; and VP19, F2, VP66 contains a major determinant involved in viral infectivity since all neutralizing antibodies tested, including a monoclone, precipitated this protein.     

Evolutionary relationships among the gnat-transmitted orbiviruses that cause African horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences.

The amino acid sequences of four major capsid proteins of African horse sickness virus (serotype 4, AHSV-4) have been compared with those of Bluetongue virus of sheep. Epizootic hemorrhagic disease virus of deer, and the phylogenetic relationships established. Complete nucleotide sequence analysis of three RNA segments (L2, L3, and M6) of AHSV-4 and their encoded products, VP2, VP3, and VP5, together with previously published data for VP7 (Roy et al., 1991), have revealed that of the four capsid proteins the innermost protein, VP3, is the most conserved, and the outermost protein, VP2, is the most variable. Some 57-58% of the aligned BTV-10 and EHDV-1 VP3 amino acids are identical with those of AHSV-4. This compares to an identity of 79% between the BTV and EHDV VP3 sequences. For the VP7 proteins 64% of the aligned amino acids are identical between BTV-10 and EHDV-1, while they share 44-46% amino acid residues with the aligned VP7 protein of AHSV-4. By contrast, the VP2 proteins of the three viruses share only 19-24% identical amino acids. Various other comparative analyses of the proteins indicate that the VP2 species of the three orbiviruses are similar. Unlike VP2, the other outer capsid protein, VP5 is more conserved among the three viruses. On alignment, the VP5 of AHSV-4 has some 43-45% identical amino acids with that of BTV-10 and EHDV-1. Between BTV and EHDV, 62% of the aligned sequences are identical.     

The structural proteins of equine arteritis virus.

Equine arteritis virus (EAV) grown in Vero, BHK-21, and RK-13 cells was purified by pelleting, Sepharose 6B chromatography, and sucrose gradient centrifugation. Analysis of whole gradients by polyacrylamide slab gel electrophoresis and subsequent autoradiography revealed a large number of proteins in all fractions. Comparison of protein patterns of virus grown in the different cell systems showed that only three proteins with molecular weights of 12,000 (VP1), 14,000 (VP2), and 21,000 (VP3) were virus specific. VP1 is a phosphorylated core protein, while VP3 is a glycoprotein. These findings, together with data obtained earlier about morphology and RNA of the virion, lend further support to inclusion of EAV in the family Togaviridae with a possible relationship to lactic dehydrogenase virus.     

Respiratory syncytial virus polypeptides: their location in the virion.

Purified respiratory syncytial (RS) virus contains, in addition to the six to seven polypeptides previously reported (S. Levine, 1977, J. Virol., 21, 427–431), a large polypeptide (VPO), MW > 160,000. Treatment of purified virus with trypsin removes the major glycoproteins, VP1 and 2. Treatment of purified virus with 2% Triton X-100 in HBSS (equivalent to 0.15 M NaCl) solubilizes the glycoproteins VP1 and 2 and a nonglycosylated protein, VP5, MW 28,000, which suggests that VP5 is an M protein. Treatment with 2% Triton X-100 in 0.4 M NaCl solubilizes all the virion proteins except VPO and VP3, which are also not solubilized in 0.8 M NaCl. The results suggest that VP3, MW 44,000, is the major nucleocapsid protein, and that VPO is not a superficial contaminant of the virus preparation, but instead is closely associated with the nucleocapsid. Only VP3 is present in nucleocapsids isolated from RS virus-infected cells by isopycnic centrifugation in CsCl.     

Production and characterization of the neutralization antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector

DNA representing RNA segment 2 of bluetongue virus (BTV) serotype 10, corresponding to the gene that codes for the BTV neutralization antigen VP2, has been inserted into a baculovirus transfer vector in lieu of the 5' coding region of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). After cotransfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the derived recombinant transfer vector DNA, polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with one of these recombinant viruses, a protein that was similar in size and antigenic properties to the BTV VP2 protein was synthesized. Antibodies raised in mice or rabbits to the baculovirus expressed VP2 protein neutralized the infectivity of BTV-10 virus and to lesser extents BTV serotype 11 and 17 viruses but not BTV-13 virus.     

Expression of hepatitis A virus capsid sequences in insect cells

A cDNA coding for hepatitis A virus (HAV) VP1 and portions of the flanking VP3 and P2 sequences was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter and translational start codon. Cells infected with recombinant virus produced high levels of a 55 kDa protein, identified as containing HAV VP1 by reactivity with anti-VP1 serum. The expressed protein also reacted on immunoblots with human HAV convalescent sera as well as sera from rabbits immunized with intact HAV. This protein was found predominantly in the cytoplasm of infected insect cells, probably as an insoluble aggregate.     

Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus.

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes.     

Isolation of capsid proteins of foot-and-mouth disease virus by chromatofocusing

A method for the isolation of foot-and-mouth disease virus (FMDV) capsid proteins was developed. The FMDV capsid proteins VP1, VP2, VP3 and VPO were isolated from sucrose gradient purified virus by chromatofocusingin apH 7.4-4.0 gradient on Polybuffer exchanger PBE 94. Under the conditions used the proteins eluted in the sequence VP1, VP2, VP0 (when present) and VP3. Capsid protein VP4did not elute and could not be isolated by this method. Protein concentration in the eluate was monitored by the use of a radiolabelled marker and recoveries of approximately 50% of the input marker could be achieved when using up to 15 mg of virus and a 30-ml column. The high capacity and relative simplicity of chromatofocusing make it a useful alternative to other methods of purifying proteins.     

鸡贫血病毒VP2基因的克隆、序列分析及表达

目的：为探讨鸡贫血病毒的分子生物学特征。方法：采用ＰＣＲ方法获得允许贫血病毒的ＶＰ２基因,采用平端连接将其克隆到ＰＵＣ１１９载体上进行测序,采用粘端连接将其亚克隆到ＰＥＴ载体上并进行原核表达。结果：发现ＣＡＶ－ＳＪ１株的ＶＰ２基因共有６５１个碱基,同围外ＴＫ５８０３,２６Ｐ４,Ｃｕｓ－１、８２０２毒株的ＶＰ２基因相比分别有６、７、８、７个碱基的差别,其中３个是ＳＪ１株特有的碱基变化。由基因序列推导