Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells

DNA sequence coding for a portion of DNA binding protein (amino acids 3-58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.     

Altered tropism of recombinant bovine adenovirus type-3 expressing chimeric fiber

Recombinant bovine adenovirus-3 (BAV-3) has been used as a gene delivery vector for vaccination of calves. However, its usefulness as a vector for non-bovine species is limited due to poor transduction efficiency. To develop BAV-3 based vector for non-bovine species, we determined the feasibility of making targeted BAV-3 vector by modifying its natural tropism. We constructed a chimeric virus, BAV600, in which the knob region of the BAV-3 fiber protein was replaced with that from human adenovirus type 5 (HAV-5). Unmodified BAV-3 vector (BAV304) was able to transduce and direct the expression of green fluorescent protein (GFP) in non-bovine cells, with low efficiency. In contrast, the transduction efficiency of BAV600 in these cells was increased by 3-67-fold. Although, expression of early and late genes was detected in non-human cells, no progeny virus (BAV600) was detected in these cells. Our results suggest that it is possible to develop BAV-3 vectors with tropism for non-bovine species.     

A movement protein and three capsid proteins are all necessary for the cell-to-cell movement of apple latent spherical cheravirus

Summary. Immunoblot analysis of apple latent spherical cheravirus (ALSV)-infected leaves using a polyclonal antibody against the 21 C-terminal amino acids of a 53 K/42 K movement protein (MP) showed that a protein with an M_r of 42 kDa (42KP) is the dominant form found in vivo, which could indicate that the second AUG is used as an initiation codon of a ORF in RNA2. Co-expression of GFP with 42KP in tobacco epidermal cells showed that 42KP is able to facilitate cell-to-cell trafficking of GFP that is expressed in the same cells. The analysis of deletion mutants on each of MP, Vp24, Vp20, or Vp25 using an ALSV vector that stably expresses GFP indicated that an MP and three capsid proteins are all indispensable for the cell-to-cell movement of the virus. In ultrathin sections of infected leaves, a file of virus-like particles passing through the plasmodesmata connecting neighboring cells and tubular structures containing virus-like particles extending into the cytoplasm were observed. These results show that ALSV moves from cell to cell as virus particles.     

Rubella virus capsid protein induces apoptosis in transfected RK13 cells

Rubella virus is an enveloped positive-strand RNA virus that can cause mild to severe birth defects or death in an infected fetus. RV induction of programmed cell death, demonstrated in cell culture, has been implicated in the pathogenesis. The timing of apoptosis, 48 h p.i., suggested that accumulation of RV structural proteins might induce cell death in infected cells. Expression of RV structural proteins, capsid, envelope glycoproteins E1 and E2, in transiently transfected RK13 cells was as potent an inducer of cell death as RV infection. Immunofluorescence microscopy revealed that RV structural protein transfected cells exhibited the condensed nuclei typical of apoptotic cell death. Transfection with the capsid protein construct, but not E2 and E1, resulted in as much cell death as joint expression of all three RV structural proteins. Capsid required a membrane-anchoring domain to induce cell death, but a heterologous polypeptide fused to the capsid membrane anchor did not cause apoptosis. Deletion mutants demonstrated that the apoptosis-inducing activity resides in the N-terminal 170 amino acids of capsid. Though apoptosis-inducing capsid constructs appear to have an ER sub-cellular localization, disruption of the ER calcium storage capacity does not correlate with cell death. Mechanisms consistent with these results are discussed.     

Role of the nuclear matrix in the growth of herpes simplex virus type 2

Nuclear matrix was prepared from Vero cells infected with herpes simplex virus type 2. In the early stage of infection, both 155K and 110K viral proteins were associated with the nuclear matrix, while in the late stage, 155K protein, presumably a viral capsid protein, was predominantly associated with the matrix. Electron microscopic study showed that empty capsids were bound to the filamentous networks of the nuclear matrix of the late stage. Neither viral DNA nor viral DNA polymerase activity was associated with the nuclear matrix. These results may indicate that the nuclear matrix plays some role in the growth of herpes simplex virus, especially during the morphogenesis.     

A capsid-associated protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata: genetic location, sequence, transcriptional mapping, and immunocytochemical characterization

Two lambda gt11 clones containing overlapping DNA inserts encoding portions of a structural protein gene from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) were identified by their immunoreactivity with polyclonal antisera produced against purified polyhedra-derived virus. Sequence analysis of a 3.6-kb region of the baculovirus genome (map units 69.1-71.6) from which the lambda gt11 inserts originated revealed an open reading frame of 1872 nt (624 amino acids) encoding a predicted protein of 70.6 kDa. Northern blot, primer extension, and 3' S1 analysis of this ORF indicated that an mRNA of approximately 2100 nt was transcribed from this gene. The mRNA appears to initiate from a late promoter/mRNA start site consensus sequence GTAAG and is expressed at late times postinfection. A gene fusion containing the C-terminal 368 amino acids of the gene was constructed using a bacterial trpE expression vector. Rabbit antiserum made against the purified fusion protein reacted with a protein of 87 kDa on Western blots of infected cell extracts at 24 hr p.i. and thereafter. The p87 protein was shown to be a component of both budded and polyhedra-derived virus and purified capsids. Immunofluorescence analysis indicated that p87 is expressed late in infection and concentrated in infected cell nuclei.     

Alterations in virus protein synthesis and capsid production in infection with DI particles of herpesvirus

High multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (rho = 1.724 g/ml) than the DNA (rho = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al. 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.     

The 113th and 117th Charged Amino Acids in the 5th Alpha-Helix of the HBV Core Protein are Necessary for pgRNA Encapsidation

Although the structure–function of Hepatitis B virus (HBV) core protein has been investigated by numerous HBV core mutants, functions of many regions in the core protein are still remained to be identified. In this report, it was found that point mutations in the 113th and 117th negative-charged amino acids in the 5th helix region of the HBV core strongly affect pregenomic RNA (pgRNA) encapsidation. These mutations were introduced by site-directed mutagenesis. The following results were obtained from analyses of the mutants. First, endogenous polymerase activity (EPA) was assayed and activity was not detected only in the two mutants, E113K and E117K. Second, the pgRNA encapsidation level of each mutant related to a change in charge of two amino acid sites was evaluated. Mutations in the 113th and 117th amino acids into uncharged amino acids reduced pgRNA encapsidation levels. Moreover, changes of the two amino acids into positive-charged amino acids almost completely reduced pgRNA encapsidation levels. To test whether the mutant core proteins assembled into normal capsid particles, the assembly of the mutant core proteins was seen. However, none of the changes in the 113th and 117th amino acids affected capsid formation. From this data, it can be inferred that the above two amino acids in the 5th alpha-helix in the HBV core protein are important for pgRNA encapsidation.     

Subcellular localization of rev-gene product in visna virus-infected cells

The 1.4-kb mRNA of visna lentivirus is expressed early during the lytic infection of sheep choroid plexus cell cultures. It encodes for visna early gene 1 (VEG1) product, since renamed rev gene product (or Rev), based on significant amino acid sequence homologies between this protein and the proteins of simian immunodeficiency virus of macaque and human immunodeficiency virus type 2. In this report, we examined the subcellular localization and time course appearance of the Rev protein in visna virus-infected cells. Immunoprecipitation assays of [35S]methionine-labeled cell lysates with antisera raised against the Rev protein revealed a polypeptide of 19 kDa (p19rev). This protein was predominant early in the viral replication cycle and accumulated preferentially in the cytoplasmic/membrane fraction of infected cells. Indirect immunofluorescence staining of infected cells confirmed the cytoplasmic location of visna Rev protein and could reveal in some stained cells a higher concentration of Rev at the cellular plasma membrane. The regulating protein, still present late in the viral lytic cycle, is packaged into mature viral particles along with the structural gag and env gene products.     

Pathway of assembly of herpesvirus capsids: An analysis using DNA+ temperature-sensitive mutants of pseudorabies virus

It was reported previously that nine different DNA⁺ is mutants of Pr virus were all defective in the processes leading to the cleavage of concatemeric viral DNA and to the formation of nucleocapsids. Six of these mutants were found to assemble capsids at the nonpermissive temperature; three were capsid^? (Ladin et al., 1980). In the present communication we have characterized these mutants further. Each of the mutants was rescued by one specific restriction fragment of wild type virus DNA indicating that none of these mutants is altered in more than one locus. Many of the mutants complemented one another well at a frequency indicating that they are probably mutated in different genes. Cells infected at the nonpermissive temperature (41°) with the DNA⁺ mutants synthesized all the virus proteins synthesized by wild type-infected cells (as detected by PAGE) with two notable exceptions: (1) ts1-infected cells synthesized a thermolabile 142K capsid protein. (2) In ts1, ts109, and tsJ (all capsid^? mutants)-infected cells, detectable amounts of 35K capsid protein did not accumulate. The 35 K capsid protein is normally processed from a precursor protein, the processed protein being detectable in the nucleus of the infected cells only. In capsid^?-infected cells processing of the 35K protein precursor did not occur. Furthermore, accumulation in the nucleus of all capsid proteins was impaired in all the capsid^?-infected cells. Because at least two of the three capsid^? mutants are mutated in different genes, we conclude that the processing of the 35K protein, capsid assembly, and accumulation of capsid proteins in the nuclei are interdependent events which occur only if all the proteins necessary for capsid assembly are functional. On the basis of these results, we propose that the 35K protein is processed during capsid assembly and that assembly of the proteins into capsids is required for the continued movement of capsid proteins to, and their accumulation in, the nuclei of infected cells.     

Mapping of the structural proteins of the hepatitis A virus

The Western blot procedure with highly specific antipeptide antibody was applied to identify the electrophoretic mobility of hepatitis A virus capsid proteins. Polypeptides with molecular weights of 33, 29 and 27 kDa proved to be VP1, VP2, and VP3 proteins as they reacted with sera generated to VP1 recombinant protein and to synthetic oligopeptides 42-62 VP2 and 62-75 VP3, respectively.     

Identification and characterization of Orf virus 050 protein proteolysis

The Orf virus 050 (ORFV050) gene is located in the core region of the ORFV genome. It is similar to Vaccinia virus (VV) Copenhagen L4R, and encodes the DNA-binding virion core protein VP8, which has structures similar to the VV P25K core protein and may undergo similar proteolytic processing during virus assembly. Three conserved Ala–Gly–X motifs at putative cleavage sites were identified in ORFV050. To investigate the proteolysis of ORFV050 and its participation in viral assembly, full-length and site-directed mutant ORFV050 recombinant proteins were constructed and expressed. Two distinct protein bands of 28.5 and 25 kDa were detected in the infected cells using anti-ORFV050 polyclonal antiserum. A potential cleavage site was identified at amino acids 30–32 of ORFV050. Mutation of AG/A to (R) in ORFV050 abolished the process of proteolysis. ORFV050 is a late gene synthesized during viral replication in the host cytoplasm. According to these results, we conclude that ORFV050 undergoes proteolysis and plays an important role in viral assembly.     

Evidence against the role of K+ in the shut-off of protein synthesis by vesicular stomatitis virus.

Intracellular K+ ion concentration and transport were measured in L cells infected with wild type VSV, which rapidly inhibits total protein synthesis in infected cells, and with a mutant, RI, defective in this function. No alterations in intracellular K+ ion concentration or transport were observed until late in infection and the late changes seen were similar for both viruses. Thus the inhibition of total protein synthesis by VSV cannot be ascribed to virus induced changes in host K+ ion concentration.     

Complete genome sequence of switchgrass mosaic virus, a member of a proposed new species in the genus Marafivirus

The complete genome sequence of a virus recently detected in switchgrass (Panicum virgatum) was determined and found to be closely related to that of maize rayado fino virus (MRFV), genus Marafivirus, family Tymoviridae. The genomic RNA is 6408 nucleotides long. It contains three predicted open reading frames (ORFs 1-3), encoding proteins of 227?kDa, 43.9?kDa, and 31.5?kDa, compared to two ORFs (1 and 2) for MRFV. The complete genome shares 76?% sequence identity with MRFV. The nucleotide sequence of ORF2 of this virus and the amino acid sequence of its encoded protein are 49?% and 77?% identical, respectively, to those of MRFV. The virus-encoded polyprotein and capsid protein aa sequences are 83?% and 74-80?% identical, respectively, to those of MRFV. Although closely related to MRFV, the amino acid sequence of its capsid protein (CP) forms a clade that is separate from that of MRFV. Based on the International Committee on Taxonomy of Viruses (ICTV) sequence-related criteria for delineation of species within the genus Marafivirus, the virus qualifies as a member of a new species, and the name Switchgrass mosaic virus (SwMV) is proposed.     

Characterization of the U(L)33 gene product of herpes simplex virus 1

The U(L)33 protein is one of six genes (including U(L)6, U(L)15, U(L)17, U(L)28, and U(L)32) required for cleavage of viral concatemeric DNA into unit-length genomes and packaging of the virus genomes into preformed capsids. The U(L)25 gene product is dispensable for cleavage of viral DNA but essential for packaging of DNA into capsids. A polyclonal antiserum was produced against an affinity-purified protein containing the full-length U(L)33 gene product of herpes simplex virus 1 fused to glutathione-S-transferase. A protein of approximate M(r) 19,000 that reacted with the antiserum was detected in immunoblots of herpes simplex virus 1-infected cellular lysates. This protein was not detected in lysates of mock-infected cells or cells infected with a mutant virus containing a stop codon in U(L)33, indicating that the 19,000 M(r) protein is the product of the U(L)33 open reading frame. The U(L)33 gene product was not detected in purified virions or capsids. Accumulation of the U(L)33 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. Indirect immunofluorescence analysis demonstrated that U(L)33 protein accumulated predominantly within replication compartments in the central domains of infected cell nuclei and within the cytoplasm. Localization of the U(L)33 gene product in replication compartments was maintained in cells infected with a variety of cleavage/packaging mutants.