Infected Tissue Culture Cells Research Articles

Detection of virus-specific RNA-dependent RNA polymerase activity in extracts from cells infected with lymphocytic choriomeningitis virus: in vitro synthesis of full-length viral RNA species.

We have developed an in vitro assay for the lymphocytic choriomeningitis virus (LCMV) RNA-dependent RNA polymerase with ribonucleoprotein complexes extracted from acutely infected tissue culture cells. The RNA products synthesized in vitro corresponded in size to the full-length genomic L and S RNAs and subgenomic NP and GP mRNAs normally produced in vivo during acute LCMV infection. In a temporal analysis spanning the first 72 h of acute infection, the in vitro polymerase activity of ribonucleoprotein complexes was maximal at 16 h and declined significantly at later times. In contrast, the intracellular levels of the viral L protein (the putative polymerase protein) appeared to be maximal at 48 to 72 h postinfection. Our results suggest that the accumulation of L protein correlates with reduced viral replication and transcription at later times in acute infection and may be involved in the transition from acute to persistent LCMV infection.

Viral DNA from Cells Infected with Porcine Circovirus

It has been demonstrated earlier that the genome of the porcine circovirus (PCV) isolated from virions is a circular single-stranded DNA (ssDNA). This report describes the isolation and characterization of PCV-specific DNA from infected cell cultures. Two types of doublestranded DNA (dsDNA) were observed which behaved like supercoiled and relaxed circular molecules, respectively. Both types of dsDNA displayed infectivity in transfection experiments and are regarded as replicative forms (RF) of the viral genome. In addition to complete single-stranded viral genomes and RF DNA molecules, PCV-specific subgenomic fractions of DNA molecules with a sedimentation coefficient of about 5 S were isolated from infected tissue culture cells. This 5 S DNA was also isolated from purified virions. It is single-stranded and represents a defined region of the viral genome.

Humoral and cytotoxic T cell immune responses to internal and external structural proteins of simian virus 5 induced by immunization with solid matrix-antibody-antigen complexes.

Monoclonal antibodies (MAbs) were complexed to solid matrices and used to purify virus proteins from simian virus 5 (SV5)-infected tissue culture cells, ideally in such a way as to bind equimolar amounts of antigen to antibody. The resulting solid matrix-antibody-antigen (SMAA) complexes were then used as immunogens and successfully induced specific humoral and cytotoxic T cell responses. By attaching more than one MAb to the solid matrix and using such complexes to purify the respective proteins multivalent immunization was achieved. Analysis of the cytotoxic T cell response of immunized animals indicated that both surface and internal SV5 structural proteins can act as target antigens. Immunization with SMAA complexes, in the absence of adjuvant, induced higher levels of antibody than the antigen alone precipitated on alum. However, immunization with SMAA complexes resulted in relatively less antibody being produced to the antigenic determinant through which the protein is coupled, via antibody, to the SMAA complex compared with the amount of antibody produced against other antigenic determinants on that protein. The particulate solid matrix used to form the SMAA complexes in most of the experiments was a 'fixed' and killed suspension of the Cowan A strain of Staphylococcus aureus, although preliminary results indicated that Protein A-Sepharose could also be used as a solid matrix. Prior immunization with S. aureus alone did not reduce the level of the immune response to the appropriate antigen on subsequent immunization with S. aureus-antibody-antigen complexes. In fact on immunization of mice with these complexes the level of antibody produced to the S. aureus matrix itself was less when S. aureus-antibody-antigen complexes were used as immunogens than when S. aureus or S. aureus-antibody complexes were used. Furthermore, rabbits immunized with S. aureus-mouse MAb-antigen complexes showed a vigorous immune response to the antigen.

Expression of herpes simplex virus type 1 (HSV-1) latency-associated transcripts and transcripts affected by the deletion in avirulent mutant HFEM: evidence for a new class of HSV-1 genes.

During latent herpes simplex virus type 1 (HSV-1) infection in the trigeminal ganglia of mice, three virus-specific transcripts, 2.0, 1.5, and 1.45 kilobases (kb), are detectable by Northern (RNA) blot analysis, but only the 2.0-kb transcript can be detected in HSV-1-infected tissue culture cells (J.G. Spivack and N. W. Fraser, J. Virol. 61:3842-3847, 1987). Since these latency-associated genes map to a diploid region of the genome, transcription from the deletion mutant HFEM, which contains only one complete copy of these genes, was investigated to determine the effect of gene dosage. The 4.1-kb HFEM deletion is located between the alpha genes ICP0 and ICP27. ICP0 mRNA and the 2.0-kb latency-associated transcript were present at normal levels during HFEM infection, but ICP27 mRNA and 0.9- and 1.1-kb transcripts that map near the deletion were not readily detectable. The levels of expression of one or more of these genes might be an important determinant of HSV-1 virulence in animal hosts. ICP27 mRNA accumulated when protein synthesis was inhibited before HFEM infection, implying that the deletion may affect ICP27 regulatory rather than coding elements. Expression of the 2.0-kb latency-associated transcript was characterized in infected CV-1 cells with metabolic inhibitors and strand-specific probes. On the basis of metabolic inhibitor studies, the gene encoding the 2.0-kb latency-associated transcript is not an alpha gene. During HSV-1 replication in infected tissue culture cells, the beta and gamma genes require the prior expression of alpha gene products. However, the latency-associated RNAs are expressed in the absence of detectable levels of alpha transcripts in latently infected mice. Thus, this latency-associated gene family appear to be regulated quite differently than alpha, beta, or gamma genes. For these reasons, and because the latency-associated genes may perform latent rather than replicative functions, we propose that they should be considered members of a new HSV-1 gene class, the lambda genes.

Characterization of a recombinant vaccinia virus expressing human melanoma-associated antigen p97.

p97 is a cell surface glycoprotein expressed at high levels in most human melanomas but present only in trace amounts in normal adult tissues. We are interested in exploring the possibility of using recombinant vaccinia virus to express a specific tumor-associated antigen as a vaccine against human cancer. To this end, we constructed a recombinant virus, v-p97NY, which contains the entire coding sequence for p97 under the control of the vaccinia virus 7.5K promoter. Upon infection of tissue culture cells, v-p97NY expressed high levels of a membrane-bound glycoprotein immunoreactive with a p97-specific monoclonal antibody. Immunization of mice with this recombinant elicited high-titered antibodies against p97. Spleen cells isolated from these mice proliferated in vitro when stimulated either with purified p97 protein or with syngeneic cells expressing p97 antigen. Delayed-type hypersensitivity was also observed in immunized mice after challenge with p97-expressing cells. These findings indicate the potential usefulness of v-p97NY and similar recombinants in tumor immunotherapy.

Detection of herpes simplex virus type 1 transcripts during latent infection in mice.

A latent infection can be established in the trigeminal ganglia of mice after corneal inoculation of herpes simplex virus type 1 (HSV-1). With a virion DNA probe, three transcripts (2.0, 1.5, and 1.45 kilobases [kb]) were detected by Northern blot (RNA blot) analysis of RNAs isolated from the ganglia of latently infected mice. All three transcripts hybridized to a nick-translated HSV-1 DNA probe from BamHI restriction fragment B (strain F). These RNAs were mapped with subfragments of BamHI-B and with strand-specific probes. They are at least partially colinear with each other, map to a 3.0-kb PstI-MluI subfragment of BamHI-B, and are transcribed from left to right. The latent HSV-1 RNAs partially overlap the 3' end of ICP0 mRNA but are transcribed in the opposite direction. The latent RNAs were not as extensively poly(A)+ as actin mRNA. The HSV-1 transcripts detected in latently infected trigeminal ganglia did not correspond with any that have been previously identified in permissively infected cells in tissue culture. However, the 2.0-kb HSV-1 RNA present during latency was detectable at reduced levels in the trigeminal ganglia of acutely infected mice and in infected tissue culture cells. The data indicate that the pattern of viral gene expression during HSV-1 latency in the trigeminal ganglia of mice does not represent restriction of the genes actively transcribed during the lytic replication cycle in tissue culture.

Analysis of the genomic l rna segment from lymphocytic choriomeningitis virus

The arenavirus genomic L RNA segment represents approximately 70% of the viral genetic material but current understanding of the organization, regulation, and functioning of the viral L products remains limited. Biological studies with reassortant viruses have implicated the L RNA segment in the lethal infection of adult guinea pigs produced by LCMV-WE but no further explanation of the pathogenic process is presently available. We have initiated a detailed molecular analysis of LCMV L products based on construction and characterization of L-specific cDNA clones and synthesis of L-specific hybridization probes. Nucleotide sequencing studies have allowed the derivation of a partial amino acid sequence for a predicted L protein and antisera raised against synthetic peptides have demonstrated an L protein in Western blotting experiments. Using this approach we have identified a single high molecular weight protein (approximately 200,000 Da) in purified virions and in viral ribonucleoprotein complexes extracted from acutely infected tissue culture cells. This L protein is translated from a nonpolyadenylated, genomic complementary L mRNA and potentially represents part or all of the viral RNA-dependent RNA polymerase.

Mapping bovine herpesvirus type 1 latency-related RNA in trigeminal ganglia of latently infected rabbits

Here we have used the bovine herpesvirus type 1 (BHV-1) rabbit model together with in situ nucleic acid hybridization to identify and map viral RNA present in latently infected neurons. Radioactively labeled cloned HindIII fragments representing most of the BHV-1 genome (Cooper strain) were individually hybridized to sections of trigeminal ganglia taken from rabbits during acute and latent stages of infection. Whereas all viral genomic fragments hybridized to lytically infected tissue culture cells and to acutely infected ganglia, only HindIII fragment D (map units 0.734 to 0.842) hybridized to latently infected ganglionic neurons. Additional in situ hybridization experiments using subcloned fragments of HindIII-D further mapped the latency-related viral RNA to a 1.9-kilobase region (map units 0.734 to 0.748) of the viral genome. These results indicate that BHV-1 gene transcription is restricted during the latent phase of infection; further, they suggest that specific viral transcription may be involved in establishment or maintenance of latent BHV-1 infection.

Expression of envelope glycoproteins of human immunodeficiency virus by an insect virus vector.

The envelope gene of human immunodeficiency virus was inserted into the genome of an insect virus vector (Autographa californica nuclear polyhedrosis virus). Upon infection of tissue culture cells, this recombinant virus produced immunoreactive polypeptides related to the envelope glycoproteins of human immunodeficiency virus. Serological survey indicates such polypeptides would be of value as antigens in diagnostics for acquired immunodeficiency syndrome.

An enzyme immunoassay for dengue antibody using infected cultured mosquito cells as antigen

A simple enzyme immunoassay (EIA) for dengue virus antibody detection used infected tissue culture cells as antigen. C6/36 Aedes albopictus cells infected with dengue virus, and uninfected control cells were fixed with 3.3% formalin. This technique eliminated microplate coating and laborious antigen preparation, and it facilitated rapid screening of large numbers of sera. Formalin also inactivated dengue virus infectivity. Microplates prepared by this technique could be stored at −20 or −70°C for at least two months. Human sera were adsorbed with C6/36 cells prior to testing in the EIA in order to reduce nonspecific binding to C6/36 cells. Dengue; Enzyme immunoassay; Mosquito cell

A detailed mutational analysis of Vmw110, a trans-acting transcriptional activator encoded by herpes simplex virus type 1.

Viral genes expressed during infection of tissue culture cells by herpes simplex virus type 1 (HSV-1) can be divided into three broad temporal groups called immediate-early (IE), early and late. Prior expression of IE gene products is required before later classes of genes can be transcribed. Using cloned copies of viral genes in plasmid vectors, it has been shown that the products of IE genes 1 and 3 (Vmw110 and Vmw175) can activate transcription from both viral and cellular promoters in short-term transfection assays. The effects of these two IE gene products are markedly synergistic in HeLa cells; the activation observed with both genes present is about 20 times that observed with Vmw175 alone. The mechanism of this activation and the properties of the IE proteins that are required are not well understood. This paper describes the construction of a large number of in-frame insertion and deletion mutations in a plasmid-encoded copy of IE gene 1. The ability of the mutant Vmw110 polypeptides to activate gene expression (in the presence of Vmw175) from the HSV-1 glycoprotein gD promoter linked to the chloramphenicol acetyl transferase gene was studied. The results show that the structural integrity of at least five regions of the polypeptide are important for its function in the presence of Vmw175.

The alpha protein ICP0 does not appear to play a major role in the regulation of herpes simplex virus gene expression during infection in tissue culture.

The herpes simplex virus type 1 (HSV-1) alpha protein ICP0 trans-activates HSV-1 early genes in transient expression assays. To investigate the function of ICP0 during HSV-1 infection, we have lowered the level of ICP0 by use of a recombinant plasmid that has been engineered to express the antisense message. Cell lines were constructed which stably carry the antisense plasmid. Total protein profiles from infected antisense cell lines showed that the level of ICP0 was reduced to less than 10% of the wild type level in two of the cell lines. However, reducing the level of ICP0 did not have a significant effect on the expression of HSV-1 early or late genes. The polypeptide patterns for the remaining infected cell polypeptides were similar in that no bands were absent although there were some quantitative differences. The level of two early proteins, glycoprotein B and glycoprotein D was reduced in one of the cell lines, however, levels were nearly equivalent to the control infection for two other cell lines tested. Virus yields were the same for the antisense cell lines and for parent cells. Decreased ICP0 levels did not lead to more restrictive phenotypes for an alpha 4 or alpha 27 mutant as protein patterns were similar for these mutants in antisense and parent cells. Therefore, while ICP0 has been demonstrated to be a strong inducer of gene expression in transient expression assays, it does not appear to have a major role as an activator during the productive infection of tissue culture cells.

The product of gene US11 of herpes simplex virus type 1 is expressed as a true late gene.

The genes of herpes simplex virus type 1 (HSV-1) can be divided into at least three temporally regulated groups termed immediate early (IE), early and late. We have studied in detail the expression of a member of the late class of genes, US11, which encodes a polypeptide of apparent molecular weight 21K. Highly specific and sensitive probes were used to monitor US11 RNA and protein synthesis during HSV-1 infection of tissue culture cells in the presence and absence of phosphonoacetic acid, an inhibitor of viral DNA replication. The results were compared with a similar study of the products of a delayed early gene, US6, encoding glycoprotein D (gD). It was found that the patterns of RNA and protein synthesis from US11 were significantly different to those of gD. US11 products appeared later and accumulated until late in infection, while gD RNA was significantly reduced at late times. In the presence of the inhibitor of DNA synthesis, US11 gene expression was reduced 50- to 100-fold while gD expression was reduced five- to tenfold. We conclude that US11 behaves as a true late gene during HSV-1 infection. However, the use of sensitive assays, which allowed the detection of very low levels of US11 gene products under conditions designed to eliminate DNA replication, brings into question the absolute requirement for DNA replication for the expression of a true late HSV-1 gene. These results are discussed in terms of current models for the regulation of late gene expression.

Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration

Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens.

Quantitation of HeLa cell monolayer invasion by Shigella and Salmonella species.

A major determinant in the virulence of Salmonella and Shigella spp. is the ability of these organisms to invade epithelial cells of the gastrointestinal mucosa and multiply intracellularly. The invasion of cell culture monolayers is a convenient experimental system to evaluate eucaryotic cell penetration and is correlated with the potential of a strain to cause human disease. We have developed an agarose-L agar overlay technique which allows for the convenient quantitation of the number of infected tissue culture cells in a monolayer. Bacterial strains were introduced onto antibiotic-free HeLa cell monolayers. Infected monolayers were washed, and noninternalized bacteria were counterselected with kanamycin (50 micrograms/ml). The number of infected HeLa cells present was determined by overlaying the monolayer with distilled water-agarose (0.5 to 1.5%) followed by an equal volume of 2X L agar. Bacterial colonies formed over infected cells in 24 h at 37 degrees C, and wells were counted with a dissecting microscope under X2 power. Bacterial colonies were not observed with noninvasive variants of Shigella spp. To obtain countable wells (20 to 200 CFU) the multiplicity of infection or invasion times were adjusted. With a 90-min invasion time, the invasive potential of a strain was reflected by the multiplicity of infection needed to produce countable wells. The quantitation of bacterium-invaded cells by using standard bacteriological methods is a convenient and rapid method to evaluate the invasive potential of bacterial strains. Additionally, parameters essential for the invasive process can easily be investigated.

Detection of reovirus type 3 in the porta hepatis of an infant with extrahepatic biliary atresia: ultrastructural and immunocytochemical study.

This report describes immunocytochemical and ultrastructural methods which led to the identification of Reovirus type 3 (Reo-3) in the porta hepatis of a patient with extrahepatic biliary atresia. The study indicates that Reo-3 antigenic sites are demonstrable by the avidin-biotinylated complex peroxidase method following formalin fixation and paraffin embedding, but are destroyed by freezing and thawing prior to fixation. Deparaffinization of the block and subsequent rembedding in epon-araldite did not alter immunoperoxidase staining. This procedure offered the advantage of higher light microscopic resolution of semithin (1 micron) sections and assisted in the selection of specific areas for ultrastructural studies. Localization of Reo-3 in extrahepatic biliary atresia was confined to a biliary remnant in which there were acutely inflammed, partially necrotic microscopic ducts. Electron microscopic examination of the immunoreactive sites revealed virus-like particles similar in appearance to Reo-3 particles in infected tissue culture cells. The observations presented here support previously reported serologic data which have shown an association between Reo-3 infection and extrahepatic biliary atresia.

Comparison of cytoskeletal organization in canine distemper virus-infected and uninfected cells.

The organization of vimentin filaments, keratin filaments, microtubules and microfilaments was compared in canine distemper virus (CDV)-infected and uninfected cells by indirect immunofluorescence. Infection of tissue culture cells with CDV caused a total reorganization of all the cytoskeletal structures with the most notable changes in the microtubules and intermediate filaments. During virus infection two different patterns of staining were observed for both the intermediate filaments and microtubules, suggesting a step-by-step reorganization of the structures. While the two types of intermediate filaments (vimentin and keratin) had quite different staining patterns, the vimentin (but not keratin) filaments had a distribution pattern similar to the microtubules in both infected and uninfected cells. These results suggest that microtubules and vimentin (but not keratin) filaments may have a close association in CDV-infected cells.

Requirements for transport of HSV-1 glycoproteins to the cell surface membrane of human fibroblasts and Vero cells.

The intracellular transport of the HSV-1 glycoproteins gA/gB, gC and gD has been followed by the indirect immunofluorescence technique (IIF). Infected tissue culture cells were stained with monoclonal antibodies made to the individual glycoproteins and with fluorochrome-coupled wheat germ agglutinin reacting specifically with Golgi apparatus of the cells. Staining of either infected, human fibroblasts or of VERO cells at 9 hours p.i. with antibodies to gA/gB showed a prominent ring-like nuclear fluorescence and distinct staining of the Golgi apparatus in the cells. Antibodies to gC and gD stained mainly the Golgi apparatus and areas close to or at the surface of the cells. By immunocytolysis of HSV-1-infected VERO cells the viral glycoproteins were demonstrable at the surface of cells but growth of infected cells in the presence of either TM or monensin inhibited the expression of most of the viral glycoproteins at the cell surface. Blocking of the glycosylation of the viral glycoproteins with tunicamycin (TM) was followed by accumulation of the core of the glycoproteins gA/gB and gD in granular structures close to the nucleus as seen by immunofluorescence microscopy. Antibodies to gC did also stain granules close to the nucleus but in addition the periphery of the cells were stained. Inhibition of intracellular transport from the Golgi apparatus by the carboxylic ionophore monensin was followed by accumulation of all the HSV-1 glycoproteins in vesicles derived from the Golgi apparatus in both human fibroblasts and VERO cells. Our data thus support the hypothesis that the HSV-1 glycoproteins are processed in the Golgi apparatus before the transport to and incorporation into the cell surface membrane of infected cells and into virion envelopes.

A common region for proviral DNA integration in MoMuLV-induced rat thymic lymphomas.

Similarly to other mammalian and avian retroviruses that lack a transforming gene, moloney murine leukaemia virus (MoMuLV) causes no morphological transformation in infected tissue culture cells. However, following injection in an appropriate animal host, MoMuLV induces mainly thymic lymphomas after a long latency period. A common characteristic of neoplasms induced by retroviruses lacking transforming genes is their clonal origin. Here we have generated MoMuLV-induced rat thymic lymphomas and confirmed their clonal nature. Furthermore, we took advantage of the clonality of these tumours to investigate the specificity of provirus integration in the tumour DNA. We reasoned that if several independently derived thymic lymphomas would contain the provirus integrated in the same region of cellular DNA, this would be a strong indication that this integration event is a contributing factor in oncogenesis. The results indicate that there is indeed a cellular DNA region (termed the MLVI-1 locus) that serves as the substrate for proviral DNA integration in 5 out of 16 tumours we examined.

Ultrastructure of arthritis induced by a caprine retrovirus.

The ultrastructure of early retrovirus-induced arthritis was studied sequentially in 20 goat kids inoculated with caprine arthritis-encephalitis virus. Synovial lesions began as intercellular edema and collagen fragmentation and continued as progressive mononuclear cell infiltration and lining cell hyperplasia, hypertrophy, and necrosis. At 18 through 45 days after the inoculation, lining cells contained small accumulations of virus-like particles similar to virus seen in infected tissue culture cells. No virus was seen budding from infected lining cell membranes.