Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry

Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Sine large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.     

DNA damage: a consequence of the combined effect of virus infection and incorporated radioactive thymidine.

Infection of the human KB cell line with Frog Virus 3 brought about a partial degradation of radioactively labeled cell DNA as determined by alkaline sucrose sedimentation analysis. Further characterisation of this phenomenon revealed that degradation only occured when a sufficiently large amount of radioactive precursors was previously incorporated into the cell DNA. Therefore DNA damage appeared to be the result of a combined effect of incorporated radioisotopes and viral infection. It is suggested that the degradation of the radioactively labeled cell DNA could be a consequence of the inhibition of cell protein synthesis by frog Virus 3 since treatment of the cells with cycloheximide also led to DNA degradation.     

Cell killing by avian leukosis viruses.

Infection of chicken cells with a cytopathic avian leukosis virus resulted in the detachment of killed cells from the culture dish. The detached, dead cells contained more unintegrated viral DNA than the attached cells. These results confirm the hypothesis that cell killing after infection with a cytopathic avian leukosis virus is associated with accumulation of large amounts of unintegrated viral DNA. No accumulation of large amounts of integrated viral DNA was found in cells infected with cytopathic avian leukosis viruses.     

Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production.

Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.     

Mass spectrometry reveals specific and global molecular transformations during viral infection

Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.     

Apoptosis: a mechanism of cell killing by influenza A and B viruses.

In previous studies, we observed that the virulent avian influenza A virus A/Turkey/Ontario/7732/66 (Ty/Ont) induced severe lymphoid depletion in vivo and rapidly killed an avian lymphocyte cell line (RP9) in vitro. In examining the mechanism of cell killing by this virus, we found that Ty/Ont induced fragmentation of the RP9 cellular DNA into a 200-bp ladder and caused ultrastructural changes characteristic of apoptotic cell death by 5 h after infection. We next determined that the ability to induce apoptosis was not unique to Ty/Ont. In fact, a variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses.     

Properties of simian adenovirus 7 after one single passage in simian marmoset lymphoblastoid cells transformed by Epstein Barr virus

Simian adenovirus 7 gave an abortive infection in simian marmoset lymphoblastoid cells, B 95-8 and M 81 (transformed by Epstein Barr Virus) whereas non transformed simian lymphocytes could not replicate this virus. Electron dense incomplete particles with a lower density than standard virus in CsCl gradients were isolated. Virus yields were low and the percentage of cells containing viral antigen as measured by immunofluorescence was 0.01% for B 95-8 cells and still less for the M 81 cells. After a single passage in either lymphoblastic cell lines, they had a reduced oncogenicity in vivo. The polypeptide pattern analysis by PAGE showed some modifications.     

Bystander killing during avian leukosis virus subgroup B infection requires TVB(S3) signaling

Cell killing by avian leukosis virus subgroup B (ALV-B) in cultures has been extensively studied, but the molecular basis of this process has not been established. Here we show that superinfection, which has been linked to cell killing by ALV-B, plays no crucial role in cell death induction. Instead, we show that signaling by the ALV-B receptor, TVB(S3), a member of the tumor necrosis factor receptor family, is essential for ALV-B-mediated cell death. TVB(S3) activated caspase-dependent apoptosis during ALV-B infection. Strikingly, apoptosis induction occurred predominantly in uninfected cells, while ALV-B-infected cells were protected against cell death. This bystander killing phenomenon was reproduced in a virus-free system by cocultivating ALV-B Env-expressing cells with TVB(S3)-expressing cells. Taken together, our results indicated that ALV-B-mediated apoptosis is triggered by ALV-B Env-TVB(S3) interactions.     

A single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with the ability to cause growth hormone deficiency syndrome.

Persistent infection of C3H/St mice with certain strains of lymphocytic choriomeningitis virus (LCMV) causes a growth hormone (GH) deficiency syndrome (GHDS) manifested as growth retardation and hypoglycemia. Infected mice show high levels of viral replication in the GH-producing cells in the anterior pituitary leading to decreased synthesis of GH mRNA and protein despite the absence of detectable virus-induced cell structural damage. Virus clones isolated from the GHDS-negative LCMV WE strain can cause the disease, while others cannot. The genetic basis of this phenotypic difference is a nucleotide substitution resulting in a single amino acid difference in the viral glycoprotein. Reassortant studies indicate that the single amino acid substitution (Ser-153 to Phe) is sufficient to allow infection of the GH-producing cells and cause GHDS. These results show that a single change in the genome can affect viral pathogenicity by altering the tropism of the virus.     

Virus susceptibility of Chinese hamster ovary (CHO) cells and detection of viral contaminations by adventitious agent testing

Biopharmaceuticals are of increasing importance in the treatment of a variety of diseases. A remaining concern associated with their production is the potential introduction of adventitious agents into their manufacturing process, which may compromise the pathogen safety of a product and potentially cause stock‐out situations for important medical supplies. To ensure the safety of biological therapeutics, regulatory guidance requires adventitious agent testing (AAT) of the bulk harvest. AAT is a deliberately promiscuous assay procedure which has been developed to indicate, ideally, the presence of any viral contaminant. One of the most important cell lines used in the production of biopharmaceuticals is Chinese hamster ovary (CHO) cells and while viral infections of CHO cells have occurred, a systematic screen of their virus susceptibility has never been published. We investigated the susceptibility of CHO cells to infection by 14 different viruses, including members of 12 families and representatives or the very species that were implicated in previously reported production cell infections. Based on our results, four different infection outcomes were distinguished, based on the possible combinations of the two factors (i) the induction, or not, of a cytopathic effect and (ii) the ability, or not, to replicate in CHO cells. Our results demonstrate that the current AAT is effective for the detection of viruses which are able to replicate in CHO cells. Due to the restricted virus susceptibility of CHO cells and the routine AAT of bulk harvests, our results provide re‐assurance for the very high safety margins of CHO cell‐derived biopharmaceuticals. Biotechnol. Bioeng. 2010;106: 598–607. © 2010 Wiley Periodicals, Inc.     

Novel paradigms of innate immune sensing of viral infections

According to the existing paradigm, cellular recognition of viral infection is mediated by molecular patterns within the virus particle or produced during virus replication. However, there are various physical cellular changes indicative of infection that could also trigger innate antiviral responses. The type-I interferon response is rapidly engaged to limit viral infection and a number of studies have shown that the interferon response, or components of it, are induced by general perturbations to cellular processes. Virus entry requires membrane and cytoskeletal perturbation, and both membrane fusion or actin depolymerising agents alone are able to activate antiviral genes. Viruses cause cellular stress and change the cellular environment, and oxidative stress or endoplasmic reticulum stress will amplify antiviral signaling. Many of these responses converge on interferon regulatory factor 3, suggesting that it plays a crucial role in determining the degree to which the cell responds. This review highlights novel paradigms of viral recognition and speculates that viral infection is sensed as a danger signal.     

Cytocidal effect caused by the envelope glycoprotein of a newly isolated avian hemangioma-inducing retrovirus.

We isolated a field strain of avian hemangioma retrovirus (AHV) which induces a cytopathic effect (CPE) on cultured avian and mammalian cells shortly after infection. The kinetics of cell killing were dependent on the multiplicity of infection. The CPE on avian and mammalian cells was independent of virus replication, because UV-irradiated virus led to cell death as well. Biochemical and genetic experiments indicated that AHV env gene products were responsible for the CPE. Partially purified AHV envelope glycoproteins (gp85), but not those of the Rous sarcoma virus Prague C strain, induced a CPE. Rous-associated virus type 1, in which the env region was replaced by the AHV gp85 region, induced a CPE on avian and mammalian cultured cells. Therefore, we suggest that CPE is induced by AHV via interaction between viral gp85 and the cell membrane. This mode of CPE is unique among avian sarcoma-leukemia viruses.     

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.     

Axin expression delays herpes simplex virus‐induced autophagy and enhances viral replication in L929 cells

Axin, a negative regulator of the Wnt signaling pathway, plays a critical role in various cellular events including cell proliferation and cell death. Axin‐regulated cell death affects multiple processes, including viral replication. For example, axin expression suppresses herpes simplex virus (HSV)‐induced necrotic cell death and enhances viral replication. Based on these observations, this study investigated the involvement of autophagy in regulation of HSV replication and found axin expression inhibits autophagy‐mediated suppression of viral replication in L929 cells. HSV infection induced autophagy in a time‐ and viral dose‐dependent manner in control L929 cells (L‐EV), whereas virus‐induced autophagy was delayed in axin‐expressing L929 cells (L‐axin). Subsequent analysis showed that induction of autophagy by rapamycin reduced HSV replication, and that inhibiting autophagy by 3‐methyladenine (3MA) and beclin‐1 knockdown facilitated viral replication in L‐EV cells. In addition, preventing autophagy with 3MA suppressed virus‐induced cytotoxicity in L‐EV cells. In contrast, HSV replication in L‐axin cells was resistant to changes in autophagy. These results suggest that axin expression may render L929 cells resistant to HSV‐infection induced autophagy, leading to enhanced viral replication.     

Differential cell killing by lymphomagenic murine leukemia viruses occurs independently of p53 activation and mitochondrial damage

Upon inoculation into AKR mice, mink cell focus-forming murine leukemia virus (MCF MLV) accelerates thymic lymphoma formation. During the preleukemic phase of disease, we observed the induction of apoptosis in thymic lymphocytes. A similar induction of apoptosis was observed for cultured mink epithelial cells after MCF13 MLV infection. In this study, the relevance of viral pathogenicity to cell killing was determined by testing the susceptibility of various cell types from different species to lymphomagenic MLVs. We observed that the cytopathic effect of lymphomagenic MLVs was restricted to mink cells. Southern blot analysis of MLV-infected cells revealed an accumulation of the linear form of unintegrated viral DNA, particularly in mink cells after MCF13 MLV infection. Thus, a strong correlation was observed between viral superinfection, which results in the accumulation of high levels of unintegrated viral DNA, and cell killing. Immunoblot analysis for MCF13 MLV-infected mink epithelial cells did not show a significant change in total p53 levels or its phosphorylated form at Ser-15 compared with that in mock-treated cells. Moreover, a time course analysis for mink epithelial cells infected with MCF13 MLV did not reveal mitochondrial depolarization or a significant change in Bax levels. These results demonstrate that MCF13 MLV induces apoptosis preferentially in cells in which superinfection occurs, and the mechanism involved is independent of p53 activation and mitochondrial damage.     

Dissociation of unintegrated viral DNA accumulation from single-cell lysis induced by human immunodeficiency virus type 1.

Acute cytopathic retroviral infections are accompanied by the accumulation, due to superinfection, of large amounts of unintegrated viral DNA in the cells. The cytopathic effects of human immunodeficiency virus type 1 (HIV-1) infection are specific for cells that express the CD4 viral receptor and consist of syncytium formation and single-cell lysis. Here we investigated the relationship between superinfection and single-cell lysis by HIV-1. Antiviral agents were added to C8166 or Jurkat lymphocytes after HIV-1 infection had occurred. Treatment with azidothymidine or a neutralizing anti-gp120 monoclonal antibody reduced or eliminated, respectively, the formation of unintegrated viral DNA but did not inhibit single-cell killing. Furthermore, in the infected Jurkat cells, the levels of unintegrated viral DNA peaked several days before significant single-cell lysis was observed. Essentially complete superinfection resistance was established before the occurrence of single-cell killing. These results demonstrate that single-cell lysis by HIV-1 can be dissociated from superinfection and unintegrated viral DNA accumulation. These results also indicate that single-cell killing may involve envelope glycoprotein-receptor interactions not accessible to the exterior of the cell.