Persistence of mumps virus in mouse L929 cells

The characteristics of a persistent infection of L929 cells with mumps virus (MuV) is presented. The persistent infection (L-MuV cells) was regulated by interferon (IFN) produced endogenously and almost all the properties showed that the carrier culture was maintained by horizontal transmission of the virus. Small-plaque mutants, but not temperature-sensitive variants, were selected during the persistent infection. MuV released from L-MuV cells (MuV-pi) replicated efficiently in L929 cells, while infection of L929 cells with the original MuV-o resulted in an abortive infection. The efficient replication of MuV-pi in L929 cells can be explained by the findings that MuV-pi induced IFN more slowly and had lower susceptibility to IFN in L929 cells than MuV-o did. M protein was synthesized to a considerable degree in MuV-pi-infected cells, while it could not be detected in MuV-o-infected cells. By contrast, MuV-pi formed small plaques in Vero cell monolayers and the yield of MuV-pi in Vero cells was lower than that of MuV-o. M protein induced by MuV-pi decayed easily in Vero cells. M protein was considered to be a limiting factor for MuV replication in both cell lines.     

High genetic stability of dengue virus propagated in MRC-5 cells as compared to the virus propagated in vero cells

This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4), including dengue virus type 4 (DEN-4) recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-free and serum-containing cultures were found to yield comparable titers of these viruses. The cloned DNA-derived DEN-4 started genetically more homogeneous was used to investigate the genetic stability of the virus propagated in Vero cells and MRC-5 cells. Sequence analysis revealed that the DEN-4 propagated in MRC-5 cells maintained a high genetic stability, compared to the virus propagated in Vero cells. Amino acid substitutions of Gly(104)Cys and Phe(108)Ile were detected at 70%, 60%, respectively, in the envelope (E) protein of DEN-4 propagated in Vero cells, whereas a single mutation of Glu(345)Lys was detected at 50% in E of the virus propagated in MRC-5 cells. Sequencing of multiple clones of three separate DNA fragments spanning 40% of the genome also indicated that DEN-4 propagated in Vero cells contained a higher number of mutations than the virus growing in MRC-5 cells. Although Vero cells yielded a peak virus titer approximately 1 to 17 folds higher than MRC-5 cells, cloned DEN-4 from MRC-5 cells maintained a greater stability than the virus from Vero cells. Serum-free microcarrier cultures of MRC-5 cells offer a potentially valuable system for the large-scale production of live-attenuated DEN vaccines.     

Parvovirus replication in normal and transformed human cells correlates with the nuclear translocation of the early protein NS1.

The parvovirus H-1 infection of the normal human diploid fibroblast strain MRC-5 produces a cytopathic effect, but no increase in infectious virus has been observed. Previously, we reported that large amounts of empty capsids are assembled in the nucleus of H-1 infected MRC-5 cells (S. Singer and S. Rhode, in D. Ward and P. Tattersall, ed., Replication of Mammalian Parvoviruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1978). The level of viral replicative-form DNA synthesis as shown by metabolic labeling is markedly reduced in these cells. Synthesis of the early protein NS1 is normal or slightly decreased, and the usual amount of the 92,000-molecular-weight (92K) posttranslationally modified NS1 was seen. The second deficient parameter that we have observed in the abortive infection is the nuclear translocation of NS1. In contrast, the simian virus 40-transformed MRC-5 cell line MRC-5 V1 and the simian virus 40-transformed human kidney cell line NB undergo a productive infection by H-1 accompanied by more efficient translocation of NS1 to the nucleus. The results indicate that there is an association between defective translocation of the NS1 rep protein to the nucleus and defective amplification of parvovirus replicative-form DNA. The nuclear translocation of specific proteins seems to be a function that is altered by development or neoplastic transformation.     

Dengue type 4 live-attenuated vaccine viruses passaged in vero cells affect genetic stability and dengue-induced hemorrhaging in mice

Most live-attenuated tetravalent dengue virus vaccines in current clinical trials are produced from Vero cells. In a previous study we demonstrated that an infectious cDNA clone-derived dengue type 4 (DEN-4) virus retains higher genetic stability in MRC-5 cells than in Vero cells. For this study we investigated two DEN-4 viruses: the infectious cDNA clone-derived DEN-4 2A and its derived 3' NCR 30-nucleotide deletion mutant DEN-4 2AΔ30, a vaccine candidate. Mutations in the C-prM-E, NS2B-NS3, and NS4B-NS5 regions of the DEN genome were sequenced and compared following cell passages in Vero and MRC-5 cells. Our results indicate stronger genetic stability in both viruses following MRC-5 cell passages, leading to significantly lower RNA polymerase error rates when the DEN-4 virus is used for genome replication. Although no significant increases in virus titers were observed following cell passages, DEN-4 2A and DEN-4 2AΔ30 virus titers following Vero cell passages were 17-fold to 25-fold higher than titers following MRC-5 cell passages. Neurovirulence for DEN-4 2A and DEN-4 2AΔ30 viruses increased significantly following passages in Vero cells compared to passages in MRC-5 cells. In addition, more severe DEN-induced hemorrhaging in mice was noted following DEN-4 2A and DEN-4 2AΔ30 passages in Vero cells compared to passages in MRC-5 cells. Target mutagenesis performed on the DEN-4 2A infectious clone indicated that single point mutation of E-Q(438)H, E-V(463)L, NS2B-Q(78)H, and NS2B-A(113)T imperatively increased mouse hemorrhaging severity. The relationship between amino acid mutations acquired during Vero cell passage and enhanced DEN-induced hemorrhages in mice may be important for understanding DHF pathogenesis, as well as for the development of live-attenuated dengue vaccines. Taken together, the genetic stability, virus yield, and DEN-induced hemorrhaging all require further investigation in the context of live-attenuated DEN vaccine development.     

Assessment of virus infection in cultured cells using metabolic monitoring

A rapid, in-process assessment of virus replication is disired to quickly investigate the effects of process parameters on virus infection, and to monitor consistency of process in routine manufacturing of viral vaccines. Live virus potency assays are generally based on plaque formation, cytopathic effect, or antigen production (TCID₅₀) and can take days to weeks to complete. Interestingly, when infected with viruses, cultured cells undergo changes in cellular metabolism that can be easily measured. These phenomena appear to be common as they has been observed in a variety of virus-host systems, e.g., in insect cells infected with baculovirus, Vero cells infected with Rotavirus, MRC-5 cells infected with Hepatitis A virus, and MRC-5 cells infected with the Varicella Zoster Virus (VZV). In this article, changes in glycolytic metabolism of MRC-5 cells as a result of CVZ infection are described. Both glucose consumption and lactate production in VZV infected MRC-5 cells are significantly elevated in comparison to uninfected cells. Based on this result, a rapid, in-process assay to follow VZV infection has been developed. The relative increase in lactate production in infected cells () increases as the infection progresses and then plateaus as the infection peaks. This plateau correlates with time of peak virus titer and could be used as a harvest triggering parameter in a virus production process.X_u = cell density of uninfected cellsX_i = cell density of infected cellsX_T = total cell densityL_i = cumulative lactate production in infected culturesL_u = cumulative lactate production in uninfected culturesq_Li = specific lactate production of infected cellsq_Lu = specific lactate production of uninfected cellsk₁, K₂ = constantsList of Symbols     

Vaccinia virus-based expression of gp120 and EGFP: survey of mammalian host cell lines

Production of recombinant proteins with the vaccinia virus expression system in five mammalian cell lines (HeLa, BS-C-1, Vero, MRC-5, and 293) was investigated for protein yield and proper posttranslational modifications. Regulatory acceptance of the host cell line was taken into consideration, where Vero, MRC-5, and 293 were considered more acceptable to the regulatory authorities. Relevant process knowledge for ease of scale-up with the particular cell type was also considered. Two proteins were expressed, enhanced green fluorescent protein (EGFP) in the cytoplasm and gp120, an HIV envelope coat protein that is secreted into the culture medium. HeLa cells produced the most EGFP at 17.2 microg/well with BS-C-1 and 293 following. BS-C-1 produced the most gp120 at 28.2 microg/mL with 293 and Vero following. Therefore, of the three most appropriate cell lines (Vero, MRC-5, and 293) for production processes, the best results were obtained with 293 cells. Although MRC-5 had a very high productivity on a per cell basis, the low cell density and slow growth rate made the overall production insufficient. Because gp120 contained a significant amount of posttranslational modification, this protein, produced by the different cell lines, was further analyzed by PNGase digestion suggesting N-linked glycosylation modifications in all cell lines tested. On the basis of these results and overall process considerations, 293 cells are recommended for further production process optimization in a serum-free suspension system.     

Evidence for a new viral late-domain core sequence, FPIV, necessary for budding of a paramyxovirus

Enveloped virus budding has been linked to both the ubiquitin-proteasome pathway and the vacuolar protein-sorting pathway of cells. We show here for the paramyxovirus SV5 that proteasome inhibitors and expression of dominant-negative VPS4(E228Q) ATPase blocks budding. The SV5 matrix (M) protein lacks previously defined late domains (e.g., P[T/S]AP, PPxY, YPDL) that recruit cellular factors. We identified a new motif for budding (core sequence FPIV) that can compensate functionally for lack of a PTAP late domain in budding human immunodeficiency virus type 1 virus-like particles (VLPs). Mutagenesis experiments suggest the more general sequence O-P-x-V. The proline residue was found to be critically important for function of this sequence, as substitution of this proline in the SV5 M protein resulted in poor budding of SV5 VLPs and failure of recombinant SV5 virus to replicate normally. Adaptation of mutant virus occurred rapidly, resulting in new proline residues elsewhere in the M protein. We hypothesize that these proline residues act to partially restore virus budding by generation of new motifs that act as suboptimal late domains.     

Reduced levels of ADP-ribosylatable elongation factor-2 in aged and SV40-transformed human cell cultures

The elongation step is involved in the regulation of protein synthesis during the cell cycle, environmental stress, ageing and transformation. Using a diphtheria toxin-mediated assay for measuring the levels of ADP-ribosylatable elongation factor EF-2, we have observed an irreversible decrease of up to 64% in the amount of ADP-ribosylatable EF-2 in normal diploid human fibroblasts MRC-5 undergoing ageing in vitro. However, a similar decrease in low serum-associated G0/G1-arrested cells is reversible both in MRC-5 cells and in their SV40-transformed counterparts. Reduced levels of ADP-ribosylatable EF-2 could account for the slowing-down of protein synthesis during cell cycle arrest and during cellular ageing in culture.     

Negative regulation of mitotic promoting factor by the checkpoint kinase chk1 in simian virus 40 lytic infection

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G(1)/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G(1)/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G(1)/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G(2). Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.     

IRF-3 activation by Sendai virus infection is required for cellular apoptosis and avoidance of persistence

Here, we report that specific manipulations of the cellular response to virus infection can cause prevention of apoptosis and consequent establishment of persistent infection. Infection of several human cell lines with Sendai virus (SeV) or human parainfluenza virus 3, two prototypic paramyxoviruses, caused slow apoptosis, which was markedly accelerated upon blocking the action of phosphatidylinositol 3-kinases (PI3 kinases) in the infected cells. The observed apoptosis required viral gene expression and the action of the caspase 8 pathway. Although virus infection activated PI3 kinase, as indicated by AKT activation, its blockage did not inhibit JNK activation or IRF-3 activation. The action of neither the Jak-STAT pathway nor the NF-kappaB pathway was required for apoptosis. In contrast, IRF-3 activation was essential, although induction of the proapototic protein TRAIL by IRF-3 was not required. When IRF-3 was absent or its activation by the RIG-I pathway was blocked, SeV established persistent infection, as documented by viral protein production and infectious virus production. Introduction of IRF-3 in the persistently infected cells restored the cells' ability to undergo apoptosis. These results demonstrated that in our model system, IRF-3 controlled the fate of the SeV-infected cells by promoting apoptosis and preventing persistence.     

Single amino acid substitution in the V protein of simian virus 5 differentiates its ability to block interferon signaling in human and murine cells

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.     

Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities.

A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.     

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

Summary Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.     

Susceptibility of human and rat neural cell lines to infection by SARS-coronavirus

Pathological characterization of autopsied tissues from patients with SARS revealed severe damage in restricted tissues, such as lung, with no apparent cell damage in other tissues, such as intestine and brain. Here, we examined the susceptibility of neural cell lines of human (OL) and rat (C6) origins to SARS-associated coronavirus. Both of the neural cell lines showed no apparent cytopathic effects (CPE) by infection but produced virus with infectivity of 10(2-5) per ml, in sharp contrast to the production by infected Vero E6 cells of >10(9) per ml that showed a lytic infection with characteristic rounding CPE. Interestingly, the infection of intestinal cell line CaCo-2 also induced no apparent CPE, with production of the virus at a slightly lower level as that of the Vero E6 cell culture. Notably, the cellular receptor for the virus, angiotensin-converting enzyme 2 was expressed at similar levels on Vero E6 and CaCo-2 cells, but at undetectable levels on OL and C6 cells.     

Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2

The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.     

Contrasting roles of endosomal pH and the cytoskeleton in infection of human glial cells by JC virus and simian virus 40

Infection of eukaryotic cells by pathogens requires the efficient use of host cell endocytic and cytoplasmic transport mechanisms. Understanding how these cellular functions are exploited by microorganisms allows us to better define the basic biology of pathogenesis while providing better insight into normal cellular functions. In this report we compare and contrast intracellular transport and trafficking of the human polyomavirus JC virus (JCV) with that of simian virus 40 (SV40). We have previously shown that infection of human glial cells by JCV requires clathrin-dependent endocytosis. In contrast, infection of cells by SV40 proceeds by caveola-dependent endocytosis. We now examine the roles of endosomal pH and the cellular cytoskeleton during infection of glial cells by both viruses. Our results demonstrate that JCV infection is sensitive to disruption of endosomal pH, whereas SV40 infection is pH independent. Infection by JCV is inhibited by treatment of glial cells with cytochalasin D, nocodazole, and acrylamide, whereas SV40 infection is affected only by nocodazole. These data point to critical differences between JCV and SV40 in terms of endocytosis and intracellular trafficking of their DNA genomes to the nucleus. These data also suggest a unique sequential involvement of cytoskeletal elements during infection of glial cells by JCV.     

Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.