Infected Monkey Cells Research Articles

Generation of a bovine cell line for gene engineering using an HIV-1-based lentiviral vector

Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors are indispensable tools for gene engineering in mammalian cells. Conversely, lentiviral vector transduction is severely inhibited in bovine cells. Previous studies demonstrated that this inhibition is caused by the anti-lentiviral host factor tripartite motif containing 5 (TRIM5), which targets incoming HIV-1 virions by interacting with the viral capsid. In this study, we investigated several methods for overcoming the limited applicability of lentiviral vectors in bovine cells. First, we demonstrated that the SPRY domain of bovine TRIM5 is the major determinant of anti-viral activity. Second, we found that mutations that allow the capsid to evade rhesus macaque TRIM5α minimally rescued HIV-1 infectivity in bovine-derived MDBK cells. Third, we found that cyclosporine A, which relieves the inhibition of HIV-1 infection in monkey cells, significantly rescued the impaired HIV-1 infectivity in MDBK cells. Lastly, we successfully generated a bovine cell line lacking intact TRIM5 using the CRISPR/Cas9 technique. This TRIM5 knockout cell line displayed significantly higher susceptibility to an HIV-1-based lentiviral vector. In conclusion, our findings provide a promising gene engineering strategy for bovine cells, thereby contributing to innovations in agriculture and improvements in animal health.

Association Between Simian Virus 40 and Human Tumors.

Simian virus 40 (SV40) is a small DNA tumor virus of monkey origin. This polyomavirus was administered to human populations mainly through contaminated polio vaccines, which were produced in naturally infected SV40 monkey cells. Previous molecular biology and recent immunological assays have indicated that SV40 is spreading in human populations, independently from earlier SV40-contaminated vaccines. SV40 DNA sequences have been detected at a higher prevalence in specific human cancer specimens, such as the brain and bone tumors, malignant pleural mesotheliomas, and lymphoproliferative disorders, compared to the corresponding normal tissues/specimens. However, other investigations, which reported negative data, did not confirm an association between SV40 and human tumors. To circumvent the controversies, which have arisen because of these molecular biology studies, immunological researches with newly developed indirect ELISA tests were carried out in serum samples from patients affected by the same kind of tumors as mentioned above. These innovative indirect ELISAs employ synthetic peptides as mimotopes/specific SV40 antigens. SV40 mimotopes do not cross-react with the homologous human polyomaviruses, BKPyV, and JCPyV. Immunological data obtained from indirect ELISAs, using SV40 mimotopes, employed to analyze serum samples from oncological patients, have indicated that these sera had a higher prevalence of antibodies against SV40 compared to healthy subjects. The main data on (i) the biology and genetics of SV40; (ii) the epidemiology of SV40 in the general population, (iii) the mechanisms of SV40 transformation; (iv) the putative role of SV40 in the onset/progression of specific human tumors, and (v) its association with other human diseases are reported in this review.

Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector.

A Wolf in Sheep's Clothing: SV40 Co-opts Host Genome Maintenance Proteins to Replicate Viral DNA

Simian virus 40 (SV40) was discovered in 1960 as a contaminant in early polio vaccines. Its discovery coincided with an explosion of knowledge in the new field of molecular biology, and SV40 was quickly adopted as a model to study eukaryotic genome structure, expression, replication, and cell growth regulation in cultured cells [1]. With a genome of only 5.2 kbp, SV40 relies heavily on host cell machinery to propagate, affording investigators a powerful tool to discover key host proteins that the virus manipulates. Indeed, a single multifunctional viral protein, the large tumor (T) antigen (Tag) (Figure 1A), is sufficient to orchestrate the replication of the viral mini-chromosome in infected monkey cells [2], [3]. The origin DNA binding domain of Tag binds specifically to the viral origin of DNA replication, and the C-terminal helicase domain of Tag unwinds parental DNA at SV40 replication forks. The development of a cell-free reaction containing purified Tag and primate cell extract enabled the identification of ten evolutionarily conserved host proteins that are necessary and sufficient, together with Tag, to replicate SV40 DNA in vitro [3], [4]. Figure 1 Assembly and activation of the SV40 pre-replication complex in vitro.

High-throughput cell-based screen for chemicals that inhibit infection by simian virus 40 and human polyomaviruses.

We developed a high-throughput, cell-based screen to identify chemicals that inhibit infection by the primate polyomaviruses. The screen is based on the detection of compounds that inhibit the ability of a replication-defective simian virus 40 (SV40)-based viral vector to cause growth arrest in HeLa cells by repressing the expression of the endogenous human papillomavirus E7 oncogene in these cells. We identified two compounds, ellagic acid and spiperone, that suppressed the ability of the SV40 recombinant virus to inhibit cellular DNA synthesis. These compounds caused a marked reduction of the ability of wild-type SV40 to productively infect permissive monkey cells, even when the compounds were added several hours after infection. The fraction of cells expressing SV40 large T antigen and the levels of T antigen mRNA were reduced in infected human and monkey cells treated with ellagic acid and spiperone, suggesting that these compounds block a step in the virus life cycle prior to SV40 early gene expression. Ellagic acid and spiperone also inhibited large T antigen expression by BK virus and JC virus, two important, pathogenic human polyomaviruses.

Effects of Simian Virus 40 Large and Small Tumor Antigens on Mammalian Target of Rapamycin Signaling: Small Tumor Antigen Mediates Hypophosphorylation of eIF4E-Binding Protein 1 Late in Infection

We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen.

Subclasses of Simian-Virus-40 Large Tumor Antigen

Two major subclasses of simian virus 40 tumor antigen were prepared from productively infected monkey cells. These subclasses can be distinguished by their sedimentation properties: one tumor antigen form sediments at 5-6S and the other at 14-16S. The DNA-binding properties of these subclasses were investigated by two different experimental procedures. In the first procedure, the DNA binding of subclasses of crude tumor antigen, separated by zone velocity sedimentation, were assayed by immunoprecipitation of the DNA-protein complexes. In the second procedure, the two tumor antigen forms were partially purified by column chromatography and DNA binding was tested in a filter binding assay. Both procedures gave comparable results. (a) The 5-6-S and the 14-16-S tumor antigen bound specifically to a DNA restriction fragment containing the viral genome control regions. (b) At low salt concentrations, both subclasses bound to specific and to nonspecific DNA sequences; competition experiments in the presence of nonspecific DNA showed, however, that the affinity of both tumor antigen forms for the viral genome control region was at least 10-fold higher than their affinity for nonspecific DNA sequences. (c) The binding of the 5-6-S subclass to viral control region DNA was optimal at 60-80 mM NaCl while specific DNA binding of the 14-16-S form was optimal at 150-200 mM NaCl; however, binding of the 14-16-S form to nonspecific DNA sequences was also more resistant to high salt concentrations than that of the 5-6S form. (d) Both tumor antigen forms bound well to specific and to nonspecific DNA at pH 6-6.5; with increasing pH values, binding to nonspecific DNA decreased while binding to specific DNA reached an optimum at pH 7-7.5. Binding of the 14-16-S form to viral origin DNA was more resistant to pH values above 7.5 than binding of the 5-6-S form.

Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59

The murine coronavirus [murine hepatitis virus (MHV)] is limited to infection of susceptible mice and murine cell lines by the specificity of the spike glycoprotein (S) for its receptor, murine carcinoembryonic antigen cell adhesion molecule 1a (mCEACAM1a). We have recently shown that 21 aa substitutions and a 7-aa insert in the N-terminal region of S are associated with the extended host range of a virus variant derived from murine cells persistently infected with the A59 strain of MHV (MHV-A59). We used targeted RNA recombination (TRR) to generate isogenic viruses that differ from MHV-A59 by the 21 aa substitutions or the 7-aa insert in S. Only viruses with both the 21 aa substitutions and the 7-aa insert in S infected hamster, feline, and monkey cells. These viruses also infected murine cells in the presence of blocking anti-mCEACAM1a antibodies. Thus, relatively few changes in the N-terminal region of S1 are sufficient to permit MHV-A59 to interact with alternative receptors on murine and non-murine cells.

Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59

The murine coronavirus [murine hepatitis virus (MHV)] is limited to infection of susceptible mice and murine cell lines by the specificity of the spike glycoprotein (S) for its receptor, murine carcinoembryonic antigen cell adhesion molecule 1a (mCEACAM1a). We have recently shown that 21 aa substitutions and a 7-aa insert in the N-terminal region of S are associated with the extended host range of a virus variant derived from murine cells persistently infected with the A59 strain of MHV (MHV-A59). We used targeted RNA recombination (TRR) to generate isogenic viruses that differ from MHV-A59 by the 21 aa substitutions or the 7-aa insert in S. Only viruses with both the 21 aa substitutions and the 7-aa insert in S infected hamster, feline, and monkey cells. These viruses also infected murine cells in the presence of blocking anti-mCEACAM1a antibodies. Thus, relatively few changes in the N-terminal region of S1 are sufficient to permit MHV-A59 to interact with alternative receptors on murine and non-murine cells.

The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys.

Host cell barriers to the early phase of immunodeficiency virus replication explain the current distribution of these viruses among human and non-human primate species. Human immunodeficiency virus type 1 (HIV-1), the cause of acquired immunodeficiency syndrome (AIDS) in humans, efficiently enters the cells of Old World monkeys but encounters a block before reverse transcription. This species-specific restriction acts on the incoming HIV-1 capsid and is mediated by a dominant repressive factor. Here we identify TRIM5alpha, a component of cytoplasmic bodies, as the blocking factor. HIV-1 infection is restricted more efficiently by rhesus monkey TRIM5alpha than by human TRIM5alpha. The simian immunodeficiency virus, which naturally infects Old World monkeys, is less susceptible to the TRIM5alpha-mediated block than is HIV-1, and this difference in susceptibility is due to the viral capsid. The early block to HIV-1 infection in monkey cells is relieved by interference with TRIM5alpha expression. Our studies identify TRIM5alpha as a species-specific mediator of innate cellular resistance to HIV-1 and reveal host cell components that modulate the uncoating of a retroviral capsid.

Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.

Simian virus 40 as a vector: recombinant viruses expressing individual polyoma T antigens

We constructed simian virus 40 (SV40)/polyomavirus recombinants by replacing in SV40 the T antigen coding region with polyoma early region sequences, either cDNAs encoding small, middle or large T antigen or the wild-type sequence coding all three proteins. The recombinants maintained the SV40 late region and origin of replication and were propagated in COS cells yielding recombinant virus preparations with titers of 10 6–10 7 infectious particles per milliliter. These viruses were characterized in productive infections of COS cells by analyzing early and late mRNA levels and by following synthesis of polyoma early proteins. In the absence of viral DNA replication, i.e. in infected monkey or mouse cells, expression of the polyoma T antigens was weak. Further experiments indicated that this was mostly due to high genomic instability during amplification, to lower levels of cDNA transcripts as compared to spliced mRNA, and possibly also to lower infectivity of the recombinant virions. It remains to be determined, whether these handicaps are unique to SV40/polyoma recombinants or whether SV40 is in general inadequate as a viral vector.

MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection.

Phosphopeptide analyses of the simian virus 40 (SV40) large tumor antigen (LT) in SV40-transformed rat cells, as well as in SV40 lytically infected monkey cells, showed that gel-purified LT that was not complexed to p53 (free LT) and p53-complexed LT differed substantially in their phosphorylation patterns. Most significantly, p53-complexed LT contained phosphopeptides not found in free LT. We show that these additional phosphopeptides were derived from MDM2, a cellular antagonist of p53, which coprecipitated with the p53-LT complexes, probably in a trimeric LT-p53-MDM2 complex. MDM2 also quantitatively bound the free p53 in SV40-transformed cells. Free LT, in contrast, was not found in complex with MDM2, indicating a specific targeting of the MDM2 protein by SV40. This specificity is underscored by significantly different phosphorylation patterns of the MDM2 proteins in normal and SV40-transformed cells. Furthermore, the MDM2 protein, like p53, becomes metabolically stabilized in SV40-transformed cells. This suggests the possibility that the specific targeting of MDM2 by SV40 is aimed at preventing MDM2-directed proteasomal degradation of p53 in SV40-infected and -transformed cells, thereby leading to metabolic stabilization of p53 in these cells.

Defective Processing of Human Adenovirus 2 Late Transcription Unit mRNAs during Abortive Infections in Monkey Cells

Growth of human adenoviruses is severely restricted in monkey cells. We examined the synthesis of mRNAs from the Ad2 late transcription unit (LTU) in abortively infected monkey cells at late times in infection. All L2, L3, and L5 mRNAs were absent or drastically reduced in abortive infections. Most L1 and L4 mRNAs were also greatly decreased in abortive infections; however, a single large messenger was produced from each of the L1 and L4 families, at levels approaching those found in productive infections. The pattern of i -leader containing mRNAs was also changed in abortive infections. These defects could be corrected in monkey cells by the presence of SV40 T antigen or an altered adenoviral DNA binding protein. These defects in late gene expression in abortive infections could not be attributed to differences in transcription along the LTU or levels of DNA replication. In abortively infected cells, nuclear levels of L5 mRNA were decreased 2 to 6 fold, while cytoplasmic levels were decreased over 200-fold. These findings imply a general defect in processing of viral mRNAs, most likely due to defective splicing and/or transport, during abortive infections.

DNA topoisomerase activity associated with simian virus 40 nucleoprotein complexes.

Simian virus 40 (SV40) chromatin extracted from nuclei of infected monkey cells (CV1) was sedimented in neutral sucrose gradients, before and after digestion with bovine pancreatic RNase I-A or DNase I. DNA topoisomerase (TI) activity was found associated with RNase-resistant, DNase-sensitive SV40 nucleoprotein complexes. After polyacrylamide gel electrophoresis, a number of proteins with a molecular mass between 40 and 70 kDa were seen at the level of viral DNA peaks, some of which may represent catalytically active breakdown products of the TI enzyme. Large protein complexes were observed under the electron microscope in association with the viral chromosomes and appear to correspond to the SV40 DNA replication complex, including TI. Our results suggest that TI activity is indeed associated with the viral minichromosomes undergoing replication in vivo.

Phosphorylation of the Adenovirus DNA-Binding Protein and Epitope Mapping of Monoclonal Antibodies against It

To facilitate structure/function studies of the adenovirus type 2 or 5 single-stranded DNA binding protein (DBP), the epitopes of five anti-DBP monoclonal antibodies were mapped. Antibodies 37-3, 38-2, and B6 mapped between DBP residues 131-174 which corresponds to the host-range or hinge region of the molecule. The epitopes for antibodies 16-5 and 18-9 mapped between residues 56-81, and included a phosphorylated serine at position 70. Using antibodies 16-5 or 18-9 as analytical reagents, ∼1/2 of the DBP molecules present at late times in infected HeLa cells were found to be phosphorylated at serine 70. A similar pattern of serine 70 phosphorylation was observed in productively and abortively infected monkey cells as well as in an E2A-transformed, DBP expressing cell line. This study also reports the preliminary identification of two new phosphorylation sites on Ad2 DBP at serines 160 and 161 (corresponding to serine 161 on Ad5 DBP). No differences were observed in the phosphorylation of serine 160/161 in DBP isolated from infected HeLa cells or from productively and abortively infected monkey cells. Therefore, phosphorylation of these sites cannot account for the block to wild-type adenovirus growth in monkey cells.

Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.

A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.

Expression in vivo and in vitro of the major structural protein (VP73) of African swine fever virus.

The VP73 protein was produced by in vitro transcription and translation from the Xho I-Bam HI fragment located between the Cla I-N and Cla I-H fragments of the viral genome. This DNA fragment encodes a late mRNA of about 2.6 kb detected in infected MS monkey and BHK hamster cells. The transcript was initiated at a site within two bases upstream of the translation initiation codon. The in vitro synthesized polypeptide shows the same molecular weight as the in vivo synthesized polypeptide, suggesting that VP73 has no post-translational modification. There are two internal AUG initiation codons for in vitro translation, one of which is functional in vivo, as well as a possible GUG initiator codon detected by expression of the protein in E. coli cells.

Cross-Neutralizing Antibodies in Rabbits Immunized with HIV-1 gp160 Purified from Simian Cells Infected with a Recombinant Vaccinia Virus

A recombinant vaccinia virus in which the transcription of the human immunodeficiency virus type 1 (BRU isolate) env gene is driven by the 11K late vaccinia promoter yields about 10-fold higher amounts of gp160 env protein upon infection of monkey cells than does a recombinant in which gp160 is expressed using the 7.5K early-late promoter. The gp160 was purified from detergent lysates of infected cells by lentil lectin affinity chromatography followed by immunoaffinity chromatography, and was obtained in yields of 1-2 mg/10(9) cells of material estimated to be about 70% pure. Pairs of rabbits were immunized with purified gp160 using either one of five different adjuvants or an immunostimulating complex. In all cases a substantial humoral immune response was obtained after boosting, including an activity that neutralized the homologous (BRU) isolate of HIV-1. In some cases, this activity also neutralized two distantly related isolates, SF2 and MN.

Association of topoisomerases I and II with the chromatin in SV40-infected monkey cells

The subcellular localization of topoisomerase I and topoisomerase II has been compared in Simian virus (SV40) infected and uninfected TC7 monkey cells. In SV40-infected cells, both of these enzymes are preferentially associated with the chromatin. Some topoisomerase I is associated with the nuclear matrix, whereas topoisomerase II shows no such association. In uninfected TC7 cells, topoisomerase I is present in both the chromatin and nuclear matrix fractions. Toposiomerase II, on the other hand, is not detected in any of the subcellular fractions of uninfected cells. After SV40 infection, there is a marked increase in the level of chromatin-associated topoisomerase II.