Productive Adenovirus Infection Research Articles

The Mre11/Rad50/Nbs1 Complex Limits Adeno-Associated Virus Transduction and Replication

Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.

Critical Role for Arginine Methylation in Adenovirus-Infected Cells

During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection.

TRAIL and its receptors in the colonic epithelium: A putative role in the defense of viral infections

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family and induces apoptosis by cross-linking either of the 2 TRAIL receptors containing a death domain (TRAIL-R1 or TRAIL-R2). TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. The aim of this study was to investigate the expression and function of TRAIL and its receptors in normal colonic epithelium. TRAIL and TRAIL receptor expression was studied by reverse-transcriptase polymerase chain reaction and immunohistochemistry. TRAIL sensitivity of epithelial cells was determined in vitro. Normal colonic epithelial cells express TRAIL, TRAIL-R1, TRAIL-R2, and TRAIL-R4. Interestingly, TRAIL and TRAIL-R2 are coexpressed mostly in the luminal surface epithelium. Despite the expression of apoptosis-mediating TRAIL receptors, the normal colonic crypt epithelium is completely resistant to TRAIL-induced apoptosis in vitro. Using an infection model with restricted human cytomegalovirus gene expression or productive adenovirus infection in the colon carcinoma cell line Caco-2, we show that TRAIL sensitivity of colonic epithelial cells is induced on virus infection along with an up-regulation of TRAIL-R1 and TRAIL-R2 on the cell surface. We conclude that the TRAIL system may play a role in the early elimination of virus-infected epithelial cells in the normal gut.

Monitoring adenovirus infections with on-line and off-line methods.

Several known process monitoring methods were tested for their efficacy in the detection of adenovirus infections. The methods that we explored include several indirect indications of viral infections, including metabolic rate analysis, secondary gauges of respiration, cell size measurement, cell number and cell viability determination, and changes in capacitance. Direct indications of the adenovirus infection were also applied, including total viral particle and infectious particle measurements, as well as a flow cytometry method for detecting infected cells. All of the methods tested in the study provide some positive indication of an adenovirus infection. Many of the methods require repeated sampling, which may limit their utility in a manufacturing process. All of the indirect measures of viral infection may be limited by the fact that they do not uniquely identify an infection. The simplest monitoring methods appear to be detection of changes in respiration or the capacitance of the culture, both of which seem to provide a clear indication of an infection. Further work will be required to demonstrate that these indications are characteristic of only a successful and productive adenovirus infection.

Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

P53-dependent cell death/apoptosis is required for a productive adenovirus infection.

The p53 tumor suppressor protein binds to both cellular and viral proteins, which influence its biological activity. One such protein is the large E1b tumor antigen (E1b58kDa) from adenoviruses (Ads), which abrogates the ability of p53 to transactivate various promoters. This inactivation of p53 function is believed to be the mechanism by which E1b58kDa contributes to the cell transformation process. Although the p53-E1b58kDa complex occurs during infection and is conserved among different serotypes, there are limited data demonstrating that it has a role in virus replication. However, loss of p53 expression occurs after adenovirus infection of human cells and an E1b58kDa deletion mutant (Onyx-015, also called dl 1520) selectively replicates in p53-defective cells. These (and other) data indicate a plausible hypothesis is that loss of p53 function may be conducive to efficient adenovirus replication. However, wild-type (wt) Ad5 grows more efficiently in cells expressing a wt p53 protein. These studies indicate that the hypothesis may be an oversimplification. Here, we show that cells expressing wt p53, as well as p53-defective cells, allow adenovirus replication, but only cells expressing wt p53 show evidence of virus-induced cytopathic effect. This correlates with the ability of adenovirus to induce cell death. Our data indicate that p53 plays a necessary part in mediating cellular destruction to allow a productive adenovirus infection. In contrast, p53-deficient cells are less sensitive to the cytolytic effects of adenovirus and as such raise questions about the use of E1b58kDa-deficient adenoviruses in tumor therapy.

Inhibition of ICE-like Proteases Inhibits Apoptosis and Increases Virus Production during Adenovirus Infection

Interleukin-1b converting enzyme (ICE)-related cysteine proteases are required for E1A-induced, p53-dependent apoptosis in baby rat kidney (BRK) cells. Adenovirus E1B 19K protein, which is a potent inhibitor of apoptosis, inhibits activation of these proteases in BRK cells. E1A expression induces apoptosis during infection of human cells by mutant adenoviruses which contain nonfunctional E1B 19K. The question arises as to whether ICE-related proteases are involved in E1A-induced apoptosis during mutant adenovirus infection of human cells. To test the involvement of the cysteine proteases in E1A-induced apoptosis during productive adenovirus infection of HeLa cells, we examined whether Z-VAD-FMK, an inhibitor of ICE-related proteases, can inhibit apoptosis induced by mutant adenovirus which lacks functional E1B 19K. Z-VAD-FMK inhibited E1A-induced apoptosis in adenovirus-infected Hela cells, suggesting that the ICE family proteases are involved in this apoptosis pathway. Z-VAD-FMK also inhibited cleavage of substrates such as cysteine protease CPP32 and nuclear lamins, whereas cleavage of poly(ADP-ribose) polymerase was partially inhibited during infection with an E1B 19K mutant. Inhibition of apoptosis by Z-VAD-FMK significantly enhanced production of infectious adenovirus and attenuated virus release. Thus apoptosis may be a method for the host cell to limit virus production and release at the end of the infection cycle.

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.

Chromosomal localization of integrated adenovirus DNA in productively infected and in transformed mammalian cells

The in situ hybridization technique has been used to localize adenovirus type 12 (Ad12) genomes on specific chromosomes of Ad12-transformed hamster cells as well as on specific chromosomes of human cells productively infected with Ad12. Hamster cell lines T637 and A2497-2 contain 20 to 22 and 17 copies of Ad12 DNA, respectively, in an integrated form. The results of in situ hybridization experiments demonstrate that the Ad12 DNA molecules are predominantly located on chromosomes 2, 7, 11, to 13, and 15 in cell line T637, and on chromosomes 7, 20, and perhaps 16 to 19 in cell line A2497-2. These data further document that viral DNA is chromosomally integrated and does not persist in a huge extrachromosomal, episomal structure. There is evidence from previous work that adenovirus DNA can also be covalently linked to human cellular DNA after productive adenovirus infection of human cells. In keeping with these earlier findings, we have now been able to show by in situ hybridization experiments that early in productive infection, i.e., 2 or 6 hr after infection, Ad12 genomes are predominantly associated with a limited number of human chromosomes. It appears biologically significant that these chromosomes, which belong to groups A and C II, have been the same ones in several independent infection experiments. Other chromosomes may also carry smaller amounts of viral DNA. In situ hybridization of Ad12 DNA to chromosomes of uninfected human cells yields no significant chromosomal association of viral DNA. The signal to noise ratio of grain counts over chromosomes from Ad12-infected cells to areas devoid of chromosomes is 5.6. The results presented raise the question of whether Ad12 DNA can integrate at selective sites in human chromosomes and whether this insertion event plays an essential role in early steps of viral transcription or replication. There is no direct evidence to suggest a biologic function for viral DNA integration in the productive infection cycle.

Posttranscriptional control of DHFR gene expression during adenovirus 2 infection.

The effects of productive adenovirus infection on host gene expression were studied by using a line of methotrexate-resistant HeLa cells with amplified dihydrofolate reductase (DHFR) genes. We have previously reported that synthesis of DHFR is induced threefold early in infection and is shut off late in infection (Yoder et al., Mol. Cell. Biol. 3:819-828, 1983). These changes in DHFR protein synthesis are accompanied by changes in both the steady-state cytoplasmic levels of DHFR mRNA and in the rate of appearance of DHFR mRNA in the cytoplasm. In this report, we examined the mechanism of nuclear control of DHFR mRNA levels. Transcription of DHFR-specific sequences continued at a constant rate throughout infection, representing 0.015% of the total transcriptional activity. In contrast, nuclear steady-state levels of DHFR sequences changed in correspondence to the changing rate of appearance of DHFR mRNA in the cytoplasm. That is, nuclear levels of DHFR-specific sequences rose 2.5-fold early in infection and declined to a level below that found in uninfected cells late in infection. Thus, the relative nuclear stability of DHFR sequences changed throughout the course of infection such that during the time of induction, DHFR sequences were preferentially stabilized. This stabilization was transient, however, and was no longer observed by the time of shutoff. These data indicate that posttranscriptional nuclear events are important in the regulation of DHFR gene expression by adenovirus.

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain.

Inhibition of HeLa cell protein synthesis during adenovirus infection: Restriction of cellular messenger RNA sequences to the nucleus

The mechanism by which adenovirus type 2 inhibits HeLa cell protein synthesis has been investigated by a quantitative analysis of the synthesis and metabolism of cellular and viral hnRNA † † Abbreviations used: hnRNA, heterogeneous nuclear RNA; cDNA, complementary DNA. sequences. By the time labeling of cellular proteins is maximally inhibited, 16 hours following adenovirus infection under the conditions we have used, all of the newly made messenger RNA sequences reaching the cytoplasm are viral-specific. Thus, the gradual shift from translation of cellular to translation of viral mRNA species that begins following entry of adenovirus-infected cells into the late phase of the productive cycle, can probably be explained simply by competition between the two types of mRNA for available ribosomes. In order to investigate the mechanism(s) by which adenovirus infection exerts this dramatic change in the biogenesis of HeLa cellular mRNA sequences, the hnRNA fraction has been isolated quantitatively from infected cells after pulselabeling or a pulse-chase. Cellular and viral transcripts were then distinguished by hybridization to viral DNA. The results of these experiments reveal that the rate of synthesis of cellular hnRNA sequences remains constant throughout the course of adenovirus type 2 infection. Moreover, poly(A) is apparently added normally to cellular hnRNA sequences synthesized during the late phase of productive adenovirus infection. This newly synthesized, poly(A)-containing hnRNA cannot be distinguished from the corresponding fraction isolated from mockinfected cells by hybridization to cDNA prepared using uninfected HeLa cell, cytoplasmic, poly(A)-containing RNA as template: it also appears to be metabolized with kinetics similar to those observed in uninfected cells.

Synthesis of complementary RNA sequences during productive adenovirus infection.

Liquid RNA.DNA hybridization with separated strands of adenovirus type 2 DNA revealed that late nuclear RNA can hybridize to about 85% of the 1-strand and 10-15% of the h-strand, whereas late cytoplasmic RNA hybridizes to 65-70% and 25% of the l- and h-strand, respectively. With separated strands from the six EcoRI fragments of adenovirus type 2 DNA as probes, it was shown that late nuclear RNA hydridizes to 85-90% of the l-strand from all six EcoRI fragments. Since late cytoplasmic RNA hybridizes to 40-50% of the h-strand from both fragments EcoRI-B and EcoRI-C, complementary viral RNA sequences are synthesized during adenovirus infection. Complementarity between nuclear and cytoplasmic RNA could also be demonstrated by showing that late cytoplasmic RNA which had been preincubated with late nuclear RNA hybridized to a smaller fraction of the h-strand of fragment EcoRI-C than without preincubation. Double-stranded RNA which contains sequences that correspond to at least 60% of the viral genome was isolated from infected cells. However, less than 2% of the newly synthesized late RNA became double-stranded after incubation under annealing conditions, which suggests that RNA derived from one of the strands is present at a low concentration. Accordingly, it was shown that nearly all viral cytoplasmic RNA which is synthesized late after infection is derived from the l-strand.

Transcription and transport of virus-specific ribonucleic acids in African green monkey kidney cells abortively infected with type 2 adenovirus.

The techniques of deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization and immunological precipitation were used to compare the synthesis of adenovirus-specific macromolecules in African green monkey kidney (AGMK) cells infected with adenovirus, an abortive infection, and coinfected with both adenovirus and simian virus 40 (SV40), which renders the cells permissive for adenovirus replication. When viral protein synthesis was proceeding at its maximum rate, the incorporation of (14)C-amino acids into adenovirus structural proteins was about 90 times greater in the doubly infected cells than in cells infected only with adenovirus. However, the rates of synthesis of virus-specific ribonucleic acid appeared to be comparable in the two infections at all times measured. A time-dependent increase in the rate of RNA synthesis observed late in the abortive infection was dependent upon the prior replication of viral DNA. Moreover, all virus-specific RNA species that are normally made late in a productive adenovirus infection (i.e., the true late and class II early RNA species) were also detected in the abortive infection. Adenovirus-specific RNA was detected by molecular hybridization in both the cytoplasm and nuclei of abortively infected cells. Comparable amounts of viral RNA were found in the cytoplasmic fractions of AGMK cells infected either with adenovirus or with both adenovirus and SV40. The results of hybridization-inhibition experiments clearly showed that there was a class of virus-specific RNA molecules, representing about 30% of the total, in the nucleus that was not transported to the cytoplasm. This class of RNA was also identified in similar amounts in productively infected human KB cells. The difference in the abilities of cytoplasmic and nuclear RNA to inhibit the hybridization of virus-specific RNA from whole cells was shown not to be due to a difference in the molecular size of the RNA species from the two cell fractions or to the specific loss of a cytoplasmic species during RNA extraction procedures.