HeLa cell adenovirus infection

Fig. l.A sample plate from a screen to identify small molecules that upregulate the expression of a cyclin-luciferase reporter protein. HeLa cells were infected with an adenovirus expressing cyclin-luciferase, as described in the methods section. The graph shows the activity of all wells from a single 384-well plate. Several compounds that upregulate cyclin-luciferase expression were identified. 

While the poly (A) sequences do seem to be involved in the transport of mRNA s from the nucleus, this does not seem to be the sole function of the poly (A) tract for example, adenovirus DNA appears to lack a DNA sequence complementary to poly (A) but replicates in the nucleus of the mammalian cell and appears to have a poly (A) tract added to the viral mRNA by host-cell mechanisms for transport of the adenovirus mRNA to the cytoplasm (Philipson et ah, 1971). As with cellular messages, cordycepin blocks both the labeling of the poly (A) tracts and the appearance of adenovirus-specific RNA in the cytoplasm of infected cells (Philipson et ah, 1971). In contrast, vaccinia virus replicates exclusively in the cytoplasm of cells it infects and still contains poly(A) sequences (Kates, 1970). Since no role in transport is involved here, it suggests that some mRNAs may require a poly (A) sequence for proper translation. Further, not all mammalian mRNAs contain poly (A) and still are transported to the cytoplasm for translation. Specifically, the 9 S histone message isolated by Adesnik and Darnell (1972) from HeLa cells lacks any detectable poly (A) sequence of any significant length. These workers have also shown that the exit time of the histone mRNA molecule from the nucleus is shorter than that of other messenger RNA s. 

Hodge LD, Mancini P, Davis FM, Hey wood P (1977) Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle. J Cell Biol 72 194-208... 

Type 5 adenovirus has been extensively studied, particularly in human HeLa cells, in which it was recognized quite early that they can cause two distinct cytopathic effects. Pereira and Kelly (1957) first described a late effect as a manifestation of infection with low... 

Pereira, H. G., and Kelly, B., 1957, Dose-response curves of toxic and infective actions of adenovirus in HeLa cells, J. Gen. Microbiol. 17 517. 

Fig. 4. Morphological changes in type C adenovirus-infected HeLa cells. (A) Uninfected HeLa cells in monolayer culture. Magnification, x 1.2 insert, x 52. (B) HeLa cell culture 3 days after Ad5 infection. Magnification, x 1.2 insert, x 52. From Periera (1958).

Adenovirus infection of rapidly growing permissive cells, for example, HeLa cells in suspension culture, leads to the cessation of cell division in 10-12 hr (Green and Daesch, 1961). Nevertheless, such infected cells do continue macromolecular synthesis and have been reported to contain twice the mass, compared to uninfected cells, of DNA and protein by 32 hr after infection (Green and Daesch, 1961 ... 

Fig. 7. Protein synthesis in adenovirus-infected cells. Ad5-infected HeLa cells were labeled with [ HJleucine for 2-hr periods starting at the times after infection shown. M = mock infected. Cytoplasmic proteins are shown in this autoradiogram. Modified from Castiglia and Flint (1983).

Each of these four mRNA species is stable in uninfected HeLa cells, with a half-life of at least several hours (Babich et al., 1983). Their high concentration in the cytoplasm, despite the inhibition of appearance in the cytoplasm of newly-synthesized RNA complementary to the four clones (Babich et al., 1983) is, therefore, not surprising and is in line with arguments given in the previous paragraph. The same result was obtained when the steady-state levels of actin mRNA in uninfected and infected cells were examined (Babich et al., 1983) Moreover, actin mRNA extracted 15 hr after infection was translated efficiently in a reticulocyte lysate. These results establish beyond any reasonable doubt that the host cells translational machinery becomes modified by the late phase of adenovirus infection such that only viral mRNA species can be recognized. 

Beltz, G., and Flint, S. J., 1979, Inhibition of HeLa cell protein synthesis during adenovirus infection Restriction of cellular messenger RNA sequences to the nucleus, J. Mol. Biol. 131 353. 

Castiglia, C. L., and Flint, S. J., 1983, Effects of adenovirus infection on rRNA synthesis and maturation in HeLa cells. Mol. Cell Biol. 3 662. 

Ross, S. R., Levine, A. J., Galos, R. S., Williams, J., and Shenk, T., 1980, Early viral proteins in HeLa cells infected with adenovirus type 5 host range mutants. Virology 163 475. 

Pereira, H. G., 1960, A virus inhibitor produced in HeLa cells infected with adenovirus, Virology 11 590. 

A detailed analysis of the involvement of microtubules in cytopathic effects was made by Ebina et al. (1978) who infected cells with poliovirus, Sendai virus, adenovirus, and herpesvirus in order to examine the effect of each virus on the formation of microtubular par-acrystals induced by vinblastine sulfate in HeLa-S3 cells. In polio-virus-infected cells, the cytopathic effect (cell rounding) and inhibition of paracrystal formation were both noted at 4 hr postinfection, proceeding in parallel. In the case of Sendai virus infection, no effect on paracrystal formation could be noted despite a syncytial cytopathic effect. In adenovirus- and herpesvirus-infected cells, inhibition of paracrystal formation occurred well before the cytopathic effect and was not blocked by UV irradiation or nucleic acid analogues but was by inhibition of protein synthesis. These findings led Ebina et al. (1978) to the hypothesis that early viral proteins are responsible for inhibition of microtubule formation and the cytopathic effect (cell rounding) except that Sendai virus did not cause this type of cytopathology. 

The adenovirus productive cycle has most usually been studied following infection of established lines of human cells, such as HeLa, with sufficiently high multiplicities of a human serotype to achieve a... 

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