Adenovirus replication cycle

Figure 49-1 provides a schematic diagram of the replicative cycle of a DNA virus (A) and an RNA virus B). DNA viruses include poxviruses (smallpox), herpesviruses (chickenpox, shingles, oral and genital herpes), adenoviruses (conjunctivitis, sore throat), hepadnaviruses (hepatitis B virus [HBV]), and papiUomaviruses (warts). Typically, DNA viruses enter the host cell nucleus, where the viral DNA is transcribed into messenger RNA (mRNA) by host cell polymerase and mRNA is translated into virus-specific proteins. 

Esimone and coworkers, in their increasing study of West African lichen species, identified the utility of R. farinacea derivatives against lentiviruses and adenoviruses. Esimone et al. (2005) initially showed that the ethyl-acetate-soluble fraction ET4) from the lichen Ramalina farinacea inhibited the infectivity of lentiviral and adenoviral vectors, as well as wild-type HIV-1. Recorded antiviral activity was about 20 pg/ml. Preliminary mechanistic studies based on the addition of the extracts at different time points in the viral infection cycle (kinetic studies) led to the suggestion that early steps in the lentiviral or adenoviral replication cycle could be the major target of ET4. Inhibition of wild-type HIV-1 was also observed at a tenfold lower concentratiOTi of the extract. 

Indeed, the whole question of the variable responses of mammalian cells to different human adenovirus serotypes is poorly understood, despite the fascinating range of interactions displayed. The variable abilities of cells derived from different species, or different tissues, to support adenovirus replication is generally interpreted in terms of the presence, or lack of, cellular factors that the virus needs to complete various steps in the replication cycle. More precisely, putative factors that permit human adenovirus replication in human cells may be present in, say, murine cells, but sufficiently divergent that they fail to interact optimally with the relevant viral components. Thus, an understanding in molecular terms of the steps in the viral replication cycle that are blocked in non- or semipermissive cells should provide important information about the host cell components utilized by the virus and, thus, the molecular interactions among viral and cellular products. 

It seems only reasonable to suppose that the ability of adenoviruses to induce cellular DNA synthesis and entry into the cell cycle in mammalian cells is of advantage to the virus replication cycle such a property could hardly have evolved for any other purpose. Bellett and colleagues (see, for example, Murray et al., 1982a) have pointed out the particular relevance of this ability to the natural conditions of infection. By contrast to the artificial laboratory situation in which the virus is usually provided with cells that are partially transformed (they are immortal) and undergoing rapid growth and division, most cells encountered by the virus in the natural host are likely to be arrested at the Gl/GO boundary. Thus, it would seem to be of considerable advantage to the virus to possess a mechanism that, soon after an adenovirus enters such a relatively inactive host cell, induces that cell to enter its most active biosynthetic state and, thus, provide maximal quantities of those cellular proteins upon which viral DNA synthesis and gene expression depend. 

Fig. 14.1 The life cycle of coxsackievirus B3. CVB3 starts its life cycle by attaching to its receptor CAR and coreceptor DAF. Internalized virus releases its viral RNA, which can be used as the template for translation of polyprotein or transcription by RNA-dependent RNA polymerase 3D to replicate its genome. The polyprotein is self-cleaved by virus-encoded proteases to release structural proteins and nonstructural proteins. Later, structural proteins and viral RNA will assemble into progeny virions to be released from infected cell. Abbreviations CVB3, coxsackievirus B3 DAF, decay accelerating factor CAR, coxsackievirus and adenovirus receptor 3Dpo1, RNA-dependent RNA polymerase.

A purified Ad5EGF-4 virus seed was propagated through three consecutive rounds of plaque purification. A final plaque was then expanded and purified by anion-exchange chromatography, sterilized by filtration and stored at -70°C 10°C. This virus stock was checked for sterility and absence of measurable replication competent adenovirus (RCA). A MVB was then created by one additional cycle of propagation in serum-free suspension culture of HEK 293 cells, and this virus was purified, aliquoted, and frozen. The MVB was tested and confirmed to be free of RCA as well as adventitious agents. The MVB was stored at-70°C 10°C. 

The antiviral activity of a triterpene saponin isolated from angallis arvensis, was studied in vitro against several vimses including HSV-1, adenovirus type 6, vaccinia, vesicular stomatitis and poliovirus (Amoros et al. 1987). The drug was found to inhibit the replication of HSV-1 and poliovirus type 2 as shown by inhibition of cytopathic effect and reduction of virus production. The action was not due to a virucidal effect but might involve inhibition of virus-host cell attachment. Single cycle experiments indicated that saponins interfered with both early and late events of herpes virus replication (Amoros et al. 1987). 

The best known response of cellular DNA synthesis to adenovirus infection must be the inhibition typically seen when permissive human cells are infected. In growing cells, cellular DNA synthesis is inhibited by more than 50% by 6-8 hr and completely by 10-13 hr after an adenovirus infection (Ginsberg et al., 1967), concomitant with the initiation and acceleration of viral DNA synthesis. Unfortunately, very few studies have concentrated on elucidation of this inhibitory process. Hodge and Scharff (1969) presented some evidence to suggest that initiation of new rounds of cellular DNA synthesis was prevented by adenovirus infection. Thus, when synchronized human cells were infected at a time in their cell cycle that placed viral DNA replication in the G1 phase, neither premature, nor normal S phase, initiation of cellular DNA synthesis were observed. Alternatively, infection to place initiation of viral DNA synthesis after the onset of the host cells S phase permitted completion of those rounds of cellular DNA synthesis begun before the inhibitory mechanism exerted... 

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