The adenovirus replication cycle Early events

Adenovirus enters the cell via receptor-mediated endocytosis in a manner analogous to that of papillomaviruses. Cellular receptors interact with the virion fiber proteins to initiate infection. Adenovirus DNA with a specific terminal protein bound to each 5' end is released into the nucleus where it associates with cellular histones. In order to initiate gene expression, adenovi-ruses must stimulate the infected cell to transcribe and replicate its genes. This is accomplished by expression of the spliced mRNAs encoding the immediate-early (or "pre-early") gene E1A and E1B protein "families." The promoters for these are enhanced and can act in the cell in

Early

I I I I I J I I I I I I I I I J I I I I I I I I I J I I I I I I I I I J I I I I I I I I I J I I I I I I I I I J I I I I I I I I I J I I I I I I I I I J I I I I I I I I I J I I II I I I I I J II I I I

Late

"] (polymerase) Terminal protein

72KDBP

60 70

Fig. 16.7 The genetic and transcription map of the 36-kbp adenovirus genome. There are three kinetic classes of transcripts. The E1 transcripts are controlled by enhanced promoters and require no modification of the host cell because some functions of their expression are similar to those of T antigen in SV40 virus replication. These functions include stimulating cellular transcriptional activity and cell replication. Early in infection, only early transcripts are expressed. These include mRNAs encoding viral DNA polymerase and terminal proteins. There are a number of early promoters and transcription units. The E2 transcription unit also has a 72-kd DNA-binding protein (72KDBP) that shuts off early transcription. Two primary transcripts, E2A and E2B, are expressed from the same E2 promoter. The mRNA for the DNA-binding protein continues to be expressed late because there is a second promoter upstream of the E2 promoter that is not shut off by the DNA-binding protein. The major late promoter at map position 15 is always "on," but polyadenylation and splicing patterns change markedly as infection proceeds. Late in infection, the late transcription unit extends to one of five polyadenylation signals and differential splicing results in generation of a myriad of late mRNAs encoding structural proteins as well as proteins involved in host cell modification and virus maturation.

the absence of any viral modification (like the SV40 early promoter). The E1A gene products block the ability of the p53 and Rb growth suppressor genes to suppress cell division, while one or several E1B proteins inhibit apoptosis in the stimulated cell. Thus, these two proteins work in concert in a manner similar to that of polyomavirus large T antigen.

Stimulation of the infected cell's transcriptional machinery leads to expression of the four early pre-mRNAs that are spliced in various ways to produce early proteins, including a DNA polymerase protein (140-kd pol), a terminal protein, and a 72-kd DNA-binding protein (DBP). The latter shuts off most early promoters, but the E2 region is not shut off because a second promoter becomes active at times when 72-kd DBP is at high levels. Interestingly, the major late promoter is "on" early in infection, but only the L1 region is expressed as mRNA because all transcripts are terminated at the polyadenylation signal at 40 map units. This termination is due to the inhibition of splicing downstream of the L1 region through binding of cellular splice factors. Further, late transcripts downstream of L1 are not transported from the nucleus.

Adenovirus DNA replication

Adenovirus genome replication takes place via an unusual mechanism that involves formation of ssDNA as intermediates; the process is shown in Fig. 16.8. Adenovirus DNA replication begins at either or both ends of the DNA and uses as a primer an 80,000-dalton precursor of the 50,000-dalton viral genome-bound terminal protein. The large priming terminal protein is proteolytically cleaved to the smaller terminal protein found in capsid-associated genomes during packaging. This is the only known instance where DNA replication initiates without a short RNA primer. However, the terminal protein does contain a covalently bound cytosine residue from which DNA replication proceeds. Note that replication utilizes the adenovirus-encoded DNA polymerase and is continuous — there are no short Okazaki fragments seen. The process can liberate the other strand as ssDNA, which can become circular by association of inverted repeat sequences at the end, and replication proceeds. Thus, adenovirus DNA replication can proceed via two routes shown in Fig. 16.8. If DNA synthesis initiates at both ends of the genome about the same time, type I replication occurs. If only one end of the genome is used to initiate a round of DNA synthesis, then type II replication occurs.

Late gene expression

With the increase in levels of early 72-kd DBP, much early gene expression shuts off. At the same time, E4 protein interferes with the inhibition of splicing downstream of the L1 region, effectively this results in differential polyadenylation site usage changes so that transcription from the major late promoter generates transcripts covering as much as 24,000 bases. Differential polyadenylation and splicing generate the five families of late mRNAs that are translated into the structural proteins that will make up the capsids. Other late proteins alter aspects of cellular structure and metabolism to ensure efficient virus assembly and release. In addition to altering splicing patterns, some species of E4 protein actively mediate the transport of late mRNA from the nucleus to the cytoplasm.

VA transcription and cytopathology

The complex interaction between human adenovirus infection and the host cell requires that the cell remains functional for a long period following infection. This precludes extensive virus-induced shutoff of host cell function; hence, virus-induced cytopathology is slow and cell death takes a long time. During this period, the cell can mount defenses against viral gene expression such as the induction of interferons, cellular gene products that can render neighboring cells resistant to virus infection (see Chapter 8, Part II). The human virus gets around this problem by synthesis of VA RNA, which is a short, highly structured RNA molecule that interferes with the cell's ability to produce interferon and, most likely, other defense mechanisms. Indeed, this

New protein

Type I replication Type I replication

dC

Type II replication dC

dC rTTTTTTTTTTTTTTTTTTT

o dC rTTTTTTTTTTTTTTTTTTT

dC dC

Type I and II replication o 5_3;

^C rTTTTTTTTTTTTTTTTTTT

Type I and II replication

dC 1 1 1 1 1 1 1 1 1 111 1 1 1 1 1 1 1 1 Maturation of

3' 5' dC terminal protein

Fig. 16.8 Adenovirus DNA replication. The 5' ends of the viral genome have 50,000-dalton terminal proteins bound to them. Adenovirus does not have discontinuous strand synthesis, and exhibits other features that are at variance with the general scheme for viral DNA replication outlined in Chapter 14. Of major interest is the fact that there is no discontinuous strand synthesis. The process is marked by the accumulation of a large amount of single-stranded DNA (unusual in eukaryotic DNA replication). Further, the initial priming event requires the first nucleotide of the new DNA strand being covalently bound to the 80,000-dalton precursor of the 50,000-dalton terminal protein. Following complete second strand synthesis, the precursor end proteins are proteolytically cleaved to form the mature terminal proteins. TBP = precursor to terminal binding protein.

RNA molecule has many features of siRNAs, which were discussed briefly in Chapter 8. VA RNA is expressed via cellular RNA pol III, which is the same polymerase used to transcribe cellular amino acid transfer RNAs (tRNAs). Interestingly, while the human Epstein-Barr herpesvirus expresses an analogous transcript (see Chapter 18), suggesting that this is an important feature in virus-mediated immune evasion, a number of adenoviruses of domesticated animals do not express a homologue to VA RNA.

A second aspect of the interaction between adenovirus and the host is reminiscent of papil-lomavirus replication. Adenovirus remains associated with the host for long periods of time as a persistent infection, especially in the epithelium of the adenoidal tissue and the lungs. The virus infects basement cells, but initiates DNA replication and viral assembly only in terminally differentiated cells. The virus actually induces an acceleration of apoptosis of these differentiated cells. One apparent advantage of eliminating dying infected cells is that more room is made available for the differentiation and growth of basement cells. This provides a ready and continuing source of cells in which the virus can initiate new rounds of replication. This stimulation of apoptosis presumably occurs because the relative levels of E1A and E1B are different in critical cells as compared to cells in which apoptosis is blocked by the latter viral protein.

Transformation of nonpermissive cells by adenovirus

As with SV40, infection of nonpermissive cells by at least some adenovirus types can lead to cell transformation and tumor formation. While there is currently no evidence for any involvement of adenovirus infection in human cancers, transformation seems to be accomplished by mechanisms very similar to those outlined for papovaviruses. Indeed, under some conditions, adenovirus gene products can substitute for early papovavirus gene products in mixed infections.

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