Replication of papillomaviruses

Cell transformation by SV40 appears to be a laboratory phenomenon, and many of the tumors caused by polyomaviruses can be thought of as dead-end artifacts of virus infection. In such infections, persistence appears to be due to the stability of histone-associated viral genomes in nonrep-licating cells marked by occasional episodes of low level viral replication as a result of an immune crisis or other events that lead to changes in the transcriptional environment of the host cell.

By contrast, a related group of viruses, papillomaviruses, follow a natural replication scheme in their host that requires the formation of tumors, usually benign tumors, in their replication cycle. In this strategy of virus replication, persistence is a consequence of the continued replication of cells bearing viral genomes!

Papillomavirus replication combines some aspects of both the abortive and productive schemes just discussed. These viruses cause warts or papillomas, and there are many different types, with most showing no antigenic cross-reactivity with each other. Infections with most papillomavirus types are completely benign (although irritating or occasionally painful), but some can be spread by sexual intercourse, leading to persistent genital infections, especially in females. Statistical analyses comparing the incidence of cervical carcinoma and the patterns of

T-Ag protein

Fig. 16.4 Representation of the two steps in transformation of a nonpermissive cell by SV40. The infection begins as described in Fig. 16.2 and early mRNA is expressed into early proteins. The infection is abortive in that DNA replication and late gene expression cannot occur in the nonpermissive cell. Still, the large T antigen (T-Ag) is able to interfere with cellular growth control (tumor suppressor) proteins, leading to cell replication. Stable transformation requires a second step, the integration of the viral DNA. This is a random (stochastic) occurrence with SV40, and integration is random throughout the genome. A similar path is followed in the transformation of nonpermissive cells by other polyomaviruses. t-Ag=small t antigen.

persistent infection by some of these papillomaviruses [including human papillomaviruses 16 and 18 (HPV-16 and HPV-18)] demonstrate a highly significant correlation despite the fact that only a small number of individuals actually get the disease. Thus, these viruses are clearly human cancer viruses.

The HPV-16 genome

The circular genome of HPV-16 is shown in Fig. 16.5. It is about 7900 base pairs long and is vaguely reminiscent of that of SV40 except there are many more early ORFs. Note that the region marked "LCR" corresponds to the promoter/origin region of SV40. Since the replication of papillomaviruses is difficult to study in cultured cells, a full characterization of the splicing patterns and transcripts expressed during infection has been and continues to be a very laborious effort. It requires analysis of DNA copies made of viral RNA using retrovirus reverse transcriptase, followed by cloning of the cDNA copies. Polymerase chain reaction (PCR) amplification of cDNA for direct sequence analysis also has been used. General methods for such analysis are covered in Chapters 11 and 12.

Sequence analysis of the bovine papillomavirus genome and the transcripts expressed indicates that early and late transcripts are expressed from a single or limited number of early and late promoters as pre-mRNAs. While the extensive splicing of pre-mRNAs is reminiscent of infections with polyomaviruses, papillomaviruses differ in that early and late promoters are found in several regions within the genome.

Virus replication and cytopathology

Formation of a wart by infection with papillomavirus is outlined in Fig. 16.6. It involves virus entering the basal cells of the epithelium (the skin in the case of warts). The virus expresses

Fig. 16.5 The human papillomavirus (HPV)-16 genome. The 7-kbp circular genome contains a number of translational reading frames that are expressed from spliced mRNAs. Unlike the related polyomaviruses, papillomaviruses encode all proteins on the same DNA strand. The actual details of mRNA expression also appear to differ among different papillomaviruses. For example, HPV-16 has only one known promoter, which appears to control expression of both early and late transcripts. The locations of cleavage/polyadenylation signals for early and late transcripts are shown. All mRNAs appear to be derived by splicing of one or two pre-mRNAs. The characterization of transcripts has required heroic efforts of isolating small amounts of RNA from infected tissue, generating cDNA clones by use of reverse transcriptase and polymerase chain reaction, and then sequence analysis. This is necessary because many are present in very small amounts in tissue and the virus does not replicate in cultured cells. The transcripts shown are three of nine that have been fully characterized, and it can be expected that others are also expressed. The region marked "LCR" encodes both the constitutive (plasmid) origin of replication and an enhancer. Location of the vegetative origin of replication is not known. Specific details of papillomavirus replication are described in the text.

Skin epithelium

- Stratum corneum

Papilloma virus infection

Papilloma virus infection

Viral DNA (episome)

Basement epithelium (non-permissive)

Benign transformation

Fig. 16.6 The formation of a wart by cell proliferation caused by infection of basement epithelial cells with human papillomavirus (HPV). Early gene expression leads to stimulation of cell division and terminal differentiation. This results in late gene expression and virus replication in a terminally differentiated, dying cell, which produces large quantities of keratin.

Differentiating cells become permissive for vegetative viral DNA replication

Differentiating cells become permissive for vegetative viral DNA replication

Cells proliferate locally, differentiate ^^ and cause wart or papilloma ,---' ^

Sloughing of virus laden keratinized epithelial cells

Virus assembly

Cells proliferate locally, differentiate ^^ and cause wart or papilloma ,---' ^

Sloughing of virus laden keratinized epithelial cells

Virus assembly

Viral DNA replication

Early viral gene expression

Viral DNA replication

Early viral gene expression

Some HPV types can cause progression to malignancy via genome integration and continued cell replication along with accumulating mutations early genes that induce cells to replicate their DNA rather more frequently than would an uninfected epithelial cell. Thus, one set of early functions is analogous to those of SV40 T antigen. But in marked contrast to SV40 replication in permissive cells where infection leads to vegetative viral genome replication and cell death, papillomavirus DNA remains in the infected cell nucleus as an episome or "mini"-chromosome where it can replicate when cell DNA replicates, but it does not replicate to the high numbers seen in viral DNA replication of a productive infection.

Such cell-linked replication is often termed plasmid-like replication. It involves the interaction of cellular DNA replication proteins with the viral origin of replication, which during persistent infection acts like an origin of cellular DNA replication and is subject to similar control. As the cells are stimulated to divide, they differentiate, and as they differentiate, they change their function and begin to produce proteins typical of terminal epithelial differentiation. For example, synthesis of K5 and K14 keratins characteristic of basal cells is terminated and keratin K1 and K10 characteristic of suprabasal skin cells are expressed. At some point in this terminal differentiation, some of these cells become fully permissive for high levels of viral DNA replication and late gene expression to generate capsid proteins. Such cells produce new virus while they die. Since this phenomenon is highly localized, and the virus infection normally just speeds up normal terminal differentiation of the epithelial cells, a benign wart is formed.

For HPV-16 and HPV-18, this growth enhancement is known to be a function of the actions of proteins encoded by the E5, E6, and E7 gene products that associate with and inactivate normal functions of the p53 and Rb proteins in a manner analogous to large-T-antigen activity in SV40. Presumably, chronic infection of cervical epithelium with either of these viruses can (rarely) generate a true cancer cell by further mutations of other control circuits in the cell. This oncogenic transformation is coincident with integration of papillomavirus DNA into cellular DNA, and it is speculated that oncogenesis involves a process similar to the transformation stabilization seen in abortive SV40 infection of the appropriate nonpermissive cell.

In such a transformed cell, no virus is produced, so formation of the cancer can be looked at as a dead-end accident induced by the continued stimulation of cell division caused by the virus's persistent infection. As these transformed cells continue to divide, they accumulate mutations that eventually allow them to spread to and invade other tissues, and form disseminated tumors (metastasis). In the case of benign warts in the skin and elsewhere, either inactivation of the p53 and Rb proteins is not so profound, or the stimulated cells are so close to death in their terminally differentiated state, that they cannot become cancerous.

We have already mentioned that HPV-16 and HPV-18 can be sexually transmitted and has a high correlation with cervical cancer in women who are persistently infected with these strains. Other strains, notably HPV-6 and HPV-11, have also been associated with this disease. As a result, a quadrivalent vaccine has been developed to protect against these four strains. The recombinant vaccine, trade name Gardasil, has been produced by Merck. The vaccine uses the major capsid protein L from each virus, produced by recombinant DNA techiniques, to form self-assembled virus-like particles (VLPs). A similar product, trade named Cervarix, is under development by GlaxoSmithKline. Gardasil was approved by the Food and Drug Administration (FDA) for general use in June of 2006.

The potential impact of this vaccine is quite large. If used prophylactically and administered to young women before they are sexually active, the vaccine could reduce the worldwide incidence of cervical cancer dramatically. This disease currently has a yearly prevalence of 16 per 1000 women, with an annual death rate of 9 per 1000, making it the third leading cause of death for women, behind breast and lung cancers. In the United States, the prevalence and mortality rates are lower, probably due to the widespread use of Pap smears for early detection. The use of this vaccine does not suggest that Pap smears can be abandoned, however, since at least 10% of cervical cancers are not linked to infection by these four viruses.

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