*6.2.2. Loss of L1 in precancerous lesions with an episomal HPV genome*

status of the host cells to allow replication in ´non dividing cells´ and the maturation of new

As long as the L1 capsid protein can be detected within the nucleus of dysplastic cells the virus was successful in this ´walk on the edge´. Despite of all viral activities the cells are still in the

L1 capsid protein negative dysplasias, however, are due to this virally induced cellular

A shift from a productive HPV infection towards a non-productive or precancerous lesion has

The reasons for this event are multifarious since the differentiation dependent expression of L1 is controlled at multiple levels. A block at any of the following steps, such as transcription (integration and / or methylation of the DNA), post-transcriptional processing and translation,

Integration of the viral DNA is considered to be of critical importance for the progression from CIN to cancer, since the frequency of HPV-16 viral integration increase in parallel with the

During the integration process of the HPV genome into the host chromosome a linearization of the ring – shaped, episomal viral DNA is required. It's easy to imagine, that this non directed event is regularly associated with a deregulation of the strictly controlled episomal DNA. As a consequence of the integration process alterations of the control region and loss or disruption

Even if the L1 gene is not affected directly, the integration of the virus with loss of transcrip‐ tional control by E2 results in over expression of E6 and E7 leading to immortalization and

As a result, the epithelial host cell remains in the cell cycle and increasingly becomes genetically

L1 genes can functionally be inactivated afterwards too, as a result of mutation, gene deletion and insertion as well as DNA methylation so that no capsid protein will be produced anymore

A dysmaturational autonomous tumor emerges in the host epithelium; a ´point of no return´

But for the background that many of the HPV-associated cancers do not even carry any integrated viral genome [31], [33] additional mechanisms have to exist to block L1 expression. In the meantime a discussion started if integration is the initial step towards cervical cancer, or maybe only the consequence of the E6/E7 induced genetic instability of the host cells.

of HPV specific proteins like the early and late proteins can be observed [31].

instable without being able to run its differentiating program.

condition to allow the normal, productive life cycle of the Human Papilloma Viruses.

106 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

deregulation processes no longer capable to produce virions.

could be responsible for the loss of L1 capsid protein.

*6.2.1. Loss of L1 due to integration of the HPV genome*

infectious virus particles.

**6.2. Transcriptional control**

severity of cervical lesions.

transformation of the cells [32].

(discussed later).

is crossed.

occurred.

Control of gene expression by epigenetic modification of distinct DNA sequences is a funda‐ mental biological process, which affects for example embryonic development, cellular differentiation and others.

One important mechanism, affecting the chromatin conformation, is the methylation of DNA, specifically at cystidine-guanidine (CpG) dinucleotides. Methylated CpG dinucleotides bind repressors, which alter the conformation of nucleosomes through their interaction with histone deacetylases in a manner unfavourable to transcription [34].

In the meantime it's known that epigenetic mechanisms play a major role in the transcription of the HPV genome as well [35], [36].

Several reports showed that the HPV genome is differentially methylated during progression from simple infected to transformed cells.

Alterations were observed particularly in the control region, and the L1 and L2 gene in high grade precancer and invasive cancer. These observations lead to the suggestion that the lack of expression of these genes may be attributed at least in part to increasing methylation of the respective parts of the viral genomes.

Kalantari et al. for example reported that methylation exceeds 50% in the case of some CpG dinucleotides within the L1 gene [37].

In addition E2 expression seems to be strictly linked to the differentiation process from normal to malignant cells, indirectly affecting L1 expression as well. Vinokurova showed that E2 binding sites are highly methylated in undifferentiated cells, inhibiting E2-bind‐ ing, and demethylation at the E2 binding sites occurs in association with the cell differen‐ tiation only [38].

That means different mechanisms are existing to prevent L1 mRNA transcription.

#### **6.3. Post transcriptional control**

Once the transcription of the late mRNA was successful, additional mechanisms have been reported that are able to control or block the L1 capsid protein expression.

### *6.3.1. Control of the stability of late mRNA*

Mori et al. [39] showed that RNA instability elements are within the L1 and L2 coding mRNAs of HPV16, which function in undifferentiated cells. Although the mechanism for RNA destabilization are still subject of further investigations this mechanism could be important for L1 expression.

#### *6.3.2. Nuclear export of late mRNAs,*

Koffa et al. [40] reported that the L1 mRNA of HPV16 was retained in the nucleus in undif‐ ferentiated W12 epithelial cells, suggesting that the nuclear export of late mRNAs was inhibited in the dividing cells, thus preventing translation of the L1 protein in the cytoplasm. The factor(s) mediating the nuclear export of late mRNAs has not been identified yet [41].

**7.2. The role of the L1 capsid protein in immune recognition**

being cytologically mild or moderate, is low.

cells.

regression [47].

clearance of the HPV infection.

positive intraepithelial lesions.

**7.3. Progression of L1 capsid protein positive cases**

is only detectable in terminally differentiated cells.

regularly if the L1 capsid protein is detectable in the dysplastic cells.

The only fully accessible antigen sources in the earlier stage of virally induced SIL to promote an activation of the immune system are free viral particles consisting of 360 L1 capsid proteins. Therefore it seems not to be surprising, that a clinical remission of the lesion is observed

HPV L1 Detection as a Prognostic Marker for Management of HPV High Risk Positive Abnormal Pap Smears

http://dx.doi.org/10.5772/55902

109

To generate an effective virus specific immune response the virus particles have to be detected by the antigen presenting cells (APC) of squamous epithelium, the Langerhans cells (LC) or Dendritic cells (DC), and armed effector cells, has to migrate back to the infected site, and destroy the infected keratinocytes leading to a spontaneous clinical remission of the lesion. If such immunologic activation mechanisms are functional, they are quite effective since with about 20% the malignant potential for these L1-positive lesions, irrespective of the dysplasia

On the other hand it is still not clear how L1 specific cytotoxic T-cells could be able to destroy the HPV reservoir in the basal cell layer to cause clinical remission, since the L1 capsid protein

In analogy to the basal cells it was shown for the L1 capsid protein negative C3 cell line, that these cells are sensitive to L1 specific cytotoxic T-cells [46]. The only explanation seems to be a L1 expression level in these cells (as well as the basal cells), lower than the detection limit used for analysis. As described earlier L1 mRNA is detectable in the nucleus of undifferentiated

A second explanation for the clinical remission of L1 capsid protein positive dysplasias could be, that the viral capsids work as a kind of abjuvance, only triggering the cell mediated response to the early proteins, principally E2 and E6, which are thought to be important for lesion

Nevertheless it was shown recently by Mehlhorn et al. [48] that the detection of antibodies against HPV16 L1 in the serum is always associated with clinical remission, if the L1 capsid protein is detectable in the smear of HPV high risk positive mild and moderate dysplasias. These L1 specific serum antibodies shouldn't be able to fight against the HPV infected cells, but it shows that a L1 specific activation of the immune system is of critical importance for the

An ineffective immune response maybe promoted by HLA incompatibilities, factors contri‐ buting to cervical cancer like tobacco smoking or the coexistence of dysplasias of different grade in the transformation zone, possibly reflecting a mixture of L1-positive and L1-negative lesions with different progressive potentials may be the reasons for a progression of some L1-

In addition Yang et al [49] identified several mutant HPV16 L1 isolates carrying genes encoding proteins that neither assemble nor activate VLP-dependent innate and adaptive immune

#### **6.4. Translational control of late mRNA**

Last but not least the rare codon usages found in L1 and L2 might also contribute to the inhibition of late gene translation [42]. In terminally differentiated cells, the altered expression ratios of tRNA species could compensate for the inhibitory effect of the rare codon usages [43].

All these steps could be of critical importance for L1 capsid protein expression. As indicated a lot of questions are still remaining and need to be answered in the future. Most probably not only a single control mechanism is responsible for the oberserved loss of L1 capsid proteins.
