**4. The HPV L1 capsid protein and the viral life cycle**

L1 or the major capsid protein is one of eight known HPV specific proteins (E1, E2, E4, E5, E6, E7, L1 and L2).

It is produced within the cytoplasm and translocated into the nucleus, clearly visible by strong nuclear immunochemical staining reaction in intermediate and superficial squamous epithe‐ lial cells.

Figure 1 : HPV life cycle

b) Virus carrier

c) productive infection

L1 Capsid-Protein

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

L1+ L1-

d) premalignant transformation CIN III

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ASCUS, CIN I/II

A particular methodical advantage of the L1 capsid protein detection is that the protein is synthesized in the cells of the superficial layer of the epithelium that are easy to obtain by

The typical L1 staining is a strong, homogenous nuclear stain (Figure 3-7), in contrast to other markers, leading to a very good interobserver reproducibility. Using histological sections Galgano et al. [17] reported for Cytoactiv a raw agreement and k of 96.9% and 0.88, respectively. With 98% raw agreement and 0.96 for kappa Mehlhorn et al. [26] reported similar results for

a) Primary infection

latent infection No intraepithelial lesion NIL

**5. Study results**

taking a smear (see Figure 2).

the use of Cytoactiv in cytological samples.

**Figure 1.** HPV life cycle, as described in the text.

The L1 protein forms an icosahedral capsid with a T=7 symmetry and a 50 nm diameter. The capsid is composed of 72 L1 pentamers, linked to each other by disulfide bonds, and associated with the minor capsid protein L2, which encapsulates the viral DNA to build new infectious viral particles that are released in the upper epithelial layer. [14]

At the same time, L1 is a ligand for a still not reliably identified surface receptor, a heparan sulfate proteoglycan, on the basal cell layer of the epithelium to provide initial virion attach‐ ment to target cells. As a general rule, the HPV gains access to the basal epithelial layers as a result of epithelial erosions or mucosal ulcerations in the transformation zone susceptible to inflammation at the cervical/endocervical junction.

Once attached, the virion enters the host cell via a L2 dependant, clathrin-mediated endocy‐ tosis, the capsid becomes decraded, the virus DNA is released and routed into the nucleus of the cell [15].

The virus genome then separately lies outside the chromosomal DNA of the host cell as a ringshaped, episomal DNA molecule.

These initial steps are not associated with cellular changes that can be detected by morpho‐ logical methods. This individually variably long so called latent or silent virus infection can only be detected with molecular biological methods.

The signals to leave the latent virus infection and to initiate the productive or permissive phase of the viral life cycle, leading to a L1 synthesis, are not identified yet. Once differentiation of the immature squamous epithelial host cells begins, the viral DNA starts to replicate to high copy numbers. In the further course and dependent on the host cell differentiation the late proteins are synthesized, and encapsidates the viral DNA. Thus, mature, infectious viruses emerge, which are released from the perishing superficial squamous epithelia [16].

Within the scope of this productive phase, morphological epithelial changes mostly occur after several weeks or months post infection, which allow the cytological diagnosis of dysplasia in the smear. The typical morphological changes like nuclear enlargement, multinucleosis, changes in the chromatin structure and cytoplasm composition as well as koilocytes or ´halo cells´ have already been described by Papanicolaou.

Upon termination of the productive phase, the viral life cycle from primary infection to the release of the virus is completed without any malignant neoplasia having occurred (Figure 1). The L1 capsid protein is detectable at that stage of the life cycle, only.

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**Figure 1.** HPV life cycle, as described in the text.

Figure 1 : HPV life cycle
