**4. Viral proteins**

## **4.1. Proteins E1 and E2**

Protein E1 is encoded by the largest ORF found in the early genomic segment of PVs. Significant homology among the different PVs has been found upon comparison of the amino acid sequences inferred for the proteins that are encoded by the E1 gene [9].

Together with viral protein E2, protein E1 recognizes the origin of replication and represents the central factor of PV replication. It is indispensable for the initiation of viral DNA replication. In addition to this main function, E1 participates in the recruitment of host cell replication proteins and exhibits intrinsic ATPase/helicase activity, which induces relaxation of the DNA coiling at the origin of replication and during the progression of the replication fork [9].

Transformation studies using BPV1 have shown that the presence of an intact E1 ORF is crucial for the maintenance of viral genome stability in cells through the presence of multiple genomic copies in episomal form [10,11,12].

However, the interaction between protein E1 and the origin of replication exhibits low specificity. Specific and efficient recognition of the origin of replication occurs exclusively through the cooperative binding of proteins E1 and E2 to sites adjacent to the origin of replication. Therefore, protein E2 participates in this mechanism as an aggregation factor that promotes the recruitment of helicase E1 to the origin of replication [13].

The BPV1 E2 ORF encodes a protein that comprise the central viral regulatory system and thus control genetic expression and viral replication. Protein E2 also modulates the transcription of the early viral promoters through its binding sites [14]. In addition, E2 participates in the maintenance of the viral genome in its episomal form by promoting binding between these genomes and mitotic chromosomes during cell division [15,16].

## **4.2. Protein E4**

late segment (L) usually contains two ORFs that are expressed in differentiated keratinocytes. A third region without ORFs has been identified in all PV genomes and is named the LCR (long control region) or URR (upstream regulatory region).This region contains the origin of

replication and elements that control transcription (Figure 2) [1].

118 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

**Figure 2.** Schematic representation of genome of bovine papillomavirus type 1 (BPV1).

sequences inferred for the proteins that are encoded by the E1 gene [9].

maturation [8].

**4. Viral proteins**

**4.1. Proteins E1 and E2**

Expression of the six most common non-structural and regulatory proteins (E1, E2, E4, E5, E6, and E7), which are encoded by the early viral genome region, occurs in basal cells or during the intermediate stages of maturation. The expression of the two viral structural proteins (L1 and L2) encoded by the late genomic segment occurs in keratinocytes in the final stage of

Protein E1 is encoded by the largest ORF found in the early genomic segment of PVs. Significant homology among the different PVs has been found upon comparison of the amino acid

Together with viral protein E2, protein E1 recognizes the origin of replication and represents the central factor of PV replication. It is indispensable for the initiation of viral DNA replication. In addition to this main function, E1 participates in the recruitment of host cell replication proteins and exhibits intrinsic ATPase/helicase activity, which induces relaxation of the DNA coiling at the origin of replication and during the progression of the replication fork [9].

The non-structural protein E4 occurs abundantly in the cytoplasm of the differentiated keratinocytes of papillomas. Therefore, although the gene that encodes this protein is located in the early viral genome region, E4 is produced later in the differentiation process. The E4 protein of HPV16 has also been associated with the collapse of cytokeratin filaments, which thus suggests an auxiliary function in the process of viral exit from cells [1].

#### **4.3. Proteins E5, E6 and E7**

In humans, the oncoproteins E5, E6, and E7 encoded by the genomes of certain HPVs, represent the primary viral factors related to the onset and progression of cervical cancer. These genetic products are able to override the negative regulation of cell growth that is mediated by host cell proteins. In addition, it is believed that these viral oncoproteins promote the genomic instability observed in HPV-related cancers [17].

Binding with proteins of the retinoblastoma family is the main mechanism by which protein E7 contributes to the escape of infected cells from the negative regulatory mechanisms of cell growth. In the case of HPV, protein E7 interacts with these cellular factors and targets them for degradation [18]. The result of such binding and degradation is the release and activation of E2F transcription factors that regulate the expression of genes during S phase of the cell cycle. Efficient interaction between E7 and these factors triggers a compensatory inhibition of cell growth and apoptosis that is mediated by the p53 tumor suppressor protein-dependent pathway [17].

The targeting of protein p53 for degradation by viral protein E6 in high-risk HPVs eliminates the inhibition of cell growth in both undifferentiated and differentiated cells [17]. The actions of the viral proteins E6 and E7 to abrogate these regulatory factors of the cell cycle allow infected cells undergoing differentiation to remain in S phase. As a result, many cell cycle checkpoints are abrogated. Consequently, an accumulation of mutations and progression into cancer occurs in cells that are persistently infected by these viruses [19].

Most of the tumors of cattle affected by enzootic hematuria express the BPV2 oncoprotein E5 [20,21,22]. The onset of cellular transformation that is triggered by E5 might occur mainly through its interaction with and activation of the platelet-derived growth factor (PDGF) β receptor. Thus, a mitogenic response is induced even in the absence of PDGF [23,24].

**Viral Proteins Approximate size**

**Table 4.** Viral proteins and their functions.

**5. Clinical conditions in cattle**

**(kDa) Function / activity**

E2 34.3 recruitment of E1 to the origin of replication / modulation of the

E4 12.5 presumed auxiliary function in the virion exit from infected cells

E6 15.8 targets p53 tumor suppressor protein E7 13.6 binds to proteins of the retinoblastoma family

L1 55.5 component of the viral capsid L2 50.5 component of the viral capsid

Infections by different BPV types are related to several clinical conditions in cattle. The occurrence of the benign skin tumors that characterize cutaneous papillomatosis might be found in several areas of the animals' bodies. Depending on the extent of lesions, the devel‐ opment of the animals might be affected, they might become predisposed to secondary infections and/or infestations, and their hidescan be damaged. These possibilities are a few of the potential consequences that might result in economic losses for the beef and, even more so, dairy industries. Papillomas affecting the udders and teats of lactating cows cause diffi‐ culties with feeding calves and manual and mechanical milking, whereas secondary bacterial infections predispose the animals to clinical and/or subclinical ascending mastitis [30].

The interaction between specific BPV types and prolonged bracken (*Pteridium aquilinum*) intake has been suggested as the cause of enzootic hematuria and upper gastrointestinal tract cancers in cattle. With regard to enzootic hematuria, it is believed that latent or subclinical infections with BPV1 or BPV2 occur first in the bladder mucosa. Because the bladder represents the main target of bracken toxins, once the virus is established, infection might be reactivated, which might induce neoplasia through the immunosuppressant and carcinogenic chemical

Although the incidence of such tumors varies among cattle raised on bracken-infested

With regard to gastrointestinal tract tumors, the immunosuppression associated with bracken intake is defining for the persistence of BPV4-induced papillomas, which might progress into

compounds present in bracken, which results in progression to malignancy [31].

pastures, it might be higher than 90% among adult animals [31,32].

E5 5.2 interacts with and activates the platelet-derived growth factor (PDGF) β

transcription of the early viral promoters

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http://dx.doi.org/10.5772/56195

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receptor

E1 68.1 recognition of origin of replication / helicase activity

#### **4.4. Proteins L1 and L2**

The viral capsid consists of the two structural proteins L1 (*ca.* 55 kDa) and L2 (*ca.* 70 kDa), being L1 the major capsid protein and representing approximately 80% of the total virus protein [25]. Virus-like particles (VLPs) can be produced using prokaryotic and eukaryotic systems to express combination of L1 and L2 or L1 alone [26,27]. Although L2 is not needed for viral assembly, it is incorporated in the VLPs when it is co-expressed with L1. Under cryoelectron microscopy, the morphology of the VLPs that contain only L1 appear identical to that of intact viral particles (Figure 3) [28]. The epitopes that induce the production of neutralizing anti‐ bodies are principally found on L1 but might also be present on L2 (Table 4) [29].

**Figure 3.** Electron micrograph of VLP sproduced through expression of BPV2 L1*.*


**Table 4.** Viral proteins and their functions.

Most of the tumors of cattle affected by enzootic hematuria express the BPV2 oncoprotein E5 [20,21,22]. The onset of cellular transformation that is triggered by E5 might occur mainly through its interaction with and activation of the platelet-derived growth factor (PDGF) β

The viral capsid consists of the two structural proteins L1 (*ca.* 55 kDa) and L2 (*ca.* 70 kDa), being L1 the major capsid protein and representing approximately 80% of the total virus protein [25]. Virus-like particles (VLPs) can be produced using prokaryotic and eukaryotic systems to express combination of L1 and L2 or L1 alone [26,27]. Although L2 is not needed for viral assembly, it is incorporated in the VLPs when it is co-expressed with L1. Under cryoelectron microscopy, the morphology of the VLPs that contain only L1 appear identical to that of intact viral particles (Figure 3) [28]. The epitopes that induce the production of neutralizing anti‐

receptor. Thus, a mitogenic response is induced even in the absence of PDGF [23,24].

120 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

bodies are principally found on L1 but might also be present on L2 (Table 4) [29].

**Figure 3.** Electron micrograph of VLP sproduced through expression of BPV2 L1*.*

**4.4. Proteins L1 and L2**
