Human papillomavirus type 18 chimeras containing the L2/L1 capsid genes from evolutionarily diverse papillomavirus types generate infectious virus
Highlights
► We constructed HPV18 chimeric genomes in which the HPV18 capsid genes were replaced with those of evolutionarily diverse PV types. ► Cell lines were generated from these chimeric genomes and each was capable of immortalization. ► Each of the chimeric genomes generated infectious virus.
Introduction
Papillomaviruses (PVs) comprise a family of small, nonenveloped, double-stranded DNA viruses that replicate only in differentiating and stratifying cutaneous or mucosal epithelia. More than 100 human PV (HPV) types and numerous animal PV types have been sequenced. HPVs have been classified into genera according to the sequence homology of the major capsid gene L1 (Bernard et al., 2006, de Villiers et al., 2004). Generally, membership in a genus also correlates with biological and pathological characteristics (Bernard et al., 2006, de Villiers et al., 2004).
The PV capsid is approximately 50–55 nm in diameter and has an icosahedral symmetry of T = 7. The capsid is composed of 360 copies of the major capsid protein L1 organized into 72 capsomeres consisting of L1 pentamers. The position, structure and number of the minor capsid protein L2 in the capsid is unknown (Chen et al., 2000, Modis et al., 2002), however it has been suggested that there may be between 12 and 72 L2 proteins per capsid (Buck et al., 2008, Finnen et al., 2003, Trus et al., 1997, Volpers et al., 1994). Studies indicate that during infection the L2 protein is translocated into the nucleus independently of L1 and accumulates at the subnuclear structure, nuclear domain 10 (ND10) (Day et al., 1998, Florin et al., 2002a), where it induces a reorganization of the ND10 (Becker et al., 2003, Florin et al., 2002a, Florin et al., 2002b). Following the reorganization of ND10 by L2, the L1 and E2 viral proteins as well as viral genomes are targeted by L2 to relocate to these subnuclear regions (Day et al., 1998, Florin et al., 2002b). It has been suggested that this co-localization increases the local concentration of all of the necessary viral components involved in virion morphogenesis (Day et al., 1998). The targeting of L2 to ND10 may also facilitate the delivery of the viral genome to ND10 during primary infection to initiate viral transcription (Day et al., 2004). It is expected that additional interactions, direct and indirect, exist between viral nonstructural and structural genes. For example it has been shown that late viral life cycle functions are affected by the viral proteins E1^E4, E5 and E7 (Fang et al., 2006, Fehrmann et al., 2003, Flores et al., 2000, McLaughlin-Drubin et al., 2005, Nakahara et al., 2005, Peh et al., 2004, Wilson et al., 2005, Wilson et al., 2007). Little is known about what cellular players may contribute to the process of virion morphogenesis except that the chaperone protein Hsc70 transiently associates with the c-terminus of L2 at the ND10 subnuclear regions (Florin et al., 2004) and HSP70i promotes the nuclear localization of L1 (Song et al., 2010), suggesting a function for these cellular proteins during viral assembly.
Roden et al. (1996), using in vitro generated virus-like particles, demonstrated that HPV16 L2 and L1 capsid proteins expressed from a Semliki Forest viral vector could self-assemble capsid particles encapsidating BPV-1 genomes. This finding suggested that the capsid proteins of one PV type were capable of encapsidating the genome of another type. However, the potential for the capsid proteins of one PV type to package the genome of another PV type had not been tested in a physiologically relevant system where the mechanisms governing native PV replication are in control.
Chimeric genetic analysis systems have been used with other viruses to study viral infectivity, replication, transforming potential, immunity, and virulence factors. Often chimeric viruses are used to compare genes from one virus with homologous genes from a related virus to attempt to ascertain their similarities and differences. Using chimeric genetic systems one can assign a particular phenotype to a specific gene or sequence. The study of chimeric viruses may reveal relatedness between exchanged sequences via the ability of the foreign sequence to provide all the needed functions and interactions for the virus to replicate. Information gained from such studies can elucidate mechanisms of viral replication and pathology, and identify common mechanisms for therapeutic targeting. Chimeric viruses have been successfully used to genetically analyze the biology of many DNA viruses, including the JC virus, SV40, BK virus, herpesviruses, and adenoviruses (Bollag et al., 1989, Daniel et al., 1996, Eberle et al., 1997, Gall et al., 1996, Gall et al., 1998, Haggerty et al., 1989, Krasnykh et al., 1996, Lynch et al., 1994, Miyazawa et al., 1999, O’Neill et al., 1992, Roy et al., 1998, Sawada et al., 1994, Tavis et al., 1994, Telling and Williams, 1994, Trowbridge and Frisque, 1993, Vacante et al., 1989, Zabner et al., 1999).
Our laboratory has previously been successful in establishing in vitro organotypic raft culture systems capable of synthesizing native infectious HPV16 (McLaughlin-Drubin et al., 2004), HPV18 (Meyers et al., 1997), HPV31 (Meyers et al., 1992), HPV39 (McLaughlin-Drubin and Meyers, 2004), HPV45 (McLaughlin-Drubin et al., 2003), and a chimeric HPV18/16 virus (Chen et al., 2010, Chen et al., 2011, Meyers et al., 2002) in differentiating and stratifying host epithelial tissue. The extension of our raft culture system to studies on HPV18/16 capsid protein chimeras demonstrated for the first time the use of a viable chimeric virus system to study PV genetics under natural host replicating conditions (Meyers et al., 2002). Although few direct interactions between early and late PV gene products have been characterized, this study showed that the early proteins and their functions in the viral life cycle of HPV18 were functional when coupled with the late genes of HPV16 (Meyers et al., 2002).
Given that HPV16 capsid proteins can interact with HPV18 early genes to generate infectious virus, we address here whether such interactions are common among all PV types or whether productive interactions are based upon the relatedness of the two PV types, a phenomenon observed in other viral families. Relatedness could be defined in several ways including; genetic and evolutionary differences, disease association, or species and anatomic site tropism. To cover these different types of relatedness we created a series of intertypic, chimeric PV genomes. We generated cell lines from the intertypic, chimeric genomes and demonstrated that each genome was capable of immortalization. Furthermore, tissues grown from these cell lines differentiated and stratified appropriately. Surprisingly, each of the chimeric genomes generated infectious virus. These findings suggest the existence of conserved viral domains that could be targeted in the development of universal papillomavirus therapeutics.
Section snippets
Plasmid construction
Using restriction digest and PCR technologies, the L2 and L1 ORFs from HPV18 were replaced with an unique BglII site creating the pHPV18L2/L1Δ construct (Meyers et al., 2002). PCR primers were designed to amplify the L2 and L1 ORFs of HPV45, 39, 33, 31, 11, 6b, 1a, CRPV, and BPV1, introducing BglII sites at both the 5′ and 3′ ends. Using the BglII sites, each L2/L1 PCR amplified sequence was ligated into the HPV18L2/L1Δ genome creating the following HPV18 L2/L1 chimeras; HPV18/45, HPV18/39,
Construction of papillomavirus chimeras
The structural genes of HPV16 and nonstructural genes of HPV18 are able to interact to produce infectious chimeric viruses (Chen et al., 2010, Chen et al., 2011, Meyers et al., 2002). This ability to interact may correlate with the sequence homology of HPV16 and HPV18 structural genes. The amino acid sequences of HPV16 L1 and L2 are 73.7% and 64.0% similar, respectively, to those of HPV18 (Table 1A). HPV16 and HPV18 are also both mucosotropic viruses and have a similar pathological spectrum.
Discussion
The study of protein chimeras can provide important information on protein structure and function, particularly as a tool to study viral infection and replication (Belnap et al., 1996, Daniel et al., 1996, Gall et al., 1996, Gall et al., 1998). For example, using chimeric JC viruses in which the JC virus early region was replaced with the corresponding sequence from the SV40 genome helped to identify functional sequences required by JC virus for host cell transformation (Bollag et al., 1989).
Acknowledgements
We thank members of the Meyers’ laboratory for many helpful discussions and Lynn R. Budgeon for histological assistance. This work was supported by grant RO1 AI 057988 (C.M.).
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- 1
Present address: Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA.
- 2
Present address: Int. Med Inf. Diseases, Yale School of Medicine, 300 Cedar St., TAC S410, New Haven, CT 06520, USA.
- 3
Authors contributed equally to the manuscript.