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Title: Large Scale RNAi Reveals the Requirement of Nuclear Envelope Breakdown for Nuclear Import of Human Papillomaviruses}
Article:
Abstract:
A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16( HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA( vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies.
Introduction:
Papillomaviruses are small, nonenveloped DNA viruses that cause mostly benign lesions[1]. Several human papillomavirus( HPV) types are, however, etiological agents for anogenital cancers, and tumors of the head and neck[1]. Among them, HPV16 is the most prevalent, and the best studied[2]. No specific drug treatment against HPV-related cancers exists, but the increasing deployment of anti-HPV vaccines will likely help to reduce the incidence rate of the vaccine types[3]. The spherical HPV particles have an icosahedral( T = 7) architecture, and a diameter of 50–55 nm. The capsid is formed by 72 homo-pentamers of the major structural protein, L1, and contains in addition 12–72 copies of the minor structural protein, L2[4]. The 8kB DNA genome is circular and double-stranded. During initial infection, HPVs enter basal stem cells or transiently amplifying cells in squamous epithelia[1]. Replication and assembly of new viruses occur in the nucleus, when the host cells differentiate into spinous and granular keratinocytes[1]. Since authentic viruses are difficult to be propagated in sufficient amount and purity, most of what is known about early interactions between HPV and host cells has been learned using virus-like particles( VLPs) and pseudoviruses( PsV). These surrogate particles have the protein composition and architecture of the authentic HPV, but do not carry the viral genome[5],[6]. Instead, the PsV contain a pseudogenome that, when delivered to cells, expresses a reporter protein[6],[7]. Binding of VLPs and PsV of HPVs to tissue culture cells depends on heparan sulfate proteoglycans( HSPG)[8]–[11]. Highly sulfated domains of the HS induce the first of several conformational changes in the incoming virion[12]–[14]. The next change is caused by cyclophilin B, a peptidylprolyl isomerase[15],[16]: The N-terminal peptide of L2 buried within the capsid is exposed so that it can be cleaved by furin[14],[15],[17],[18]. The alterations in the capsid lead to a loss of affinity to HSPGs and transfer to a secondary receptor[8],[14],[15],[19],[20], such as alpha6-integrin, annexin A2 heterotetramer, growth factor receptors, and the tetraspanin CD151[21]–[28]. However, the exact functions of these receptors in PV entry is disputed[29]. HPV entry into host cells requires endocytosis. Compared with other animal viruses, endocytic uptake is slow and asynchronous( t1/2 = 4–12 h)[8],[30],[31]. Reporter gene expression of the pseudogenome is not detected until 24–48 h postinfection( p. i.)[31]. The endocytic mechanism used by high-risk HPV16, 18, and 31 is ligand-induced, clathrin-, caveolin-, lipid raft-, and dynamin-independent, and critically dependent on actin dynamics[27],[31],[32]. The viruses are internalized in small vesicles, transported to late endosomes or endolysosomes, and the Golgi complex[31],[33],[34]. The subsequent steps are poorly understood. It is known, however, that a late step involving penetration into the cytosol or initial uncoating of the capsid requires exposure of the virus to low pH[31]. Also, the major capsid protein L1 dissociates from a subviral complex composed of the viral DNA( vDNA) and L2 probably within endosomal compartments[33],[35]. In transit to the nucleus, the subviral complex is routed to the Golgi complex[33],[34]. After penetration into the cytosol[36], the vDNA moves complexed with L2 into the nucleus and eventually localizes to ND10 nuclear domains[37],[38]. How nuclear import of this subviral complex occurs is not known. Of note, entry of HPV16 requires progression of cells through the cell cycle into early mitosis[39]. However, it is unclear which entry step is influenced by cell cycle progression. In this study, we used a large-scale RNA interference( RNAi) screen to identify host cell factors and processes involved in HPV entry. Such screens have emerged as a powerful tool to analyze pathogen-host interactions[40]. By focusing our follow-up studies on a cluster of genes that suppresses HPV16 infectivity, we uncovered a crucial role for mitosis in HPV16 nuclear entry. Mitosis was important, since nuclear import of the HPV16 vDNA genome requires changes in nuclear envelope( NE) permeability facilitated by nuclear envelope breakdown( NEB). These changes allowed association of the subviral complex with host cell chromatin aligned on the metaphase plate.
Discussion:
To screen for cell factors that influence entry and other early events in the HPV infection cycle, we employed a two-step strategy with a total of six siRNAs per gene. We relied on a robust, image-based infection assay, supervised classification of phenotypes and an improved bioinformatic pipeline[41],[45]. To avoid some of the pitfalls inherent in the RNAi approach[40], we focused our validation on gene clusters rather than individual hits. The primary screen involved 7000 human genes targeted in the so-called druggable library of siRNAs( Qiagen). Of the 315 potential hits, 291 were followed up in a secondary screen with additional siRNAs, and 162 could be confirmed. Our confidence in the results was strengthened by the identification of several factors previously described to be involved in HPV16 infection. The results of the follow-up studies demonstrated that not only genes that give reduced infectivity after silencing can lead to novel insights but also those hits that show enhanced infection. A recent genome-wide RNAi-screen for HPV16 PsV infection in HeLa-S3 cells revealed the importance of retrograde membrane trafficking to the Golgi complex and a role for the retromer as an important determinant in HPV16 entry into cells[34]. Out of the 261 and 162 hits identified by Lipovsky et al. and by us, respectively, four were common( Figure S9). This low number of overlapping genes is not unusual when RNAi screens are compared, and the overlap typically increases significantly, when clusters of genes are compared[40],[41],[70]. This was also the case for the two HPV screens when significantly enriched functional annotation clusters were compared( Figure S9). For example, both studies identified the importance of a cluster associated with the Golgi complex. A more thorough comparison was not possible, because the results of the Lipovsky screen are only partially published. Besides the Golgi cluster, the bioinformatic analysis of our screen revealed a number of clusters with multiple hits associated with known pathways or complexes. The most prominent was a set of genes involved in the regulation of mitosis and the cell cycle. This cluster has not been observed in screens performed with other viruses by us or published by others([40],[45],[71], unpublished data), and may be unique to papillomaviruses. Most of the genes in the cluster were inhibitory, i. e. they showed enhanced infection after silencing suggesting that they function to limit infection. Moreover, it was striking that the hit list failed to include nuclear import factors or nuclear pore components that have been seen as prominent hits in screens against other viruses including those that replicate in the nucleus[40],[45]. This suggested that, unlike these other viruses, HPV may not be using the nuclear pores and nuclear transport receptors for accessing the nucleus during the host cell entry. Our results showed that the virus was efficiently internalized by endocytosis, routed to the perinuclear area, and partially uncoated in interphase cells. However, the viral DNA failed to enter the nucleus. Therefore no transcription of the marker protein GFP occurred. Mitosis was the key step that allowed infection to proceed. The expression of GFP from the vDNA correlated perfectly with mitosis; the GFP became detectable 4. 5±0. 4 h after mitosis was completed. Moreover, since late steps in mitosis such as chromosome separation and cytokinesis were not required, the critical process could be narrowed down to one of the early steps. That the critical step was the disruption of the NE was indicated by two observations. First, co-infection experiments in interphase cells showed that transient NE perforation by parvovirus H1 allowed HPV infection. This implied that the viruses were able to reach a stage in their entry program where they were ready to enter the nucleus productively, but could only do so if the NE integrity was compromised. Secondly, analysis of some of the hits from the siRNA screen indicated that mitosis provided a kinetic bottleneck for the nuclear import of viral genomes. The average time during mitosis when the NE is disrupted is about 45–60 min in HeLa cells. After depletion of INCENP, ANAPC, and AURKB, the time window was extended 3–5 fold resulting in a doubling or more in infection. This suggested that the longer the period without a functional membrane barrier, the better the chances for the virus to be productively included when daughter nuclei were formed. We also noted that while the addition of HPV16 itself did not promote division in the cell population, it did cause a significant increase in the fraction of cells that displayed prolonged mitosis, a process that appears to depend on the amount of L2 associating with host cell chromatin. This may be a mechanism for the virus to promote vDNA import into nuclei, a difficult step in the entry program. The results were consistent with observations of Pyeon and coworkers, who showed that cell cycle progression into early mitosis is required for HPV16 PsV infection[39]. Our data allowed us to define the key step as the breakdown of the NE barrier. Since raft-derived HPV16, as well as HPV6 and 18 PsV exhibted similar requirements, it is likely that cell cycle progression into early mitosis represents a general requirement for papillomavirus infection. We propose that after virus internalization, trafficking, conformational alterations, and penetration into the cytosol, L2/vDNA complexes wait in the perinuclear space until NEB occurs. When the nuclear components are rendered accessible, the subviral complexes associate with the condensed chromatin, and are included in newly forming daughter cell nuclei. Such a mechanism has not been observed for other viruses that replicate in the nucleus except for certain retroviruses that also depend on mitosis for provirus integration[57],[63]. Most other DNA viruses make use of the nuclear import machinery and the nuclear pore complexes. The inclusion of the HPV16 genome into the newly forming nuclei involves the participation of the minor capsid protein L2, since it is the only viral protein that accompanies the vDNA into the nucleus[17],[35],[38]. L2 contains two functional nuclear localization signals( NLSs)[67]. While these are necessary for nuclear import during virus assembly, they are hardly important for nuclear inclusion during virus entry. In fact, the N-terminal NLS is cleaved off by the cellular protein convertase furin in an earlier stage of entry[11],[17],[18]. The C-terminal NLS is involved in binding both to dynein light chains and to the vDNA[43],[72],[73]. Strikingly, L2 associated with condensed host cell chromatin on the metaphase plate, which may indicate an active recruitment of the subviral complex during mitosis to ensure enclosure of the vDNA into newly forming nuclei. It is tempting to speculate that L2 interacts specifically with cell proteins or nuclear structures that facilitate nuclear localization. Such a role has been observed for the p12 protein of murine leukemia virus and the E2 protein of HPV[74],[75]. It is interesting to note that HPV appear to encode two proteins that secure vDNA localization to daughter nuclei during cell division. Whereas E2 links the replicated, episomal vDNA to chromosomes by cellular adapter molecules during cytokinesis in suprabasal layers, L2 may fulfill a similar role during entry. The association of HPV16 vDNA with host cell chromatin and its enclosure into newly forming nuclei is efficient, since the majority of vDNA located on host chromosomes during metaphase and in the nucleus after cytokinesis. That HPV16 entry did not induce mitosis or nuclear envelope breakdown suggested that nuclear entry of the vDNA is dependent on physiologically occurring cell divisions. This provides an explanation for the narrow cell specificity of HPV; it exclusively infects basal stem cells or transiently amplifying cells in human skin or mucosa[1]. Only these are mitotically active. One possible advantage may be that, since the transcription and replication of HPV vDNA is tightly coupled to the differentiation program of keratinocytes[76], it may be beneficial to limit viral transcription and replication during G1 phase, and thus avoid immune detection. In addition, it may be favorable to restrict productive entry to basal cells in order to provide a reservoir of persistently infected cells. The advantage may outweigh the risks that the prolonged viral entry process would trigger innate immune responses, which might lead to viral clearance during entry.
Certain human papillomaviruses ( HPV ) are the etiological cause of cervical cancers and other epithelial tumors . Recent advances in the development of anti-HPV vaccines and their increasing deployment provide hope for a significant decrease of these cancers in the future . However , many details of the transmission of HPV between infected and uninfected individuals are still not well understood . In particular , the multistep process of entry into host cells is only partially characterized . Here , we used a systematic RNAi silencing approach to identify host cell proteins required during HPV16 infection , the most prevalent cancer-causing HPV . Our results indicated that HPV entry requires cells to divide . Cell division is important , since it removes the barrier between the nucleoplasm and the cytosol , so that the virus can access the nucleus for transcription and replication . Our study provides new mechanistic insights into the entry process of an important pathogen , and explains why it can only infect the stem cells or transiently amplifying cells of human skin or mucosa . This work also highlights a novel nuclear import strategy for DNA viruses , which typically use the nuclear import machinery to access the nucleoplasm for infection , Thus , HPV entry displays another unique aspect uncommon to most other viruses .