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A recent manuscript published by our lab (Bienkowska-Haba et al) described the transcriptomic changes of primary keratinocytes after infection with HPV16 and was selected for the spotlight section of the Journal of Virology (https://www.ncbi.nlm.nih.gov/pubmed/31748387)

In 2019, the Sapp lab was awarded an NIH R56 grant by the NIAID to continue studies into late viral trafficking of incoming viral genome.

Dr. Lucile Guion successfully defended her dissertation titled “Defining the late stages of human papillomavirus entry: microtubule trafficking and role of promyeolocytic leukemia nuclear bodies”.

Research

Infection with certain human papillomavirus (HPV) types such as HPV16 and HPV18 is responsible for more than 5% of all human cancers worldwide. Despite the introduction of a prophylactic vaccine in 2006, HPV will continue to cause significant morbidity and mortality in the human population due to the long latency period between infection and disease, limited vaccine coverage, and type-restricted neutralizing immune responses. Therefore, further studies into the understanding of the biology of this virus group are warranted.

HPV are nonenveloped viruses harboring a circular double-stranded DNA genome of approximately 8,000 bp. They are highly tissue-specific and only replicate in keratinocytes of the skin and mucosa. Their life cycle begins by establishing infection in the basal cell of this stratified epithelia, gaining access through abrasions, wounds and microlesions. Once infection is established in the basal keratinocytes, the virus goes latent with a restricted viral replication and transcription program. When HPV harboring keratinocytes enter into the terminal differentiation program, the productive phase of the life cycle begins finally resulting in the generation of progeny virions in the uppermost layers of this stratified epithelium. This may result in the formation of skin, oral and genital warts. In rare cases of persistency, infection with high-risk HPV types can induce the development of tumors such as cervical, anogenital, and oropharyngeal squamous cell carcinoma.

While we do have a good understanding of the transformation processes at the mechanistic levels and the role viral oncoproteins play, we have an incomplete understanding of immediate early events of the viral life cycle. This is partially due to the complexity of the viral life cycle, the difficulty to generate infectious viruses and to infect primary keratinocytes in cell culture models. Our research interest is focused on these immediate early events. To this end, we have developed a number of surrogate reagents, assays, and more recently cell culture models to study binding, entry and trafficking of virions in immortalized and primary keratinocytes, respectively. This allowed us to uncover novel cellular pathways manipulated and exploited by the virus to achieve establishment of infection. By identifying cellular factors and pathways involved, we hope to identify novel prophylactic and therapeutic targets.

Late Intracellular trafficking of HPV virions – We have recently demonstrated that the minor capsid protein L2 traverses the endocytic membrane after acidification of endosomes and partial uncoating. This process requires the cellular chaperone cyclophilin B and a membrane-destabilizing domain at the L2 carboxyl terminus for uncoating and membrane penetration (Fig 1). The cytosolic portion of L2 then mediates interaction with transport factors such as retromer complexes for trafficking to the trans golgi network (TGN). Once arriving at the TGN, HPV requires nuclear envelope break down and mitosis for nuclear translocation. We recently demonstrated that transport vesicles harboring HPV dissociate from the TGN and are transported to the microtubule organizing center and further on to mitotic chromosomes. The transport is again mediated by the cytosolic domains of the L2 protein. We are currently investigating the role of microtubules and motor proteins in the late trafficking of HPV using live cell imaging, inhibitor studies, and knock out approaches.

One of the most striking observations we recently made was that HPV is delivered to the nucleus during mitosis residing in membrane-bound transport vesicles (Fig 2). Egress of viral genome from the transport vesicles occurs in early G1 phase. These are paradigm-shifting observations as it has been assumed that nuclear membrane-bound vesicles do not exist. Since we do not observe accumulation of vesicles in the nucleus, our data indirectly point to the existence of a quality control pathway that discards of membrane-bound vesicles ending up in the nucleus. We are currently exploring putative roles of nuclear lipases in this process using release of HPV genome from transport vesicles as biological read-out.

PML nuclear bodies and establishment of HPV infection – It is well established that HPV associated with PML nuclear bodies after infectious delivery and require PML protein for efficient transcription. We recently demonstrated that PML protein play a protective role and its absence results in the loss of incoming viral genome despite efficient nuclear delivery. We now found that PML protein is recruited to incoming viral genome and completely engulfs it before egress from the transport vesicle. Interestingly recruitment of another component of PML nuclear bodies, Sp100, which functions as HPV restriction factor, is specifically delayed to HPV-harboring PML nuclear bodies. We are currently exploring a role of these subnuclear structures in the early transcriptional regulation of incoming HPV genome and are trying to unravel the innate immune pathways present in the nucleus that are capable of sensing and degrading incoming HPV genome.

Immediate early events of the HPV life cycle – Until recently, primary keratinocytes, the natural target cells of HPV, could not be efficiently infected in vitro. Therefore, most studies were limited to the use of established immortalized cell lines harboring episomal HPV genomes. Based on the extensive knowledge gained in recent years regarding binding and internalization of HPV, we have now developed an infection model that allows efficient infection of primary keratinocytes. For this, we are exploiting the preferential binding of HPV virions to extracellular depositions by pre-binding virions to extracellular matrix derived from keratinocytes prior to addition of keratinocytes. HPV virions are generated using the 293TT packaging cell line alleviating the need for any viral factors other than the capsid proteins for virion production. This allows an extensive genetic manipulation of the viral genome. We are currently exploiting this model to further our understanding of temporal viral gene expression, genome replication and segregation, and the role of viral factors involved. The model will also be helpful to compare low and high risk viruses and to analyse the host response to viral infection without the for immortalization and selection.
 

Selected Publications

1. Richards, K.F., Bienkowska-Haba, M., Dasgupta, J., Chen, X. S., and Sapp, M. (2013): Multiple heparan sulfate binding site engagements are required for the infectious entry of human papillomavirus type 16. J Virol 87:11426-37. (featured in spotlight section)
2. Richards, K.F., Mukherjee, S., Bienkowska-Haba, M., Pang, J., and Sapp, M. (2014): Human papillomavirus species-specific interaction with the basement membrane-resident non-heparan sulfate receptor. Viruses 6(12):4856-79.
3. DiGiuseppe, S., Keiffer, T., Bienkowska-Haba, M., Luszczek, W., Guion, L.G.M., and Sapp, M. (2015): Topography of the human papillomavirus minor capsid protein L2 during vesicular trafficking of infectious entry. J Virol 89: 10442-52. (featured in spotlight section)
4. DiGiuseppe, S., Luszczek, W., Keiffer, T., Bienkowska-Haba, M., Guion, L.G.M., and Sapp, M. (2016): Incoming HPV16 genome resides in a vesicular compartment throughout mitosis. PNAS 113 (22) 6289-6294. PMID: 27190090
5. DiGiuseppe S, Bienkowska-Haba M, Sapp M. (2016) Human papillomavirus entry: hiding in a bubble. J Virol 90(18):8032-5. PMID: 27412595
6. DiGiuseppe, S., Bienkowska-Haba, M., Guion, L.G., and Sapp, M. (2016): Cruising the cellular highways: how human papillomavirus travels from the surface to the nucleus. Virus Res., doi: 10.1016/j.virusres.2016.10.015. Epub 2016 Oct 2016. PMID: 279884059
7. Bienkowska-Haba, M., Luszczek, W., Keiffer, T.R., Guion, L.G.M., DiGiuseppe, S., Scott, R.S., and Sapp, M. (2017): Incoming human papillomavirus 16 genome is lost in PML protein-deficient HaCaT keratinocytes. Cellular Microbiology doi: 10/1111/cmi.12708. Epub 2017 Jan 3017 PMID: 27860076
8. DiGiuseppe, S., Guion, L.G.M., Bienkowska-Haba, M., Keiffer, T.R., and Sapp, M. (2017): Human papillomavirus major capsid protein L1 remains associated with the incoming viral genome throughout the entry process. J. Virol. doi: 10.1128/JVI.00537-17 Epub ahead of print PMID:28566382 (featured in spotlight section)
9. Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, DiGiuseppe S, Polk P, et al. (2018) A new cell culture model to genetically dissect the complete human papillomavirus life cycle. PLoS Pathog. 2018;14(3):e1006846. Epub 2018. (highlighted by press release)
10. Guion LG, Bienkowska-Haba M, DiGiuseppe S, Florin L, Sapp M (2019): PML nuclear body-residing proteins sequentially associate with HPV genome after infectious nuclear delivery. PLoS Pathog.: e1007590. doi: 10.1371
11. Myers JE, Guidry JT, Scott ML, Zwolinska K, Raikhy G, Prasai K, Bienkowska-Haba M, Bodily JM, Sapp MJ, Scott RS (2019): Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay. Virology 537:149-156
12. Bienkowska-Haba M, Luszczek W, Zwolinska K, Scott RS, Sapp M. (2019): Genome-wide transcriptome analysis of human papillomavirus type 16 infected primary keratinocytes reveals subtle perturbations mostly due to E7 protein expression. J. Virol.: in press

We are constantly looking for talented graduate students and post-doctoral fellows to join our program. We are offering a highly collaborative work environment, competitive compensation and highly innovative research projects.

Students interested in working in our Department should visit our Graduate Program website.

Those interested in postdoctoral work should contact Dr. Sapp.