Entry of tumor viruses into cells is the first step in viral-mediated tumorigenesis. Virus entry is a complex, multistep process in which the virus particle binds to the cell surface, internalizes, disassembles, and undergoes intracellular trafficking to the site of virus replication. In addition, viruses modulate cellular metabolism to support virus entry. These events require the action of hundreds of cellular proteins. We have conducted functional genomics RNA interference screens to identify novel cellular factors required for infection by two families of DNA tumor viruses, polyomaviruses and human papillomaviruses. These factors, some of which were not previously implicated in virus infection, represent potential new targets of anti-viral approaches.
The human papillomavirus are important pathogens responsible for approximately 5% of all human cancer worldwide, including essentially all cervical cancer and a large fraction of other anogenital cancers and head and neck cancers. We conducted a genome-wide siRNA screen to identify cell factors required for infection by HPV16, the most common high-risk HPV type, and identified hundreds of cellular genes that influence the efficiency of infection. Most notably, our results demonstrated that the retrograde transport pathway plays an important role in mediating HPV16 entry and led to the discovery that HPV traffics to the Golgi apparatus en route to the nucleus. Furthermore, we identified the retromer, an intracellular retrograde trafficking machine, as an essential cellular factor required for entry of HPV16 and other HPV types (Fig 5). Biochemical experiments suggest that the incoming virus particle is directly transported by the retromer from the endosome to the Golgi apparatus. We are conducting experiments to identify the mechanism of action of the retromer and other cellular HPV entry factors, with confidence that systematic analysis will unravel the cellular pathways responsible for virus entry.
The polyomaviruses are small DNA tumor viruses, which include the common human pathogens, JC virus and BK virus, as well as the most recently recognized human tumor virus, Merkle Cell polyomavirus. We developed a high-throughput assay to screen for agents that inhibit infection by SV40, a well-studied monkey polyomavirus, and used this assay to screen a chemical library to identify natural products that inhibit SV40 entry. We also screened shRNAs to identify several cellular proteins localized in the endoplasmic reticulum that are required for SV40 infection. In particular a number of ER luminal and membrane chaperones are required for efficient virus entry, including several members of the DNAJ family of molecular co-chaperones. Physiological studies demonstrated that these proteins are required for the exit of disassembling SV40 capsids from the ER, and further mechanistic experiments are underway to determine the precise role of these factors in infection. In addition, we are studying the cell surface receptors for SV40 and examining how the interaction of the virus with these factors perturbs cellular metabolism.
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- Nelson CD, Carney DW, Derdowski A, Lipovsky A, Gee GV, O'Hara B, Williard P, DiMaio D, Sello JK, Atwood WJ: A retrograde trafficking inhibitor of ricin and Shiga-like toxins inhibits infection of cells by human and monkey polyomaviruses. MBio. 2013 Nov 12; 2013 Nov 12. PMID: 24222489
- Lipovsky A, Popa A, Pimienta G, Wyler M, Bhan A, Kuruvilla L, Guie MA, Poffenberger AC, Nelson CD, Atwood WJ, DiMaio D: Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus. Proc Natl Acad Sci U S A. 2013 Apr 30; 2013 Apr 8. PMID: 23569269
- Magaldi TG, Buch MH, Murata H, Erickson KD, Neu U, Garcea RL, Peden K, Stehle T, DiMaio D: Mutations in the GM1 binding site of simian virus 40 VP1 alter receptor usage and cell tropism. J Virol. 2012 Jul; 2012 Apr 18. PMID: 22514351
- Magaldi TG, Almstead LL, Bellone S, Prevatt EG, Santin AD, DiMaio D: Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation. Virology. 2012 Jan 5; 2011 Nov 5. PMID: 22056390
- Goodwin EC, Lipovsky A, Inoue T, Magaldi TG, Edwards AP, Van Goor KE, Paton AW, Paton JC, Atwood WJ, Tsai B, DiMaio D: BiP and multiple DNAJ molecular chaperones in the endoplasmic reticulum are required for efficient simian virus 40 infection. MBio. 2011; 2011 Jun 14. PMID: 21673190
- Goodwin EC, Atwood WJ, DiMaio D: High-throughput cell-based screen for chemicals that inhibit infection by simian virus 40 and human polyomaviruses. J Virol. 2009 Jun; 2009 Mar 18. PMID: 19297472