Mechanism of Membrane Penetration by Nonenveloped Polyomavirus and Papillomavirus

Membrane penetration represents a critical step during virus infection. As nonenveloped viruses lack a surrounding lipid bilayer, they are unable to penetrate host membranes by a membrane fusion mechanism. Consequently, nonenveloped viruses must devise alternative strategies to enter the host cell. In the case of polyomavirus SV40 and human papillomavirus (HPV), these two nonenveloped DNA tumor viruses must hijack selective host factors in order to promote their membrane penetration. Upon endocytosis, SV40 traffics through the endosomal pathway to reach the endoplasmic reticulum (ER). Here the virion is inserted into the ER membrane and is extracted into the cytosol by the cytosolic extraction machinery composed of the Hsc70, SGTA, and Hsp105 chaperones. From the cytosol, the virus is transported into the nucleus to cause infection. My thesis work unambiguously identified Hsc70 as a critical component of the cytosolic extraction machinery that ejects SV40 from the ER into the cytosol, clarified the structure-function relationship of SGTA during this process, and unveiled SGTA’s ability to negatively regulate Hsc70’s action during this the ER-to-cytosol membrane transport step. Moreover, my studies revealed that the Bag2 nucleotide exchange factor (NEF) is a new component of the cytosolic extraction machinery. In this context, Bag2 stimulates SV40 release from Hsc70, promoting successful arrival of SV40 to the cytosol, leading to infection. Collectively, my findings identify a novel component of a cytosolic extraction machinery essential during membrane penetration of a nonenveloped virus, and provide further mechanistic insights into this process. Similar to SV40, HPV membrane penetration requires host factors that are poorly characterized. After initial entry, HPV reaches the endosome, where the viral L2 minor capsid protein is inserted into the endosomal membrane. Membrane insertion of L2 is a decisive event because this step recruits the cytosolic sorting retromer complex to endosome-localized HPV, which in turn directs the virus to the Golgi apparatus. From this compartment, the virus enters the nucleus during mitosis where viral DNA is replicated. Through classic biochemical analyses, we recently reported that the transmembrane protease gamma secretase acts as a novel chaperone that binds to and inserts L2 into the endosomal membrane, an essential HPV infection step. In this thesis, we now identify the gamma secretase-binding partner delta-catenin/p120 as a new host factor that interacts with L2 and promotes HPV infection. Our analysis further suggests a model in which p120 engages HPV early in infection, delivering the virus to gamma secretase so that HPV can properly insert into the endosome membrane. In sum, my results provide fundamental insights into the infectious entry pathway of the nonenveloped SV40 and HPV by illuminating their membrane penetration mechanism.PHDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149876/1/adupzyk_1.pd

How Polyomaviruses Exploit the ERAD Machinery to Cause Infection

To infect cells, polyomavirus (PyV) traffics from the cell surface to the endoplasmic reticulum (ER) where it hijacks elements of the ER-associated degradation (ERAD) machinery to penetrate the ER membrane and reach the cytosol. From the cytosol, the virus transports to the nucleus, enabling transcription and replication of the viral genome that leads to lytic infection or cellular transformation. How PyV exploits the ERAD machinery to cross the ER membrane and access the cytosol, a decisive infection step, remains enigmatic. However, recent studies have slowly unraveled many aspects of this process. These emerging insights should advance our efforts to develop more effective therapies against PyV-induced human diseases

How Polyomaviruses Exploit the ERAD Machinery to Cause Infection

To infect cells, polyomavirus (PyV) traffics from the cell surface to the endoplasmic reticulum (ER) where it hijacks elements of the ER-associated degradation (ERAD) machinery to penetrate the ER membrane and reach the cytosol. From the cytosol, the virus transports to the nucleus, enabling transcription and replication of the viral genome that leads to lytic infection or cellular transformation. How PyV exploits the ERAD machinery to cross the ER membrane and access the cytosol, a decisive infection step, remains enigmatic. However, recent studies have slowly unraveled many aspects of this process. These emerging insights should advance our efforts to develop more effective therapies against PyV-induced human diseases

p120 catenin recruits HPV to γ-secretase to promote virus infection.

During internalization and trafficking, human papillomavirus (HPV) moves from the cell surface to the endosome where the transmembrane protease γ-secretase promotes insertion of the viral L2 capsid protein into the endosome membrane. Protrusion of L2 through the endosome membrane into the cytosol allows the recruitment of cytosolic host factors that target the virus to the Golgi en route for productive infection. How endosome-localized HPV is delivered to γ-secretase, a decisive infection step, is unclear. Here we demonstrate that cytosolic p120 catenin, likely via an unidentified transmembrane protein, interacts with HPV at early time-points during viral internalization and trafficking. In the endosome, p120 is not required for low pH-dependent disassembly of the HPV L1 capsid protein from the incoming virion. Rather, p120 is required for HPV to interact with γ-secretase-an interaction that ensures the virus is transported along a productive route. Our findings clarify an enigmatic HPV infection step and provide critical insights into HPV infection that may lead to new therapeutic strategies against HPV-induced diseases

Erratum for Duncan et al., An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

perspective drawing, B-type unit, July 198

Piericidin A1 blocks Yersinia Ysc Type III secretion system needle assembly

The type III secretion system (T3SS) is a bacterial virulence factor expressed by dozens of Gram-negative pathogens but largely absent from commensals. The T3SS is an attractive target for antimicrobial agents that may disarm pathogenic bacteria while leaving commensal populations intact. We previously identified piericidin A1 as an inhibitor of the Ysc T3SS in Yersinia pseudotuberculosis. Piericidins were first discovered as inhibitors of complex I of the electron transport chain in mitochondria and some bacteria. However, we found that piericidin A1 did not alter Yersinia membrane potential or inhibit flagellar motility powered by the proton motive force, indicating that the piericidin mode of action against Yersinia type III secretion is independent of complex I. Instead, piericidin A1 reduced the number of T3SS needle complexes visible by fluorescence microscopy at the bacterial surface, preventing T3SS translocator and effector protein secretion. Furthermore, piericidin A1 decreased the abundance of higher-order YscF needle subunit complexes, suggesting that piericidin A1 blocks YscF needle assembly. While expression of T3SS components in Yersinia are positively regulated by active type III secretion, the block in secretion by piericidin A1 was not accompanied by a decrease in T3SS gene expression, indicating that piericidin A1 may target a T3SS regulatory circuit. However, piericidin A1 still inhibited effector protein secretion in the absence of the T3SS regulator YopK, YopD, or YopN. Surprisingly, while piericidin A1 also inhibited the Y. enterocolitica Ysc T3SS, it did not inhibit the SPI-1 family Ysa T3SS in Y. enterocolitica or the Ysc family T3SS in Pseudomonas aeruginosa. Together, these data indicate that piericidin A1 specifically inhibits Yersinia Ysc T3SS needle assembly. IMPORTANCE The bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood. Deciphering how some natural products, such as the microbial metabolite piericidin, inhibit type III secretion can provide important insight into how the T3SS functions or is regulated. Taking this approach, we investigated the ability of piericidin to block T3SS function in several human pathogens. Surprisingly, piericidin selectively inhibited the Ysc family T3SS in enteropathogenic Yersinia but did not affect the function of a different T3SS within the same species. Furthermore, piericidin specifically blocked the formation of T3SS needles on the bacterial surface without altering the localization of several other T3SS components or regulation of T3SS gene expression. These data show that piericidin targets a mechanism important for needle assembly that is unique to the Yersinia Ysc T3SS