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.

γ-Secretase promotes membrane insertion of the human papillomavirus L2 capsid protein during virus infection.

Despite their importance as human pathogens, entry of human papillomaviruses (HPVs) into cells is poorly understood. The transmembrane protease γ-secretase executes a crucial function during the early stages of HPV infection, but the role of γ-secretase in infection and the identity of its critical substrate are unknown. Here we demonstrate that γ-secretase harbors a previously uncharacterized chaperone function, promoting low pH-dependent insertion of the HPV L2 capsid protein into endosomal membranes. Upon membrane insertion, L2 recruits the cytosolic retromer, which enables the L2 viral genome complex to enter the retrograde transport pathway and traffic to the Golgi en route for infection. Although a small fraction of membrane-inserted L2 is also cleaved by γ-secretase, this proteolytic event appears dispensable for HPV infection. Our findings demonstrate that γ-secretase is endowed with an activity that can promote membrane insertion of L2, thereby targeting the virus to the productive infectious pathway.

Bag2 Is a Component of a Cytosolic Extraction Machinery That Promotes Membrane Penetration of a Nonenveloped Virus.

During entry, the nonenveloped polyomavirus (PyV) simian virus 40 (SV40) traffics from the cell surface to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol; the virus is then transported into the nucleus to cause infection. Although a coherent understanding of SV40's host entry is emerging, how the virus is ejected from the ER into the cytosol remains mysterious. Our previous analyses revealed that the cytosolic Hsc70-SGTA-Hsp105 complex binds to SV40 and extracts it from the ER into the cytosol. We now report that the nucleotide exchange factor (NEF) Bag2 stimulates SV40 release from Hsc70, thereby enabling successful virus arrival at the cytosol, which leads to infection. Hsp105, another NEF of Hsc70, displays a function overlapping that of Bag2, underscoring the importance of this release reaction. Our findings identify a new component of an extraction machinery essential during membrane penetration of a nonenveloped virus and provide further mechanistic insights into this process.IMPORTANCE How a nonenveloped virus penetrates a biological membrane to cause infection is a mystery. For the nonenveloped polyomavirus SV40, transport across the ER membrane to reach the cytosol is an essential virus infection step. Here, we identify a novel component of a cytosolic Hsc70-dependent chaperone complex called Bag2 that extracts SV40 from the ER into the cytosol. Bag2 does this by triggering SV40 release from Hsc70, thus ensuring that the virus reaches the cytosol en route for productive infection.

SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum.

Membrane penetration by nonenveloped viruses remains enigmatic. In the case of the nonenveloped polyomavirus simian virus 40 (SV40), the virus penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and then traffics to the nucleus to cause infection. We previously demonstrated that the cytosolic Hsc70-SGTA-Hsp105 complex is tethered to the ER membrane, where Hsp105 and SGTA facilitate the extraction of SV40 from the ER and transport of the virus into the cytosol. We now find that Hsc70 also ejects SV40 from the ER into the cytosol in a step regulated by SGTA. Although SGTA's N-terminal domain, which mediates homodimerization and recruits cellular adaptors, is dispensable during ER-to-cytosol transport of SV40, this domain appears to exert an unexpected post-ER membrane translocation function during SV40 entry. Our study thus establishes a critical function of Hsc70 within the Hsc70-SGTA-Hsp105 complex in promoting SV40 ER-to-cytosol membrane penetration and unveils a role of SGTA in controlling this step.IMPORTANCE How a nonenveloped virus transports across a biological membrane to cause infection remains mysterious. One enigmatic step is whether host cytosolic components are co-opted to transport the viral particle into the cytosol. During ER-to-cytosol membrane transport of the nonenveloped polyomavirus SV40, a decisive infection step, a cytosolic complex composed of Hsc70-SGTA-Hsp105 was previously shown to associate with the ER membrane. SGTA and Hsp105 have been shown to extract SV40 from the ER and transport the virus into the cytosol. We demonstrate here a critical role of Hsc70 in SV40 ER-to-cytosol penetration and reveal how SGTA controls Hsc70 to impact this process.

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.

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.

An NF-κB-based high-throughput screen identifies piericidins as inhibitors of the Yersinia pseudotuberculosis type III secretion system.

The type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibited Yersinia pseudotuberculosis from triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As the Yersinia T3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of the Y. pseudotuberculosis T3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growth in vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics in Yersinia. In addition, when Yersinia was incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS.