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

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Allison Dupzyk ◽  
Jeffrey M. Williams ◽  
Parikshit Bagchi ◽  
Takamasa Inoue ◽  
Billy Tsai
Keyword(s):  
Endoplasmic Reticulum ◽  
Biological Membrane ◽  
Critical Role ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Membrane Penetration ◽  
Critical Function ◽  
Nonenveloped Virus ◽  
Er Membrane

ABSTRACT 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.

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

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Allison Dupzyk ◽  
Billy Tsai
Keyword(s):  
Biological Membrane ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Productive Infection ◽  
Membrane Penetration ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Nonenveloped Virus ◽  
Chaperone Complex ◽  
Er Membrane

ABSTRACT 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.

scholarly journals The Endoplasmic Reticulum Membrane J Protein C18 Executes a Distinct Role in Promoting Simian Virus 40 Membrane Penetration

2015 ◽  
Vol 89 (8) ◽  
pp. 4058-4068 ◽  
Author(s):  
Parikshit Bagchi ◽  
Christopher Paul Walczak ◽  
Billy Tsai
Keyword(s):  
Endoplasmic Reticulum ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Penetration ◽  
Virus Family ◽  
Successful Infection ◽  
J Domain ◽  
Sv40 Infection ◽  
Er Membrane

ABSTRACTThe nonenveloped simian virus 40 (SV40) hijacks the three endoplasmic reticulum (ER) membrane-bound J proteins B12, B14, and C18 to escape from the ER into the cytosol en route to successful infection. How C18 controls SV40 ER-to-cytosol membrane penetration is the least understood of these processes. We previously found that SV40 triggers B12 and B14 to reorganize into discrete puncta in the ER membrane called foci, structures postulated to represent the cytosol entry site (C. P. Walczak, M. S. Ravindran, T. Inoue, and B. Tsai, PLoS Pathog10:e1004007, 2014). We now find that SV40 also recruits C18 to the virus-induced B12/B14 foci. Importantly, the C18 foci harbor membrane penetration-competent SV40, further implicating this structure as the membrane penetration site. Consistent with this, a mutant SV40 that cannot penetrate the ER membrane and promote infection fails to induce C18 foci. C18 also regulates the recruitment of B12/B14 into the foci. In contrast to B14, C18's cytosolic Hsc70-binding J domain, but not the lumenal domain, is essential for its targeting to the foci; this J domain likewise is necessary to support SV40 infection. Knockdown-rescue experiments reveal that C18 executes a role that is not redundant with those of B12/B14 during SV40 infection. Collectively, our data illuminate C18's contribution to SV40 ER membrane penetration, strengthening the idea that SV40-triggered foci are critical for cytosol entry.IMPORTANCEPolyomaviruses (PyVs) cause devastating human diseases, particularly in immunocompromised patients. As this virus family continues to be a significant human pathogen, clarifying the molecular basis of their cellular entry pathway remains a high priority. To infect cells, PyV traffics from the cell surface to the ER, where it penetrates the ER membrane to reach the cytosol. In the cytosol, the virus moves to the nucleus to cause infection. ER-to-cytosol membrane penetration is a critical yet mysterious infection step. In this study, we clarify the role of an ER membrane protein called C18 in mobilizing the simian PyV SV40, a PyV archetype, from the ER into the cytosol. Our findings also support the hypothesis that SV40 induces the formation of punctate structures in the ER membrane, called foci, that serve as the portal for cytosol entry of the virus.

scholarly journals A Nucleotide Exchange Factor Promotes Endoplasmic Reticulum-to-Cytosol Membrane Penetration of the Nonenveloped Virus Simian Virus 40

2015 ◽  
Vol 89 (8) ◽  
pp. 4069-4079 ◽  
Author(s):  
Takamasa Inoue ◽  
Billy Tsai
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Membrane Penetration ◽  
Nucleotide Exchange ◽  
Virus Family ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Er Membrane

ABSTRACTThe nonenveloped simian polyomavirus (PyV) simian virus 40 (SV40) hijacks the endoplasmic reticulum (ER) quality control machinery to penetrate the ER membrane and reach the cytosol, a critical infection step. During entry, SV40 traffics to the ER, where host-induced conformational changes render the virus hydrophobic. The hydrophobic virus binds and integrates into the ER lipid bilayer to initiate membrane penetration. However, prior to membrane transport, the hydrophobic SV40 recruits the ER-resident Hsp70 BiP, which holds the virus in a transport-competent state until it is ready to cross the ER membrane. Here we probed how BiP disengages from SV40 to enable the virus to penetrate the ER membrane. We found that nucleotide exchange factor (NEF) Grp170 induces nucleotide exchange of BiP and releases SV40 from BiP. Importantly, this reaction promotes SV40 ER-to-cytosol transport and infection. The human BK PyV also relies on Grp170 for successful infection. Interestingly, SV40 mobilizes a pool of Grp170 into discrete puncta in the ER called foci. These foci, postulated to represent the ER membrane penetration site, harbor ER components, including BiP, known to facilitate viral ER-to-cytosol transport. Our results thus identify a nucleotide exchange activity essential for catalyzing the most proximal event before ER membrane penetration of PyVs.IMPORTANCEPyVs are known to cause debilitating human diseases. During entry, this virus family, including monkey SV40 and human BK PyV, hijacks ER protein quality control machinery to breach the ER membrane and access the cytosol, a decisive infection step. In this study, we pinpointed an ER-resident factor that executes a crucial role in promoting ER-to-cytosol membrane penetration of PyVs. Identifying a host factor that facilitates entry of the PyV family thus provides additional therapeutic targets to combat PyV-induced diseases.

scholarly journals Ubqln4 Facilitates Endoplasmic Reticulum-to-Cytosol Escape of a Nonenveloped Virus during Infection

2020 ◽  
Vol 94 (11) ◽  
Author(s):  
Xiaofang Liu ◽  
Billy Tsai
Keyword(s):  
Endoplasmic Reticulum ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Independent Manner ◽  
New Host ◽  
Host Membrane ◽  
J Proteins ◽  
Nonenveloped Virus ◽  
Infectious Entry ◽  
Er Membrane

ABSTRACT The nonenveloped polyomavirus simian virus 40 (SV40) must penetrate the host endoplasmic reticulum (ER) membrane to enter the cytosol in order to promote infection. How this is accomplished is not entirely clear. Here, we demonstrate that the cytosolic chaperone Ubiquilin4 (Ubqln4) binds directly to the ER membrane J proteins B12 and B14. Strategically localized at the ER-cytosol interface, Ubqln4 captures SV40 emerging from the ER, thereby facilitating escape of the virus from the ER into the cytosol, which leads to infection. Strikingly, Ubqln4 engages the J proteins in a J-domain-independent manner, in contrast to the previously reported Hsc70-Hsp105-SGTA-Bag2 cytosolic complex that also mediates SV40 ER-to-cytosol transport. Our results also reveal that the H domain and STI1 motif (1-2) of Ubqln4 support J protein binding, essential for SV40 infection. Together, these data further clarify the molecular basis by which a nonenveloped virus escapes a host membrane during infectious entry. IMPORTANCE How a nonenveloped virus escapes from a host membrane to promote infection remains enigmatic. In the case of the nonenveloped polyomavirus SV40, penetration of the ER membrane to reach the cytosol is a decisive virus infection step. In this study, we found a new host factor called Ubqln4 that facilitates escape of SV40 from the ER into the cytosol, thereby providing a path for the virus to enter the nucleus to cause infection.

scholarly journals ERdj5 Reductase Cooperates with Protein Disulfide Isomerase To Promote Simian Virus 40 Endoplasmic Reticulum Membrane Translocation

2015 ◽  
Vol 89 (17) ◽  
pp. 8897-8908 ◽  
Author(s):  
Takamasa Inoue ◽  
Annie Dosey ◽  
Jeffrey F. Herbstman ◽  
Madhu Sudhan Ravindran ◽  
Georgios Skiniotis ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Disulfide Isomerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Membrane Penetration ◽  
Structural Rearrangements ◽  
Membrane Translocation ◽  
Disulfide Isomerase ◽  
Protein Disulfide ◽  
Er Membrane

ABSTRACTThe 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 before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40's disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in the unfolding of SV40, to fully stimulate membrane penetration. Negative-stain electron microscopy of ER-localized SV40 suggests that ERdj5 and PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling of SV40 from BAP31 traps the virus in ER subdomains called foci, which likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation and establishes a functional connection between lumenal and membrane events driving this process.IMPORTANCEPyVs are established etiologic agents of many debilitating human diseases, especially in immunocompromised individuals. To infect cells at the cellular level, this virus family must penetrate the host ER membrane to reach the cytosol, a critical entry step. In this report, we identify two ER lumenal factors that prepare the virus for ER membrane translocation and connect these lumenal events with events on the ER membrane. Pinpointing cellular components necessary for supporting PyV infection should lead to rational therapeutic strategies for preventing and treating PyV-related diseases.

scholarly journals Dynein Engages and Disassembles Cytosol-Localized Simian Virus 40 To Promote Infection

2018 ◽  
Vol 92 (12) ◽  
pp. e00353-18 ◽  
Author(s):  
Madhu Sudhan Ravindran ◽  
Chelsey C. Spriggs ◽  
Kristen J. Verhey ◽  
Billy Tsai
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Viral Particle ◽  
Simian Virus 40 ◽  
Cytoplasmic Dynein ◽  
Simian Virus ◽  
Dynein Motor ◽  
Successful Infection ◽  
Nonenveloped Virus ◽  
Cellular Motor

ABSTRACTDuring entry, polyomavirus (PyV) is endocytosed and sorts to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol. From the cytosol, the virus moves to the nucleus to cause infection. How PyV is transported from the cytosol into the nucleus, a crucial infection step, is unclear. We found that upon reaching the cytosol, the archetypal PyV simian virus 40 (SV40) recruits the cytoplasmic dynein motor, which disassembles the viral particle. This reaction enables the resulting disassembled virus to enter the nucleus to promote infection. Our findings reveal how a cytosolic motor can be hijacked to impart conformational changes to a viral particle, a process essential for successful infection.IMPORTANCEHow a nonenveloped virus successfully traffics from the cell surface to the nucleus to cause infection remains enigmatic in many instances. In the case of the nonenveloped PyV, the viral particle is sorted from the plasma membrane to the ER and then the cytosol, from which it enters the nucleus to promote infection. The molecular mechanism by which PyV reaches the nucleus from the cytosol is not entirely clear. Here we demonstrate that the prototype PyV SV40 recruits dynein upon reaching the cytosol. Importantly, this cellular motor disassembles the viral particle during cytosol-to-nucleus transport to cause infection.

Role of specific simian virus 40 sequences in the nuclease-sensitive structure in viral chromatin

1985 ◽  
Vol 5 (1) ◽  
pp. 52-58
Author(s):  
R D Gerard ◽  
B A Montelone ◽  
C F Walter ◽  
J W Innis ◽  
W A Scott
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Origin Of Replication ◽  
Viral Origin ◽  
Deletion Mutations ◽  
Sensitive Region ◽  
Complete Independence ◽  
Sensitive Structure ◽  
Nuclease Sensitivity

A nuclease-sensitive region forms in chromatin containing a 273-base-pair (bp) segment of simian virus 40 DNA encompassing the viral origin of replication and early and late promoters. We have saturated this region with short deletion mutations and compared the nuclease sensitivity of each mutated segment to that of an unaltered segment elsewhere in the partially duplicated mutant. Although no single DNA segment is required for the formation of a nuclease-sensitive region, a deletion mutation (dl45) which disrupted both exact copies of the 21-bp repeats substantially reduced nuclease sensitivity. Deletion mutations limited to only one copy of the 21-bp repeats had little, if any, effect. A mutant (dl135) lacking all copies of the 21- and 72-bp repeats, while retaining the origin of replication and the TATA box, did not exhibit a nuclease-sensitive region. Mutants which showed reduced nuclease sensitivity had this effect throughout the nuclease-sensitive region, not just at the site of the deletion, indicating that although multiple determinants must be responsible for the nuclease-sensitive chromatin structure they do not function with complete independence. Mutant dl9, which lacks the late portion of the 72-bp segment, showed reduced accessibility to BglI, even though the BglI site is 146 bp away from the site of the deletion.

scholarly journals Elongation of the Cytoplasmic Tail Interferes with the Fusion Activity of Influenza Virus Hemagglutinin

1998 ◽  
Vol 72 (5) ◽  
pp. 3554-3559 ◽  
Author(s):  
Masanobu Ohuchi ◽  
Christian Fischer ◽  
Reiko Ohuchi ◽  
Astrid Herwig ◽  
Hans-Dieter Klenk
Keyword(s):  
Intracellular Transport ◽  
Critical Role ◽  
Simian Virus 40 ◽  
Cytoplasmic Tail ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Simian Virus ◽  
Cell Surface Expression ◽  
Erythrocyte Membranes ◽  
Fusion Pores

ABSTRACT The hemagglutinin (HA) of fowl plague virus was lengthened and shortened by site-specific mutagenesis at the cytoplasmic tail, and the effects of these modifications on HA functions were analyzed after expression from a simian virus 40 vector. Elongation of the tail by the addition of one to six histidine (His) residues did not interfere with intracellular transport, glycosylation, proteolytic cleavage, acylation, cell surface expression, and hemadsorption. However, the ability to induce syncytia at a low pH decreased dramatically depending on the number of His residues added. Partial fusion (hemifusion), assayed by fluorescence transfer from octadecylrhodamine-labeled erythrocyte membranes, was also reduced, but even with the mutant carrying six His residues, significant transfer was observed. However, when the formation of fusion pores was examined with hydrophilic fluorescent calcein, transfer from erythrocytes to HA-expressing cells was not observed with the mutant carrying six histidine residues. The addition of different amino acids to the cytoplasmic tail of HA caused an inhibitory effect similar to that caused by the addition of His. On the other hand, a mutant lacking the cytoplasmic tail was still able to fuse at a reduced level. These results demonstrate that elongation of the cytoplasmic tail interferes with the formation and enlargement of fusion pores. Thus, the length of the cytoplasmic tail plays a critical role in the fusion process.

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