Rotavirus NSP1 inhibits interferon induced non-canonical NFκB activation by interacting with TNF receptor associated factor 2

AbstractTNF receptor associated factor 2 (TRAF2) plays a very important role in cellular innate immune as well as inflammatory responses. Previous studies have reported TRAF2 mediated regulation of TNF and Interferon (IFN) induced canonical and non-canonical activation of NFκB. In this study, we show that rotavirus NSP1 targets TRAF2 to regulate IFN induced non-canonical NFκB activation. Here we found that rotavirus Non-Structural Protein-1 (NSP1) interacts with TRAF2 and degrades it in a proteasome dependent manner. C-terminal part of NSP1 was sufficient for interacting with TRAF2 but it alone could not degrade TRAF2. This inhibition of interferon mediated non-canonical NFκB activation by NSP1 may modulate inflammatory cytokine production after rotavirus infection to help the virus propagation

The molecular chaperone heat shock protein-90 positively regulates rotavirus infection

AbstractRotaviruses are the major cause of severe dehydrating gastroenteritis in children worldwide. In this study, we report a positive role of cellular chaperone Hsp90 during rotavirus infection. A highly specific Hsp90 inhibitor, 17-allylamono-demethoxygeldanamycin (17-AAG) was used to delineate the functional role of Hsp90. In MA104 cells treated with 17-AAG after viral adsorption, replication of simian (SA11) or human (KU) strains was attenuated as assessed by quantitating both plaque forming units and expression of viral genes. Phosphorylation of Akt and NFκB observed 2–4 hpi with SA11, was strongly inhibited in the presence of 17-AAG. Direct Hsp90–Akt interaction in virus infected cells was also reduced in the presence of 17-AAG. Anti-rotaviral effects of 17-AAG were due to inhibition of activation of Akt that was confirmed since, PI3K/Akt inhibitors attenuated rotavirus growth significantly. Thus, Hsp90 regulates rotavirus by modulating cellular signaling proteins. The results highlight the importance of cellular proteins during rotavirus infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies

Host Cell–Virus Interaction 2.0: Viral Stratagems of Immune Evasion, Host Cellular Responses and Antiviral Counterattacks

As rightly stated by the author Mira Grant in her novel Countdown, “There is nothing so patient, in this world or any other, as a virus searching for a host” [...

A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane

<div><p>Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.</p></div

miRNAs in Herpesvirus Infection: Powerful Regulators in Small Packages

microRNAs are a class of small, single-stranded, noncoding RNAs that regulate gene expression. They can be significantly dysregulated upon exposure to any infection, serving as important biomarkers and therapeutic targets. Numerous human DNA viruses, along with several herpesviruses, have been found to encode and express functional viral microRNAs known as vmiRNAs, which can play a vital role in host–pathogen interactions by controlling the viral life cycle and altering host biological pathways. Viruses have also adopted a variety of strategies to prevent being targeted by cellular miRNAs. Cellular miRNAs can act as anti- or proviral components, and their dysregulation occurs during a wide range of infections, including herpesvirus infection. This demonstrates the significance of miRNAs in host herpesvirus infection. The current state of knowledge regarding microRNAs and their role in the different stages of herpes virus infection are discussed in this review. It also delineates the therapeutic and biomarker potential of these microRNAs in future research directions

Primers used in this study.

<p>Primers used in this study.</p

Hsp105 is essential for polyomavirus infection.

<p><b>A</b>. CV-1 cells were transfected with a ctrl siRNA, or <i>Hsp105</i> siRNA #1 or #2 for 24 h, and the resulting WCE were immunoblotted with the indicated antibodies (top panel) or RT-PCR analysis was performed to observe the <i>XBP1</i> splicing (bottom panel). Cells treated with DTT were used as a positive control. <b>B</b>. Cells in (A) were infected with SV40 (MOI ~0.5) for 24 h, fixed, and immunostained against SV40 large T antigen (TAg). Infection was scored using immunofluorescence microscopy (counting >1000 cells for each condition). Data are normalized to the ctrl siRNA. Values represent the mean ± SD (n≥3). <b>C</b>. As in (B), except cells were infected with BKV for 40 h before immunostaining for BKV TAg. <b>D</b>. Multiple sequence alignment of Hsp70 and Hsp110 family proteins from yeast and humans. Only the relevant sequences are shown. The highlighted regions indicate the amino acid(s) that were altered to generate the Hsp105 mutants (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005086#sec009" target="_blank">Methods</a>). <b>E</b>. The indicated F-tagged proteins were purified from 293T cells, and their purity analyzed by SDS-PAGE followed by staining with Brilliant Blue R250. Hsc70 was obtained from commercial source (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005086#sec009" target="_blank">Methods</a>). The asterisk indicates an antibody heavy chain band. <b>F</b>. Purified proteins in (E) were incubated with ATP conjugated-agarose beads. Unbound and bound proteins were analyzed by immunoblotting using a FLAG antibody. <b>G</b>. CV-1 cells expressing the indicated F-tagged proteins were immunoprecipitated, and the eluted samples were analyzed by immunoblotting. <b>H</b>. Thin layer chromatography was used to determine the level of radiolabeled ADP that remain bound to Hsc70 after the indicated purified protein was incubated with radiolabeled ADP-Hsc70. The black line indicates that an intervening lane has been spliced out of the same film. <b>I</b>. CV-1 cells were reverse transfected with ctrl or <i>Hsp105</i> siRNA #1 for 24 h prior to transfection with the indicated tagged constructs for 24 h. Cells were then infected with SV40 (MOI ~0.5) for 24 h, fixed, and stained with anti-FLAG/S and anti-TAg antibodies. The percentage of TAg positive cells were counted only in cells expressing the indicated tagged protein by immunofluorescence microscopy (right graph). Values represent mean ± SD (n≥3). The protein expression levels of endogenous Hsp105, as well as transfected Hsp105 WT-F and GFP-F, in cell extracts derived from control and Hsp105-depleted cells (transfected with either <i>GFP</i>-F or <i>Hsp105</i> WT-F) are shown (left panels). The three lanes in the immunoblot correspond to the first three bars in the right graph.</p

The cytosolic Hsp105 interacts with the ER membrane J-protein B14

<p><b>A</b>. Expression of B14-3xF and endogenous B14 in Flp-In 293 T-Rex cell lysates were analyzed by immunoblotting against B14. A corresponding molecular weight marker in kDa is shown on the left. <b>B</b>. B14-3xF was immunopurified from Flp-In 293 T-Rex cells infected with SV40 MOI ~50 (‘+’) or uninfected (‘-’). Bound proteins were eluted with 3x FLAG peptide, and the samples separated by SDS-PAGE followed by silver staining. Bands (indicated on the right) were excised and subjected to mass spectrometry analysis. Protein identities of the bands are listed on the right side of the gel. <b>C</b>. Samples in (B) were immunoblotted with the indicated antibodies. Uninfected HEK 293T cells not expressing B14-3xF were used as a control. <b>D</b>. CV-1 cells were cross-linked with DSP, lysed, the endogenous B14 immunoprecipitated, and the precipitated samples subjected to immunoblotting using the indicated antibodies. Where indicated, cells were infected with SV40. <b>E</b>. Cells expressing F-B14 or F-B14 H136Q were cross-linked, lysed, and the FLAG-tagged proteins immunoprecipitated followed by immunoblotting using the indicated antibodies. <b>F</b>. CV-1 cells treated with a control (ctrl) or <i>Hsc70</i> siRNA were transfected with <i>Hsp105</i> WT-F, and the FLAG-tagged protein immunoprecipitated followed by immunoblotting using the indicated antibodies. <b>G</b>. As in F, except <i>SGTA</i> siRNA was used. <b>H</b>. The S-tagged protein in CV-1 cells were affinity purified and immunoblotted using the indicated antibodies.</p