Ubiquitin-Mediated Regulation of Autophagy During Viral Infection.

Purpose of Review: Virus infections skew the host autophagic response to meet their replication and transmission demands by tapping into the critical host regulatory mechanisms that control the autophagic flux. This review is a compendium of previous reports highlighting the mechanisms that viruses adapt to hijack the host ubiquitination machinery to repurpose autophagy for their sustenance. Recent Findings: Emerging evidence suggests a critical role of host ubiquitin machinery in the manifestation of the antiviral or proviral functions of autophagy. Lately, more emphasis has been laid to identify specific host E3 ubiquitin ligases, their targets (viral or host), and characterizing corresponding ubiquitin linkages by biochemical or genome-wide genetic screening approaches. Summary: Here, we highlight how viruses ingeniously engage and subvert the host ubiquitin-autophagy system to promote virus replication and antagonize intracellular innate immune responses.

Chandipura virus induces cell death in cancer cell lines of human origin and promotes tumor regression in vivo.

Chandipura virus (CHPV) is an emerging human pathogen of great clinical significance. In this study, we have investigated the susceptibility pattern of both normal and cancer cell lines of human origin to wild-type (wt) CHPV in order to explore the possibility of developing CHPV as an oncolytic vector (OV). Marked cytopathic effect along with enhanced virus output was observed in cancer cell lines (HeLa, A549, U-138, PC-3, and HepG2) in comparison to normal human adult dermal fibroblast (HADF) cells. At an MOI of 0.1, cancer cell lines were differentially susceptible to CHPV, with cells like HeLa and U-138 having pronounced cell death, while the PC-3 were comparatively resistant. All cell lines used in the study except U-138 restricted CHPV infection to varying degrees with IFN-ß pre-treatment and supplementation of interferon (IFN) could neither activate the IFN signaling pathway in U-138 cells. Finally, U-138 tumor xenografts established in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice showed significant delay in tumor growth in the CHPV-challenged animals. Thus, targeted cytopathic effect in cancer cells at a very low dose with restricted replication in normal cells offers a rationale to exploit CHPV as an oncolytic vector in the future.

Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization.

The human complement system is an important part of the innate immune system. Its effector pathways largely mediate virus neutralization. Vesicular stomatitis virus (VSV) activates the classical pathway of the complement, leading to virus neutralization by lysis. Two host-derived membrane-associated regulators of complement activation (RCA), CD55 and CD46, which are incorporated into the VSV envelope during egress, confer protection by delaying/resisting complement-mediated neutralization. We showed previously that CD55 is more effective than CD46 in the inhibition of neutralization. In this study, we identified that, at the protein level, VSV infection resulted in the down-regulation of CD46 but not CD55. The mRNA of both the RCAs was significantly down-regulated by VSV, but it was delayed in the case of CD55. The immunoblot analysis of the levels of RCAs in the progeny virion harvested at three specific time intervals, points to an equal ratio of its distribution relative to viral proteins. Besides reconfirming the dominant role of CD55 over CD46 in shielding VSV from complement, our results also highlight the importance of the subtle modulation in the expression pattern of RCAs in a system naturally expressing them.

A Factor I-Like Activity Associated with Chikungunya Virus Contributes to Its Resistance to the Human Complement System.

Chikungunya virus (CHIKV) is an emerging pathogen capable of causing explosive outbreaks. Prior studies showed that exacerbation in arthritogenic alphavirus-induced pathogenesis is attributed to its interaction with multiple immune components, including the complement system. Viremia concomitant to CHIKV infection makes exposure of the virus to complement unavoidable, yet very little is known about CHIKV-complement interactions. Here, we show that CHIKV activated serum complement to modest levels in a concentration- and time-dependent manner, but the virus effectively resisted complement-mediated neutralization. Heat-inactivated serum from seropositive donors could actively neutralize CHIKV due to the presence of potent anti-CHIKV antibodies. Deposition of key complement components C3 and C4 did not alter the resistance of CHIKV to complement. Further, we identified a factor I-like activity in CHIKV that limited complement by inactivating C3b into inactive C3b (iC3b), the complement component known to significantly contribute to disease severity in vivo, but this activity had no effect on C4b. Inactivation of C3b by CHIKV was largely dependent on the concentration of the soluble host cofactor factor H and the virus concentration. A factor I function-blocking antibody had only a negligible effect on the factor I-like activity associated with CHIKV, suggesting that this activity is independent of host factor I and could be of viral origin. Thus, our findings suggest a complement modulatory action of CHIKV which not only helps the virus to evade human complement but may also have implications in alphavirus-induced arthritogenic symptoms.IMPORTANCE Chikungunya virus is a vector-borne pathogen of global significance. The morbidity associated with chikungunya virus (CHIKV) infection, neurovirulence and adaptability to Aedes albopictus, necessitates a deeper understanding of the interaction of CHIKV with the host immune system. Here, we demonstrate that CHIKV is resistant to neutralization by one of the potent barriers of the innate immune arm, the complement system. Chikungunya virus showed marked resistance to complement despite activation and deposition of complement proteins. Interestingly the C3 component associated with the virion was found to be inactive C3b (iC3b), a key factor implicated in the pathogenesis and disease severity in the mouse model of Ross River virus infection. CHIKV also had an associated unique factor I-like activity that mediated the inactivation of C3b into iC3b. We have unraveled a smart strategy adopted by CHIKV to limit complement which has serious implications in viral dissemination, pathogenesis, and disease.

Complement-Mediated Neutralization of a Potent Neurotropic Human Pathogen, Chandipura Virus, Is Dependent on C1q.

Among the innate immune sentinels, the complement system is a formidable first line of defense against pathogens, including viruses. Chandipura virus (CHPV), a neurotropic vesiculovirus of the family Rhabdoviridae, is a deadly human pathogen known to cause fatal encephalitis, especially among children. The nature of interaction and the effect of human complement on CHPV are unknown. Here, we report that CHPV is a potent activator of complement and, thus, is highly sensitive to complement proteins in normal human serum (NHS). Utilizing a panel of specific complement component depleted/reconstituted human serum, we have demonstrated that CHPV neutralization is C3, C4, and C1q dependent and independent of factor B, suggesting the importance of the classical pathway in limiting CHPV. Employing a range of biochemical approaches, we showed (i) a direct association of C1q to CHPV, (ii) deposition of complement proteins C3b, C4b, and C1q on CHPV, and (iii) virus aggregation. Depletion of C8, an important component of the pore-forming complex of complement, had no effect on CHPV, further supporting the finding that aggregation and not virolysis is the mechanism of virus neutralization. With no approved vaccines or treatment modalities in place against CHPV, insights into such interactions can be exploited to develop potent vaccines or therapeutics targeting CHPV.IMPORTANCE Chandipura virus is a clinically important human pathogen of the Indian subcontinent. The rapidity of death associated with CHPV infection in addition to the absence of an effective vaccine or therapeutics results in poor clinical prognosis. The biology of the virus and its interaction with the host immune system, including the complement system, are understudied. Our investigation reveals the susceptibility of CHPV to fluid phase complement and also dissects the pathway involved and the mechanism of virus neutralization. Direct binding of C1q, an important upstream component of the classical pathway of complement to CHPV, and the strong dependency on C1q for virus neutralization highlight the significance of identifying such interactions to better understand CHPV pathogenesis and devise strategies to target this deadly pathogen.