Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

Viral detection by electron microscopy: past, present and future.

International audienceViruses are very small and most of them can be seen only by TEM (transmission electron microscopy). TEM has therefore made a major contribution to virology, including the discovery of many viruses, the diagnosis of various viral infections and fundamental investigations of virus-host cell interactions. However, TEM has gradually been replaced by more sensitive methods, such as the PCR. In research, new imaging techniques for fluorescence light microscopy have supplanted TEM, making it possible to study live cells and dynamic interactions between viruses and the cellular machinery. Nevertheless, TEM remains essential for certain aspects of virology. It is very useful for the initial identification of unknown viral agents in particular outbreaks, and is recommended by regulatory agencies for investigation of the viral safety of biological products and/or the cells used to produce them. In research, only TEM has a resolution sufficiently high for discrimination between aggregated viral proteins and structured viral particles. Recent examples of different viral assembly models illustrate the value of TEM for improving our understanding of virus-cell interactions

Persistent Expression of Hepatitis C Virus Non-Structural Proteins Leads to Increased Autophagy and Mitochondrial Injury in Human Hepatoma Cells

HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol

IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity

Innate Antiviral Immune Responses to Hepatitis B Virus

Hepatitis B virus (HBV) is a major cause of acute and chronic hepatitis in humans. As HBV itself is currently viewed as a non-cytopathic virus, the liver pathology associated with hepatitis B is mainly thought to be due to immune responses directed against HBV antigens. The outcome of HBV infection is the result of complex interactions between replicating HBV and the immune system. While the role of the adaptive immune response in the resolution of HBV infection is well understood, the contribution of innate immune mechanisms remains to be clearly defined. The innate immune system represents the first line of defense against viral infection, but its role has been difficult to analyze in humans due to late diagnosis of HBV infection. In this review, we discuss recent advances in the field of innate immunity to HBV infection

Host Cell Restriction Factors of Paramyxoviruses and Pneumoviruses

The paramyxo- and pneumovirus family includes a wide range of viruses that can cause respiratory and/or systemic infections in humans and animals. The significant disease burden of these viruses is further exacerbated by the limited therapeutics that are currently available. Host cellular proteins that can antagonize or limit virus replication are therefore a promising area of research to identify candidate molecules with the potential for host-targeted therapies. Host proteins known as host cell restriction factors are constitutively expressed and/or induced in response to virus infection and include proteins from interferon-stimulated genes (ISGs). Many ISG proteins have been identified but relatively few have been characterized in detail and most studies have focused on studying their antiviral activities against particular viruses, such as influenza A viruses and human immunodeficiency virus (HIV)-1. This review summarizes current literature regarding host cell restriction factors against paramyxo- and pneumoviruses, on which there is more limited data. Alongside discussion of known restriction factors, this review also considers viral countermeasures in overcoming host restriction, the strengths and limitations in different experimental approaches in studies reported to date, and the challenges in reconciling differences between in vitro and in vivo data. Furthermore, this review provides an outlook regarding the landscape of emerging technologies and tools available to study host cell restriction factors, as well as the suitability of these proteins as targets for broad-spectrum antiviral therapeutics