Influenza viruses are enveloped, negative stranded, segmented RNA viruses belonging to Orthomyxoviridae family. Each virion consists of three major subviral components, namely (i) a viral envelope decorated with three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and M2, (ii) an intermediate layer of matrix protein (M1), and (iii) an innermost helical viral ribonucleocapsid [vRNP] core formed by nucleoprotein (NP) and negative strand viral RNA (vRNA). Since complete virus particles are not found inside the cell, the processes of assembly, morphogenesis, budding and release of progeny virus particles at the plasma membrane of the infected cells are critically important for the production of infectious virions and pathogenesis of influenza viruses as well. Morphogenesis and budding require that all virus components must be brought to the budding site which is the apical plasma membrane in polarized epithelial cells whether in vitro cultured cells or in vivo infected animals. HA and NA forming the outer spikes on the viral envelope possess apical sorting signals and use exocytic pathways and lipid rafts for cell surface transport and apical sorting. NP also has apical determinant(s) and is probably transported to the apical budding site similarly via lipid rafts and/or through cortical actin microfilaments. M1 binds the NP and the exposed RNAs of vRNPs, as well as to the cytoplasmic tails (CT) and transmembrane (TM) domains of HA, NA and M2, and is likely brought to the budding site on the piggy-back of vRNP and transmembrane proteins.
Budding processes involve bud initiation, bud growth and bud release. Presence of lipid rafts and assembly of viral components at the budding site can cause asymmetry of lipid bilayers and outward membrane bending leading to bud initiation and bud growth. Bud release requires fusion of the apposing viral and cellular membranes and scission of the virus buds from the infected cellular membrane. The processes involved in bud initiation, bud growth and bud scission/release require involvement both viral and host components and can affect bud closing and virus release in both positive and negative ways. Among the viral components, M1, M2 and NA play important roles in bud release and M1, M2 and NA mutations all affect the morphology of buds and released viruses. Disassembly of host cortical actin microfilaments at the pinching-off site appears to facilitate bud fission and release. Bud scission is energy dependent and only a small fraction of virus buds present on the cell surface is released. Discontinuity of M1 layer underneath the lipid bilayer, absence of outer membrane spikes, absence of lipid rafts in the lipid bilayer, as well as possible presence of M2 and disassembly of cortical actin microfilaments at the pinching off site appear to facilitate bud fission and bud release. We provide our current understanding of these important processes leading to the production of infectious influenza virus particles.
Herpesviruses acquire their envelope by budding into the lumen of cytoplasmic membrane vesicles. This process is initiated by component(s) on viral particles, which recognize the budding site where the viral glycoproteins are present and recruit cellular cargo transport and sorting machinery to the site to complete the budding process. Proteins in the tegument layer, connecting capsid and envelope, are candidates for the recognition of budding sites on vesicle membrane and induction of budding and final envelopment. We examined several outer and matrix tegument proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) and found that ORF45 associates with lipid rafts (LRs) of cellular membrane. LRs are membrane micro-domains, which have been implicated as relay stations in intracellular signaling and transport including viral entry and virion assembly. The ability of ORF45 to target LR is dependent on the mono-ubiquitylation of ORF45 at Lys297 as the mutation at Lys297 (K297R) abolished LR-association of ORF45. The K297R mutation also impairs ORF45 and viral particle co-localization with trans-Golgi network and endosomes, but facilitates ORF45 and viral particles co-localizing with lysosomes. More importantly, the recombinant KSHV carrying ORF45 K297R mutant (BAC-K297R) was found severely defective in producing mature and infectious virion particles in comparison to wild type KSHV (BAC16). Taken together, our results reveal a new function of KSHV tegument protein ORF45 in targeting LR of host cell membrane, promoting viral particles co-localization with trans-Golgi and endosome vesicles and facilitating the maturation and release of virion particles, suggesting that ORF45 plays a role in bringing KSHV particles to the budding site on cytoplasmic vesicle membrane and triggering the viral budding process for final envelopment and virion maturation.
Most enveloped viruses acquire their envelope membrane by budding into cellular membrane. Although viruses utilize cellular cargo transport and sorting machinery for their budding into luminal vesicles or at plasma membrane, the budding process is initiated and controlled by viral component(s). For some viruses, a single viral protein possesses all the information necessary for virus budding to generate virus-like particles (VLP) in the absence of other viral components. Herpesviruses also gain their membrane envelope through budding into cytoplasmic membrane vesicles (trans-Golgi and endosome vesicles) and the underlying mechanism is much less understood in comparison to that of RNA viruses. We searched for herpesviral components that initiate or orchestrate viral budding in final envelopment and egress. We found that a tegument protein of Kaposi’s sarcoma-associated herpesvirus (KSHV), namely ORF45, associates with lipid rafts of cell membranes and this association is regulated by a mono-ubiquitylation of ORF45 at Lys297. The ubiquitylated ORF45 determines the association with trans-Golgi and endosome vesicles and facilitates the final virion maturation and production. These data suggest that ORF45 may function in recognition of budding site and recruiting cellular transport and sorting machinery for KSHV budding, envelopment and mature virion release.
Interferon-induced BST2/Tetherin prevents budding of vpu-deficient HIV-1 by tethering mature viral particles to the plasma membrane. BST2 also inhibits release of other enveloped viruses including Ebola virus and Kaposi's sarcoma associated herpesvirus (KSHV), indicating that BST2 is a broadly acting antiviral host protein. Unexpectedly however, recovery of human cytomegalovirus (HCMV) from supernatants of BST2-expressing human fibroblasts was increased rather than decreased. Furthermore, BST2 seemed to enhance viral entry into cells since more virion proteins were released into BST2-expressing cells and subsequent viral gene expression was elevated. A significant increase in viral entry was also observed upon induction of endogenous BST2 during differentiation of the pro-monocytic cell line THP-1. Moreover, treatment of primary human monocytes with siRNA to BST2 reduced HCMV infection, suggesting that BST2 facilitates entry of HCMV into cells expressing high levels of BST2 either constitutively or in response to exogenous stimuli. Since BST2 is present in HCMV particles we propose that HCMV entry is enhanced via a reverse-tethering mechanism with BST2 in the viral envelope interacting with BST2 in the target cell membrane. Our data suggest that HCMV not only counteracts the well-established function of BST2 as inhibitor of viral egress but also employs this anti-viral protein to gain entry into BST2-expressing hematopoietic cells, a process that might play a role in hematogenous dissemination of HCMV.
Human Cytomegalovirus (HCMV) persistently infects a large proportion of the human population without causing any symptoms. The establishment and maintenance of HCMV in infected individuals is thought to be facilitated by the ability of HCMV to modulate innate and adaptive immune responses by the host. BST2, aka Tetherin, was recently shown to be an innate immune response molecule that is induced by the antiviral cytokine interferon. BST2 has been shown to prevent the release of many different viruses, including the human immunodeficiency virus and Ebola virus, from infected cells by tethering the viral envelope to the host cell membrane. Unexpectedly however, we observed that BST2 had the opposite effect on infection by HCMV. Cells expressing BST2 became more susceptible to infection with HCMV. Thus, HCMV seems to use this antiviral protein to gain access to cells that naturally express high levels of BST2 such as macrophages.
The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVΔE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15−/− cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVΔE3L caused significant disease and mortality, an effect not observed in VVΔE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVΔE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor.
Modification of proteins by ubiquitin (UB) and ubiquitin-like proteins (UBL) represents a key regulatory process of innate and adaptive immune responses. Interferon-stimulated gene product 15 (ISG15) is a member of UBL molecules that can reversibly be conjugated to proteins mediating considerable antiviral response. In turn, many viruses, including poxviruses, have evolved strategies to block the antiviral and inflammatory effects of innate immune responses to keep cells alive until virus replication is completed. Here, a novel viral immune evasion mechanism that inhibits ISG15-dependent antiviral pathway is described. Vaccinia virus (VACV) pathogenesis in ISG15+/+ versus ISG15−/− mice is linked to the virus E3 protein, blocking the activity of ISG15 through its C-terminal domain. This effect was independent of PKR activation. ISG15 controls the inflammatory response regulating cytokine levels. Our findings support a new strategy for poxviruses to evade the host antiviral response through interaction of the virus E3 protein with ISG15.
Virus budding is a complex, multistep process in which viral proteins make specific alterations in membrane curvature. Many different viral proteins can deform the membrane and form a budding virion, but very few can mediate membrane scission to complete the budding process. As a result, enveloped viruses have developed numerous ways of facilitating membrane scission, including hijacking host cellular scission machinery and expressing their own scission proteins. These proteins mediate scission in very different ways, though the biophysical mechanics underlying their actions may be similar. In this review, we explore the mechanisms of membrane scission and the ways in which enveloped viruses use these systems to mediate the release of budding virions.
virus budding; ESCRT; membrane curvature; lipid phase segregation; line tension
The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process.
The type I interferon (IFN) signaling pathway restricts infection of many divergent families of RNA and DNA viruses by inducing hundreds of IFN-stimulated genes (ISGs), some of which have direct antiviral activity. We screened 813 short hairpin RNA (shRNA) constructs targeting 245 human ISGs using a flow cytometry approach to identify genes that modulated infection of West Nile virus (WNV) in IFN-β-treated human cells. Thirty ISGs with inhibitory effects against WNV were identified, including several novel genes that had antiviral activity against related and unrelated positive-strand RNA viruses. We also defined one ISG, activating signal cointegrator complex 3 (ASCC3), which functioned as a negative regulator of the host defense response. Silencing of ASCC3 resulted in upregulation of multiple antiviral ISGs, which correlated with inhibition of infection of several positive-strand RNA viruses. Reciprocally, ectopic expression of human ASCC3 or mouse Ascc3 resulted in downregulation of ISGs and increased viral infection. Mechanism-of-action and RNA sequencing studies revealed that ASCC3 functions to modulate ISG expression in an IRF-3- and IRF-7-dependent manner. Compared to prior ectopic ISG expression studies, our shRNA screen identified novel ISGs that restrict infection of WNV and other viruses and defined a new counterregulatory ISG, ASCC3, which tempers cell-intrinsic immunity.
West Nile virus (WNV) is a mosquito-transmitted virus that continues to pose a threat to public health. Innate immune responses, especially those downstream of type I interferon (IFN) signaling, are critical for controlling virus infection and spread. We performed a genetic screen using a gene silencing approach and identified 30 interferon-stimulated genes (ISGs) that contributed to the host antiviral response against WNV. As part of this screen, we also identified a novel negative regulatory protein, ASCC3, which dampens expression of ISGs, including those with antiviral or proinflammatory activity. In summary, our studies define a series of heretofore-uncharacterized ISGs with antiviral effects against multiple viruses or counterregulatory effects that temper IFN signaling and likely minimize immune-mediated pathology.
Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15−/− macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.
Modification of proteins by ubiquitin (UB) and ubiquitin-like proteins (UBLs) are key regulatory processes of the innate and adaptive immune response. Interferon (IFN) stimulated gene product 15 (ISG15) is an ubiquitin-like protein modifier, which is reversibly conjugated to different viral and cellular proteins mediating considerable antiviral responses. In turn, many viruses, including poxviruses, have evolved strategies to block the antiviral and inflammatory effects of the innate immune responses to keep cells alive until virus replication is completed. Here, we describe a novel function of ISG15 in the control of macrophages activation, phagocytosis and apoptosis in response to viral infection. These processes are essential for the self-defense mechanism to protect animals from infectious disease and could be crucial to understand the ISG15 antiviral activity described in animal models.
Nipah virus (NiV) is a recently emerged paramyxovirus capable of causing fatal disease in a broad range of mammalian hosts, including humans. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the family Paramyxoviridae. Recombinant expression systems have played a crucial role in studying the cell biology of these Biosafety Level-4 restricted viruses. Henipavirus assembly and budding occurs at the plasma membrane, although the details of this process remain poorly understood. Multivesicular body (MVB) proteins have been found to play a role in the budding of several enveloped viruses, including some paramyxoviruses, and the recruitment of MVB proteins by viral proteins possessing late budding domains (L-domains) has become an important concept in the viral budding process. Previously we developed a system for producing NiV virus-like particles (VLPs) and demonstrated that the matrix (M) protein possessed an intrinsic budding ability and played a major role in assembly. Here, we have used this system to further explore the budding process by analyzing elements within the M protein that are critical for particle release.
Using rationally targeted site-directed mutagenesis we show that a NiV M sequence YPLGVG is required for M budding and that mutation or deletion of the sequence abrogates budding ability. Replacement of the native and overlapping Ebola VP40 L-domains with the NiV sequence failed to rescue VP40 budding; however, it did induce the cellular morphology of extensive filamentous projection consistent with wild-type VP40-expressing cells. Cells expressing wild-type NiV M also displayed this morphology, which was dependent on the YPLGVG sequence, and deletion of the sequence also resulted in nuclear localization of M. Dominant-negative VPS4 proteins had no effect on NiV M budding, suggesting that unlike other viruses such as Ebola, NiV M accomplishes budding independent of MVB cellular proteins.
These data indicate that the YPLGVG motif within the NiV M protein plays an important role in M budding; however, involvement of any specific components of the cellular MVB sorting pathway in henipavirus budding remains to be demonstrated. Further investigation of henipavirus assembly and budding may yet reveal a novel mechanism(s) of viral assembly and release that could be applicable to other enveloped viruses or have therapeutic implications.
Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.
To replicate efficiently in their hosts, viruses must avoid antiviral cellular defenses that comprise part of the innate immune system. Tetherin, an antiviral membrane protein that inhibits the release of several enveloped viruses from infected cells, is antagonized by the HIV-1 Vpu protein. The K5 protein of the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV) modulates the cell surface levels of several host proteins including tetherin. We show that KSHV release is sensitive to tetherin, and that K5 expression is required for efficient virus production in tetherin-expressing cells. K5 is also capable of rescuing Vpu-defective HIV-1 virus release from tetherin. K5 expression induces a down-regulation of cell-surface tetherin levels and degradation in late endosomes, which depends on a single lysine residue in the tetherin cytoplasmic tail. Finally, we show that the ESCRT pathway, which promotes the trafficking of cell surface receptors for degradation, is required for K5-mediated tetherin removal from the plasma membrane. Thus, we demonstrate that herpesviruses are sensitive to the antiviral effects of tetherin and that KSHV has evolved a mechanism for its destruction. These findings extend the list of viruses sensitive to tetherin, suggesting that tetherin counter-measures are widespread defense mechanisms amongst enveloped viruses.
The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a “submarine-like” budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular “rocket-like” protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses.
The filoviruses, Marburg and Ebola, cause lethal hemorrhagic fever and are highest-priority bioterrorism agents. Filovirus particles contain a rod-like nucleocapsid and are normally filamentous, though other shapes are seen. It is poorly understood how such large filamentous particles are assembled and released from infected cells. Here we have studied Marburg virus production in infected cells using electron tomography. This technique allows virus particles to be visualized in three dimensions at different stages during assembly. We find that in early stages of virus production, highly infectious filamentous viruses are produced, whereas after prolonged infection poorly infectious spherical viruses are released. We also define the sequence of steps in filamentous virus release. The intracellular nucleocapsid first travels to the plasma membrane of the cell, where it binds laterally along its whole length. One end is then wrapped by the plasma membrane and wrapping proceeds rapidly until the virus protrudes vertically from the cell surface. The rear end of the virus particle then pinches off from the cell. We propose that other important filamentous and rod-shaped viruses also follow this series of steps of assembly and budding.
The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.
Viruses hijack the cellular machinery to complete their life cycle. In the case of HIV-1, the endosomal sorting complex required for transport (ESCRT) is recruited by nascent viruses to release themselves from infected cells. Currently, there has been an intense amount of research on how the ESCRT machinery induces viral release. Using super-resolution imaging with endogenous ESCRT proteins or ESCRT proteins containing a small tag, we are able to provide insight into how ESCRT leads to budding of HIV-1. Super resolution imaging of the early ESCRT factors Tsg101 and ALIX, as well as later factors CHMP4B and CHMP2A, also showed condensed, circular structures with diameters of roughly 60 to 90 nm. The cluster sizes were significantly smaller than that of the HIV-1 bud and the distribution of cluster sizes that colocalized with nascent HIV-1 assembly sites were narrower than for non-colocalizing structures. This indicates that the point of interaction between the ESCRT machinery and the HIV-1 assembly site is in the bud neck.
During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process.
IMPORTANCE Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.
The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.
The release of an enveloped virus particle from an infected cell requires the separation of the viral and cell membranes. Many enveloped viruses accomplish this by parasitizing a set of cellular proteins, termed the ESCRT pathway, that normally separates cellular membranes from each other. In some cases, viral structural proteins encode peptides motifs that bind directly to, and thereby recruit, the ESCRT machinery. Alternatively, viruses can recruit enzymes, termed ubiquitin ligases, that bind to other proteins, and catalyze the addition of ubiquitin to them. It has, heretofore, been somewhat unclear precisely how the recruitment of ubiquitin ligases leads to the engagement of the ESCRT machinery. We show that the simple recruitment of a fragment of a ubiquitin ligase that is responsible for the addition of ubiquitin to other proteins is sufficient to drive virus particle release, even when it is not possible to attach ubiquitin to viral proteins. Paradoxically, we also found that simple attachment of ubiquitin to the same viral protein can also drive particle release. These results show that there is flexibility in the ways in which the ESCRT machinery can be recruited and how ubiquitin can be co-opted to enable this.
Promyelocytic leukemia protein (PML), a major component of PML nuclear bodies (also known as nuclear domain 10), is involved in diverse cellular processes such as cell proliferation, apoptosis, gene regulation, and DNA damage response. PML also acts as a restriction factor that suppresses incoming viral genomes, therefore playing an important role in intrinsic defense. Here, we show that PML positively regulates type I interferon response by promoting transcription of interferon-stimulated genes (ISGs) and that this regulation by PML is counteracted by human cytomegalovirus (HCMV) IE1 protein. Small hairpin RNA-mediated PML knockdown in human fibroblasts reduced ISG induction by treatment of interferon-β or infection with UV-inactivated HCMV. PML was required for accumulation of activated STAT1 and STAT2, interacted with them and HDAC1 and HDAC2, and was associated with ISG promoters after HCMV infection. During HCMV infection, viral IE1 protein interacted with PML, STAT1, STAT2, and HDACs. Analysis of IE1 mutant viruses revealed that, in addition to the STAT2-binding domain, the PML-binding domain of IE1 was necessary for suppression of interferon-β-mediated ISG transcription, and that IE1 inhibited ISG transcription by sequestering interferon-stimulated gene factor 3 (ISGF3) in a manner requiring its binding of PML and STAT2, but not of HDACs. In conclusion, our results demonstrate that PML participates in type I interferon-induced ISG expression by regulating ISGF3, and that this regulation by PML is counteracted by HCMV IE1, highlighting a widely shared viral strategy targeting PML to evade intrinsic and innate defense mechanisms.
For productive viral infection, virus needs to overcome successive host defenses including intrinsic defense and innate and acquired immunity. Promyelocytic leukemia protein (PML) has been shown to play an important role in intrinsic defense by acting as a nuclear restriction factor that suppresses incoming viral genomes. In this study, we demonstrate that PML also positively regulates type I interferon response by promoting transcription of interferon-stimulated genes (ISGs). Therefore, PML is a key player in both intrinsic and innate host defenses. We further show that this regulation by PML in type I interferon response is inhibited by human cytomegalovirus (HCMV) IE1 protein, which forms a complex with PML, STAT1, STAT2, and HDACs in virus-infected cells. By analyzing mutant viruses, we demonstrate that IE1 inhibits ISG transcription by sequestering interferon-stimulated gene factor 3 (ISGF3) in a manner requiring its binding of PML and STAT2, but not of HDACs. Our findings reveal that PML is a regulator of ISGF3 in type I interferon response and that this PML activity is counteracted by HCMV IE1. Our study explains why PML targeting activity is widely conserved among many viruses.
Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 μm in length and excluded the PM marker, and tubulin-positive extensions that were >10 μm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites.
IMPORTANCE Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.
Mammalian cells respond to virus infections by eliciting both innate and adaptive immune responses. One of the most effective innate antiviral responses is the production of alpha/beta interferon and the subsequent induction of interferon-stimulated genes (ISGs), whose products collectively limit virus replication and spread. Following viral infection, interferon is produced in a biphasic fashion that involves a number of transcription factors, including the interferon regulatory factors (IRFs) 1, 3, 7, and 9. In addition, virus infection has been shown to directly induce ISGs in the absence of prior interferon production through the activation of IRF3. This process is believed to require virus replication and results in IRF3 hyperphosphorylation, nuclear localization, and proteasome-mediated degradation. Previously, we and others demonstrated that herpes simplex virus type 1 (HSV-1) induces ISGs and an antiviral response in fibroblasts in the absence of both interferon production and virus replication. In this report, we show that the entry of enveloped virus particles from diverse virus families elicits a similar innate response. This process requires IRF3, but not IRF1, IRF7, or IRF9. Following virus replication, the large DNA viruses HSV-1 and vaccinia virus effectively inhibit ISG mRNA accumulation, whereas the small RNA viruses Newcastle disease virus, Sendai virus, and vesicular stomatitis virus do not. In addition, we found that IRF3 hyperphosphorylation and degradation do not correlate with ISG and antiviral state induction but instead serve as a hallmark of productive virus replication, particularly following a high-multiplicity infection. Collectively, these data suggest that virus entry triggers an innate antiviral response mediated by IRF3 and that subsequent virus replication results in posttranslational modification of IRF3, such as hyperphosphorylation, depending on the nature of the incoming virus.
Influenza A virus causes seasonal epidemics, sporadic pandemics and is a significant global heath burden. Influenza virus is an enveloped virus that contains a segmented negative strand RNA genome. Assembly and budding of progeny influenza virions is a complex, multistep process that occurs in lipid raft domains on the apical membrane of infected cells. The viral proteins hemagglutinin (HA) and neuraminidase (NA) are targeted to lipid rafts, causing the coalescence and enlargement of the raft domains. This clustering of HA and NA may cause a deformation of the membrane and the initiation of the virus budding event. M1 is then thought to bind to the cytoplasmic tails of HA and NA where it can then polymerize and form the interior structure of the emerging virion. M1, bound to the cytoplasmic tails of HA and NA, additionally serves as a docking site for the recruitment of the viral RNPs and may mediate the recruitment of M2 to the site of virus budding. M2 initially stabilizes the site of budding, possibly enabling the polymerization of the matrix protein and the formation of filamentous virions. Subsequently, M2 is able to alter membrane curvature at the neck of the budding virus, causing membrane scission and the release of the progeny virion. This review investigates the latest research on influenza virus budding in an attempt to provide a step-by-step analysis of the assembly and budding processes for influenza viruses.
Influenza Virus; Viral Like Particle; Assembly; Budding; Filamentous Virion; Membrane Curvature
The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.
Herpesviruses are large DNA viruses associated with human and animal diseases. After viral DNA replication, the herpesviral nucleocapsids egress through the nuclear membrane for subsequent cytoplasmic virion maturation. However, the mechanism by which the virus regulates the nuclear membrane and cellular machinery involved in this process remained elusive. The cellular endosomal sorting complex required for transport (ESCRT) machinery is known to participate in the biogenesis of multivesicular bodies, cytokinesis and the release of enveloped viruses from cytoplasmic membranes. Here, we show that functional ESCRT machinery is required for the maturation of Epstein-Barr virus (EBV). ESCRT proteins are redistributed close to the nucleus-associated membrane through interaction with the viral BFRF1 protein, leading to vesicle formation and structural changes of the nuclear membrane. Remarkably, inhibition of ESCRT machinery abolishes BFRF1-induced vesicle formation, and leads to the accumulation of viral DNA and capsid proteins in the nucleus. Specific interactions between BFRF1 and Alix are required for BFRF1-derived vesicle formation and crucial for the nuclear egress of EBV.
The type I interferon system plays a critical role in limiting the spread of viral infection. Viruses induce the production of interferon (IFN), which after binding to the IFN-α/β receptor (IFNAR), and triggering of the JAK/STAT signaling cascade, results in the induction of interferon-stimulated genes (ISGs). These ISGs function to inhibit viral replication and to regulate the host immune response. Among these ISGs, the ubiquitin-like molecule, ISG15, is one of the most strongly induced proteins. Similar to ubiquitin, through an IFN induced conjugation cascade, ISG15 is covalently linked to a variety of cellular proteins, suggesting regulation of different cellular processes. Studies performed over the past several years have shown that ISG15 plays a central role in the host’s antiviral response against many viruses. Mice lacking ISG15 display increased susceptibility to multiple viruses. Furthermore, several viruses have developed immune evasion strategies that directly target the ISG15 pathway. Work is now underway to determine the mechanism by which ISG15 functions as an antiviral molecule, such that therapies targeting this pathway can be developed in the future.
ISG15; interferon; antiviral; ubiquitin-like molecule
Hemorrhagic fever viruses, including the filoviruses (Ebola and Marburg) and arenaviruses (Lassa and Junín viruses), are serious human pathogens for which there are currently no FDA approved therapeutics or vaccines. Importantly, transmission of these viruses, and specifically late steps of budding, critically depend upon host cell machinery. Consequently, strategies which target these mechanisms represent potential targets for broad spectrum host oriented therapeutics. An important cellular signal implicated previously in EBOV budding is calcium. Indeed, host cell calcium signals are increasingly being recognized to play a role in steps of entry, replication, and transmission for a range of viruses, but if and how filoviruses and arenaviruses mobilize calcium and the precise stage of virus transmission regulated by calcium have not been defined. Here we demonstrate that expression of matrix proteins from both filoviruses and arenaviruses triggers an increase in host cytoplasmic Ca2+ concentration by a mechanism that requires host Orai1 channels. Furthermore, we demonstrate that Orai1 regulates both VLP and infectious filovirus and arenavirus production and spread. Notably, suppression of the protein that triggers Orai activation (Stromal Interaction Molecule 1, STIM1) and genetic inactivation or pharmacological blockade of Orai1 channels inhibits VLP and infectious virus egress. These findings are highly significant as they expand our understanding of host mechanisms that may broadly control enveloped RNA virus budding, and they establish Orai and STIM1 as novel targets for broad-spectrum host-oriented therapeutics to combat these emerging BSL-4 pathogens and potentially other enveloped RNA viruses that bud via similar mechanisms.
Filoviruses (Ebola and Marburg viruses) and arenaviruses (Lassa and Junín viruses) are high-priority pathogens that hijack host proteins and pathways to complete their replication cycles and spread from cell to cell. Here we provide genetic and pharmacological evidence to demonstrate that the host calcium channel protein Orai1 and ER calcium sensor protein STIM1 regulate efficient budding and spread of BSL-4 pathogens Ebola, Marburg, Lassa, and Junín viruses. Our findings are of broad significance as they provide new mechanistic insight into fundamental, immutable, and conserved mechanisms of hemorrhagic fever virus pathogenesis. Moreover, this strategy of targeting highly conserved host cellular protein(s) and mechanisms required by these viruses to complete their life cycle should elicit minimal drug resistance.
Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L−/− mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15−/− mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15−/− mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.
Type I interferon plays a critical role in the host defense to viral infection. Signaling through the type I IFN receptor allows for the induction of hundreds of interferon stimulated genes (ISGs) that generate an antiviral state within host cells. The ubiquitin-like molecule ISG15 has been shown to play an important role during multiple viral infections, including influenza virus infection. To date, the ability of ISG15 to protect against viral infection has been shown to be dependent on its ability to covalently bind (or conjugate) to target proteins, including the binding of viral proteins. We investigated the importance of the type I interferon response and ISG15 conjugation in a neonatal model of Chikungunya virus infection, a re-emerging human pathogen in the Indian Ocean region. Remarkably, the role of ISG15 during CHIKV infection appears to be conjugation independent, suggesting a non-classical role for ISG15 during viral infection. Our data also suggests that ISG15 plays an immunoregulatory role, as opposed to having direct antiviral function. Our CHIKV model may provide an opportunity to identify a novel mechanism by which ISG15 contributes to the innate immune response to viral infection.
The paramyxoviruses define a diverse group of enveloped RNA viruses that includes a number of important human and animal pathogens. Examples include human respiratory syncytial virus and the human parainfluenza viruses, which cause respiratory illnesses in young children and the elderly; measles and mumps viruses, which have caused recent resurgences of disease in developed countries; the zoonotic Hendra and Nipah viruses, which have caused several outbreaks of fatal disease in Australia and Asia; and Newcastle disease virus, which infects chickens and other avian species. Like other enveloped viruses, paramyxoviruses form particles that assemble and bud from cellular membranes, allowing the transmission of infections to new cells and hosts. Here, we review recent advances that have improved our understanding of events involved in paramyxovirus particle formation. Contributions of viral matrix proteins, glycoproteins, nucleocapsid proteins, and accessory proteins to particle formation are discussed, as well as the importance of host factor recruitment for efficient virus budding. Trafficking of viral structural components within infected cells is described, together with mechanisms that allow for the selection of specific sites on cellular membranes for the coalescence of viral proteins in preparation of bud formation and virion release.
virus budding; virus assembly; matrix protein; paramyxovirus; virus-like particle; polarized budding; lipid raft membranes; viral trafficking
The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.
Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.
Human rhinovirus (HRV) is a non-enveloped virus of the picornavirus family and is responsible for respiratory infections (common colds) costing billions of dollars ($) annually. There remains no vaccine or licensed drug to prevent or reduce infection. Related members of the picornavirus family include significant pathogens such as poliovirus, enterovirus 71 and foot-and-mouth disease virus, for which improved control measures are also required. A fundamental step in virus infection is the delivery of the viral genetic material through the barrier of the cellular membrane. Viruses such as HIV and influenza are enveloped in an outer membrane which can fuse with the host cell membrane to allow the viral genome to penetrate into the cytoplasm. However, non-enveloped viruses such as picornaviruses lack a membrane and the mechanisms for penetration of the membrane by these viruses remain poorly understood. The capsid protein VP4 has previously been implicated in the delivery of the picornavirus genome. In this study we demonstrate that HRV VP4 interacts with membranes to make them permeable by the formation of multimeric, size-selective membrane pores with properties consistent with the transport of viral genome through the membrane. This function of VP4 provides a novel antiviral target for this family of viruses.