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.
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.
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 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.
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.
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.
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
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.
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.
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 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.
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.
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 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
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 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.
Bst-2/Tetherin inhibits the release of HIV by tethering newly formed virus particles to the plasma membrane of infected cells. Although the mechanisms of Tetherin-mediated restriction are increasingly well understood, the biological relevance of this restriction in the natural target cells of HIV is unclear. Moreover, whether Tetherin exerts any restriction on the direct cell-cell spread of HIV across intercellular contacts remains controversial. Here we analyse the restriction endogenous Tetherin imposes on HIV transmission from primary human macrophages, one of the main targets of HIV in vivo. We find that the mRNA and protein levels of Tetherin in macrophages are comparable to those in T cells from the same donors, and are highly upregulated by type I interferons. Improved immunocytochemistry protocols enable us to demonstrate that Tetherin localises to the cell surface, the trans-Golgi network, and the macrophage HIV assembly compartments. Tetherin retains budded virions in the assembly compartments, thereby impeding the release and cell-free spread of HIV, but it is not required for the maintenance of these compartments per se. Notably, using a novel assay to quantify cell-cell spread, we show that Tetherin promotes the transfer of virus clusters from macrophages to T cells and thereby restricts the direct transmission of a dual-tropic HIV-1. Kinetic analyses provide support for the notion that this direct macrophage-T cell spread is mediated, at least in part, by so-called virological synapses. Finally, we demonstrate that the viral Vpu protein efficiently downregulates the cell surface and overall levels of Tetherin, and thereby abrogates this HIV restriction in macrophages. Together, our study shows that Tetherin, one of the most potent HIV restriction factors identified to date, can inhibit virus spread from primary macrophages, regardless of the mode of transmission.
Tetherin is a cellular protein that inhibits (or restricts) a broad range of enveloped viruses, including HIV, by physically “tethering” nascent particles to the plasma membrane of infected cells. CD4+ T cells and macrophages are the main targets of HIV in vivo, and both cell types express Tetherin. Although the mechanisms of Tetherin-mediated restriction in model cell lines and T cells are increasingly well understood, experimental data from macrophages are sparse, and partially contradict observations made in other cell types. Here we investigate the sensitivity of Tetherin expression to interferon, and the subcellular localisation of the restriction factor in primary human macrophages. We find that Tetherin inhibits HIV release by retaining nascent particles in macrophage HIV assembly compartments, and can also restrict the transmission of HIV across intercellular contacts between macrophages and T cells. Finally, we demonstrate that the HIV protein Vpu efficiently counteracts Tetherin in macrophages, and thereby ensures viral propagation. Our results, together with other published data, show that Tetherin can efficiently inhibit viral replication in both major target cell types of HIV, regardless of the mode of transmission. These data support the view that efficient counteraction of Tetherin was a crucial factor for the global spread of HIV.
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
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.
Tetherin/BST-2 is an important host restriction factor that limits the replication of HIV and other enveloped viruses. Tetherin is a type II membrane glycoprotein with a very unusual domain structure that allows it to engage budding virions and retain them on the plasma membrane of infected cells. Following the initial report identifying tetherin as the host cell factor targeted by the HIV-1 Vpu gene, knowledge of the molecular, structural, and cellular biology of tetherin has rapidly advanced. This paper summarizes the discovery and impact of tetherin biology on the HIV field, with a focus on recent advances in understanding its structure and function. The relevance of tetherin to replication and spread of other retroviruses is also reviewed. Tetherin is a unique host restriction factor that is likely to continue to provide new insights into host-virus interactions and illustrates well the varied ways by which host organisms defend against viral pathogens.
Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.
Dengue virus is the most prevalent arbovirus in the world and an increasingly significant public health problem. Development of vaccines and therapeutics has been slowed by poor understanding of viral pathogenesis. Especially, how the virus subverts the host interferon response, a powerful branch of the innate immune system remains the subject of debate and great interest. Dengue virus produces large quantities of a non-coding, highly structured viral RNA, termed sfRNA, whose function in viral replication is elusive but has been linked in related viruses to inhibition of the interferon response. Nonetheless the mechanisms involved are yet to be characterized. Here, we show that dengue virus 2 sfRNA targets and antagonizes a set of host RNA-binding proteins G3BP1, G3BP2 and CAPRIN1, to interfere with translation of antiviral interferon-stimulated mRNAs. This activity impairs establishment of the antiviral state, allowing the virus to replicate and evade the interferon response. While this particular mechanism was not conserved among other flaviviruses, we believe it is highly relevant for dengue virus 2 replication and pathogenesis. Taken together, our results highlight both new layers of complexity in the regulation of the innate immune response, as well as the diversity of strategies flaviviruses employ to counteract it.
Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.
Viral recognition by the host often triggers an antiviral state, which suppresses viral spread and imparts adaptive immunity. Like many viruses, dengue virus (DENV) defeats the host-sensing pathway within infected cells. However, in vivo studies have demonstrated a key role of innate immunity in controlling DENV infection. Here we report that sensing of DENV-infected cells by non-permissive innate immune cells, the plasmacytoid dendritic cells (pDCs), triggers a cell-contact- and TLR7-dependent activation of a strong antiviral IFN response. This cell-to-cell sensing involves transmission of viral elements that are clustered at the interface between pDCs and infected cells and is regulated by the actin network. Importantly, we revealed that uncleaved prM surface protein-containing immature particles play a key function in stimulating the innate immune response. These non-infectious immature particles are released by infected cells as a consequence of a suboptimal cleavage site, which is an evolutionarily conserved viral feature that likely favors the export of infectious virus by prevention of premature membrane fusion in the secretory pathway. Therefore our results highlight a conceptually novel trade-off between efficient infectious virus release and the production of IFN-inducing particles. This concept may have broad importance for the many viruses that, like DENV, can disable the pathogen-sensing machinery within infected cells and can release uncleaved glycoprotein-containing non-infectious particles.
Many enveloped viruses complete their replication cycle by forming vesicles that bud from the plasma membrane. Some viruses encode “late” (L) domain motifs that are able to hijack host proteins involved in the vacuolar protein sorting (VPS) pathway, a cellular budding process that gives rise to multivesicular bodies and that is topologically equivalent to virus budding. Although many enveloped viruses share this mechanism, examples of viruses that require additional viral factors and viruses that appear to be independent of the VPS pathway have been identified. Alternative mechanisms for virus budding could involve other topologically similar process such as cell abscission, which occurs following cytokinesis, or virus budding could proceed spontaneously as a result of lipid microdomain accumulation of viral proteins. Further examination of novel virus-host protein interactions and characterization of other enveloped viruses for which budding requirements are currently unknown will lead to a better understanding of the cellular processes involved in virus assembly and budding.