African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.African swine fever virus (ASFV) (61, 72) is the only known DNA-containing arbovirus and the sole member of the Asfarviridae family (24). Infection by this virus of its natural hosts, the wild swine warthogs and bushpigs and the argasid ticks of the genus Ornithodoros, results in a mild disease, often asymptomatic, with low viremia titers, that in many cases develops into a persistent infection (3, 43, 71). In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which the only available methods of disease control are the quarantine of the affected area and the elimination of the infected animals (51).The ASFV genome is a lineal molecule of double-stranded DNA of 170 to 190 kbp in length with convalently closed ends and terminal inverted repeats. The genome encodes more than 150 open reading frames, half of which lack any known or predictable function (16, 75).The virus particle, with an overall icosahedral shape and an average diameter of 200 nm (11), is organized in several concentric layers (6, 11, 15) containing more than 50 structural proteins (29). Intracellular particles are formed by an inner viral core, which contains the central nucleoid surrounded by a thick protein coat, referred to as core shell. This core is enwrapped by an inner lipid envelope (7, 34) on top of which the icosahedral capsid is assembled (26, 27, 31). Extracellular virions possess an additional membrane acquired during the budding from the plasma membrane (11). Both forms of the virus, intracellular and extracellular, are infective (8).The assembly of ASFV particles occurs in the cytoplasm of the infected cell, in viral factories located close to the cell nucleus (6, 13, 49). ASFV factories possess several characteristics similar to those of the cellular aggresomes (35), which are accumulations of aggregates of cellular proteins that form perinuclear inclusions (44).Current models propose that ASFV assembly begins with the modification of endoplasmic reticulum (ER) membranes, which are subsequently recruited to the viral factories and transformed into viral precursor membranes. These ER-derived viral membranes represent the precursors of the inner viral envelope and are the first morphological evidence of viral assembly (7, 60). ASFV viral membrane precursors evolve into icosahedral intermediates and icosahedral particles by the progressive assembly of the outer capsid layer at the convex face of the precursor membranes (5, 26, 27, 31) through an ATP- and calcium-dependent process (19). At the same time, the core shell is formed underneath the concave face of the viral envelope, and the viral DNA and nucleoproteins are packaged and condensed to form the innermost electron-dense nucleoid (6, 9, 12, 69). However, the assembly of the capsid and the internal envelope appears to be largely independent of the components of the core of the particle, since the absence of the viral polyprotein pp220 during assembly produces empty virus-like particles that do not contain the core (9).Comparative genome analysis suggests that ASFV shares a common origin with the members of the proposed nucleocytoplasmic large DNA viruses (NCLDVs) (40, 41). The reconstructed phylogeny of NCLDVs as well as the similitude in the structures and organizations of the genomes indicates that ASFV is more closely related to poxviruses than to other members of the NCLDVs. A consensus about the origin and nature of the envelope of the immature form of vaccinia virus (VV), the prototypical poxvirus, seems to be emerging (10, 17, 20, 54). VV assembly starts with the appearance of crescent-shaped structures within specialized regions of the cytoplasm also known as viral factories (21, 23). The crescent membranes originate from preexisting membranes derived from some specialized compartment of the ER (32, 37, 52, 53, 67), and an operative pathway from the ER to the crescent membrane has recently been described (38, 39). VV crescents apparently grow in length while maintaining the same curvature until they become closed circles, spheres in three dimensions, called immature virions (IV) (22). The uniform curvature is produced by a honeycomb lattice of protein D13L (36, 70), which attaches rapidly to the membranes so that nascent viral membranes always appear to be coated over their entirety. The D13L protein is evolutionarily related to the capsid proteins of the other members of the NCLDV group, including ASFV, but lacks the C-terminal jelly roll motif (40). This structural difference is probably related to the fact that poxviruses are the only member of this group without an icosahedral capsid; instead, the spherical D13L coat acts as a scaffold during the IV stage but is discarded in subsequent steps of morphogenesis (10, 28, 46, 66). Thus, although crescents in VV and precursors of the inner envelope in ASFV are the first morphogenetic stages discernible in the viral factories of these viruses, they seem to be different in nature. Crescents are covered by the D13L protein and are more akin to the icosahedral intermediates of ASFV assembly, whereas ASFV viral membrane precursors are more similar to the naked membranes seen when VV morphogenesis is arrested by rifampin treatment (33, 47, 48, 50) or when the expression of the D13L and A17L proteins are repressed during infection with lethal conditional VV viruses (45, 55, 56, 68, 74, 76).Although available evidence strongly supports the reticular origin of the ASFV inner envelope (7, 60), the mechanism of acquisition remains unknown, and the number of membranes present in the inner envelope is controversial. The traditional view of the inner envelope as formed by two tightly opposed membranes derived from ER collapsed cisternae (7, 59, 60) has recently been challenged by the careful examination of the width of the internal membrane of viral particles and the single outer mitochondrial membrane, carried out using chemical fixation, cryosectioning, and high-pressure freezing (34). The results suggest that the inner envelope of ASFV is a single lipid bilayer, which raises the question of how such a structure can be generated and stabilized in the precursors of the ASFV internal envelope. In the case of VV, the coat of the D13L protein has been suggested to play a key role in the stabilization of the single membrane structure of the crescent (10, 17, 36), but the ASFV capsid protein p72 is not a component of the viral membrane precursors. The identification and functional characterization of the proteins involved in the generation of these structures are essential for the understanding of the mechanisms involved in these early stages of viral assembly. For this reason, we are focusing our interest on the study of abundant structural membrane proteins that reside at the inner envelope of the viral particle. We have shown previously that one of these proteins, p54, is essential for the recruitment of ER membranes to the viral factory (59). Repression of protein p54 expression has a profound impact on virus production and leads to an early arrest in virion morphogenesis, resulting in the virtual absence of membranes in the viral factory.Protein p17, encoded by the late gene D117L in the BA71V strain, is an abundant structural protein (60, 65). Its sequence, which is highly conserved among ASFV isolates (16), does not show any significant similarity with the sequences present in the databases. Protein p17 is an integral membrane protein (18) that is predicted to insert in membranes with a Singer type I topology and has been localized in the envelope precursors as well as in both intracellular and extracellular mature particles (60), suggesting that it resides at the internal envelope, the only membranous structure of the intracellular particles.In this work, we analyze the role of protein p17 in viral assembly by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-dependent lethal conditional virus. The data presented indicate that protein p17 is essential for viral morphogenesis. The repression of this protein appears to block assembly at the level of viral precursor membranes, resulting in their accumulation at the viral factory.From the electron microscopy analysis of serial sections of viral factories at very early times during morphogenesis, we present experimental evidence that suggests that, during assembly, viral precursor membranes and core material organize into large helicoidal intermediates from which icosahedral particles emerge. The possible role of these structures during ASFV morphogenesis is discussed.     

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A)⁺ RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.The herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is required for productive viral infection. ICP27 interacts with a number of cellular proteins, and it binds RNA (35). One of the functions that ICP27 performs is to escort viral mRNAs from the nucleus to the cytoplasm for translation (2, 3, 5, 10, 13, 21, 34). ICP27 binds viral RNAs (5, 34) and interacts directly with the cellular mRNA export receptor TAP/NXF1 (2, 21), which is required for the export of HSV-1 mRNAs (20, 21). ICP27 also interacts with the export adaptor proteins Aly/REF (2, 3, 23) and UAP56 (L. A. Johnson, H. Swesey, and R. M. Sandri-Goldin, unpublished results), which form part of the TREX complex that binds to the 5′ end of mRNA through an interaction with CBP80 (26, 32, 41). Aly/REF does not appear to bind viral RNA directly (3), and it is not essential for HSV-1 RNA export based upon small interfering RNA (siRNA) knockdown studies (20), but it contributes to the efficiency of viral RNA export (3, 23). ICP27 also interacts with the SR splicing proteins SRp20 and 9G8 (11, 36), which have been shown to shuttle between the nucleus and the cytoplasm (1). SRp20 and 9G8 have also been shown to facilitate the export of some cellular RNAs (16, 17, 27) by binding RNA and interacting with TAP/NXF1 (14, 16, 18). The knockdown of SRp20 or 9G8 adversely affects HSV-1 replication and specifically results in a nuclear accumulation of newly transcribed RNA during infection (11). Thus, these SR proteins also contribute to the efficiency of viral RNA export. However, the overexpression of SRp20 was unable to rescue the defect in RNA export during infection with an ICP27 mutant that cannot bind RNA (11), suggesting that ICP27 is the major HSV-1 RNA export protein that links viral RNA to TAP/NXF1.ICP27 was shown previously to bind RNA through an RGG box motif located at amino acids 138 to 152 within the 512-amino-acid protein (28, 34). Using electrophoretic mobility shift assays (EMSAs), we showed that the N-terminal portion of ICP27 from amino acids 1 to 160 bound specifically to viral oligonucleotides that are GC rich and that are flexible and relatively unstructured (5). Here we report the importance of three arginine residues within the RGG box for ICP27 binding to GC-rich sequences in vitro and for viral RNA export during infection. We also performed nuclear magnetic resonance (NMR) structural analysis of the N-terminal portion of ICP27 for both the wild-type protein and an ICP27 mutant in which three arginines were replaced with lysines. The NMR data showed that the N-terminal portion of ICP27 is relatively unstructured but compact, and NMR analysis in the presence of oligonucleotide substrates to which the N-terminal portion of ICP27 binds did not show any discernible alterations in this highly flexible structure, nor did the arginine-to-lysine substitutions.     

Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation

The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4_DN) in HCMV replication. Vps4_DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1_DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101_DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4_DN or CHMP1_DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.Cellular endosomal sorting complex required for transport (ESCRT) machinery controls the evolutionarily conserved process (33) of membrane budding that is normally a component of cytokinesis (6, 46), endosome sorting and multivesicular body (MVB) formation (28). After the initial characterization in retroviruses, many enveloped viruses have been shown to rely on this machinery during envelopment and release from cells (1, 18, 35, 40, 47, 69). Other viruses, such as influenza virus, mature independent of ESCRT machinery and are believed to use an alternative virus-intrinsic pathway (7). The core of the ESCRT machinery consists of five multiprotein complexes (ESCRT-0, -I, -II, and -III and Vps4-Vta1) (27). Vacuolar protein sorting-4 (Vps4) is a critical ATPase that functions downstream of most ESCRT components. Based on sensitivity to dominant-negative (DN) inhibitors of protein function, replication of several RNA viruses, as well as of the DNA virus herpes simplex virus type 1 (HSV-1) (5, 10), have been shown to rely on Vps4 in a manner that is analogous to the formation of MVBs (endosomal compartments containing intraluminal vesicles) (10, 45). Evidence based exclusively on small interfering RNA (siRNA) methods suggested cytomegalovirus (CMV) maturation was independent of ESCRT components, although the maturation of this virus remained MVB associated (16).ESCRT machinery facilitates envelopment and release at cytoplasmic membranes and recruits cargo for sorting via any of three alternative pathways that converge on a Vps4-dependent downstream step: (i) a tumor susceptibility gene-101 (Tsg101)-dependent pathway, (ii) an apoptosis linked gene-2 interacting protein X (ALIX)-dependent pathway, and (iii) a pathway that relies on a subset of Nedd4-like HECT E3 ubiquitin ligases (35). The involvement of ESCRT in viral envelopment and egress was first observed in human immunodeficiency virus (HIV) (18, 19, 40, 60) and has been extended to equine infectious anemia virus (34, 40, 52, 60), Rous sarcoma virus (29, 70, 71), Mason-Pfizer monkey virus (20, 72), rabies virus (24), Ebola virus (23), hepatitis B virus (68), vaccinia virus (25), HSV-1 (5, 10), and several other RNA and DNA viruses (7). Structural proteins in most of these viruses carry late (L) domains characterized by conserved amino acid motifs (PTAP, PPXY, and YXXL) that mediate protein-protein interactions and facilitate recruitment of ESCRT components to facilitate virus budding. The introduction of mutations in these motifs leads to defects in viral maturation and release from cells (40).Vps4 controls the release of ESCRT complexes from membranes (18, 40). Inhibition of Vps4A and Vps4B using Vps4A_DN reduces levels of viral maturation mediated by L domains (47). For this reason, inhibition by a Vps4_DN is considered the gold standard test to establish the role of ESCRT machinery in maturation of any virus (7). Tsg101, a component of ESCRT-I, normally functions to deliver ubiquitinated transmembrane proteins to MVBs (35). HIV-1 p6 Gag PTAP domain interacts with Tsg101 (18) and directs viral cores (capsids) to sites of viral envelopment (39). Upon disruption of HIV-1 PTAP domain, particle release becomes dependent on auxiliary factors, including an ALIX-binding YXXL domain within p6 Gag (60). A minimal amino-terminal L domain of Tsg101 functions as a DN inhibitor of PTAP-mediated viral budding without inhibiting Tsg101-independent PPXY- or YXXL-dependent pathways (40). The murine leukemia virus PPXY domain recruits a subset of Nedd4-like HECT E3 ubiquitin ligases (WWP1, WWP2, and Itch) (36) that in turn recruit ESCRT-III components (35). The YXXL L domain binds to the cellular protein ALIX (60). ALIX binds to Tsg101 (38) and also with ESCRT-III protein CHMP-4B (60), thus linking ESCRT-I and ESCRT-III. Green fluorescent protein (GFP)-, red fluorescent protein, or yellow fluorescent protein (YFP)-fused CHMPs are general DN inhibitors of all natural CHMP-associated activities and cause the formation of aberrant endosomal compartments that sequester ESCRT complexes (26, 31, 60). Through the use of these DN constructs, the recruitment and assembly of ESCRT components can be inhibited to specifically disrupt different steps of the ESCRT pathway.The best evidence supporting involvement of ESCRT machinery in the life cycle of herpesviruses comes from the inhibition of HSV-1 envelopment by Vps4_DN (10), as well as by CHMP3_DN (5), together with the association of HSV-1 maturation with MVB. It was recently reported that HHV-6 also induces MVB formation that controls viral egress via an exosomal release pathway (45). After losing primary envelope acquired at the nuclear membrane, Human CMV (HCMV) undergoes a secondary, or final, envelopment step within a cytoplasmic assembly compartments (AC) (59). Secondary envelopment is thought to occur within early endosomal compartments based on diverse observations: (i) purified virions and dense bodies have a lipid composition that is similar to this compartment (64); (ii) the AC of HCMV-infected fibroblasts contain endosomal markers (11); and (iii) a number of HCMV envelope proteins, including US28 (14), UL33, US27 (15), and gB (9), colocalize with endosomal markers in infected cells. A model of HCMV egress via early endosomes has been proposed (11).The approach that we have used here employed human foreskin fibroblasts (HFs) and restricted viral replication to cells that expressed the DN or wild-type (WT) component of the ESCRT pathway by including a requirement that transfected cells complement replication of virus. Confirming expression of both DN and complementing protein in transfected cells by epifluorescence microscopy ensured that an overwhelming majority of cells coexpressed these proteins. The results were scored as inhibition of viral spread to adjacent cells as well as demonstration of late gene expression in the transfected and/or infected cell. Viral progeny is released within 48 to 72 h from CMV-infected cells (44), reducing the likelihood that nonspecific or long-term toxicity of DN-ESCRT proteins would impact our analysis. This assay has been effectively used earlier for both immediate-early gene (54) and late gene (2, 62) mutants, and similar complementation assay results have been reported in diverse systems (8, 49, 73). This assay further provided an opportunity to determine when inhibition occurred relative to the viral replication cycle. Our data implicate ESCRT machinery late during HCMV maturation, which is consistent with a role in secondary envelopment and release.     

A Targeted Analysis of Cellular Chaperones Reveals Contrasting Roles for Heat Shock Protein 70 in Flock House Virus RNA Replication

Cytosolic chaperones are a diverse group of ubiquitous proteins that play central roles in multiple processes within the cell, including protein translation, folding, intracellular trafficking, and quality control. These cellular proteins have also been implicated in the replication of numerous viruses, although the full extent of their involvement in viral replication is unknown. We have previously shown that the heat shock protein 40 (hsp40) chaperone encoded by the yeast YDJ1 gene facilitates RNA replication of flock house virus (FHV), a well-studied and versatile positive-sense RNA model virus. To further explore the roles of chaperones in FHV replication, we examined a panel of 30 yeast strains with single deletions of cytosolic proteins that have known or hypothesized chaperone activity. We found that the majority of cytosolic chaperone deletions had no impact on FHV RNA accumulation, with the notable exception of J-domain-containing hsp40 chaperones, where deletion of APJ1 reduced FHV RNA accumulation by 60%, while deletion of ZUO1, JJJ1, or JJJ2 markedly increased FHV RNA accumulation, by 4- to 40-fold. Further studies using cross complementation and double-deletion strains revealed that the contrasting effects of J domain proteins were reproduced by altering expression of the major cytosolic hsp70s encoded by the SSA and SSB families and were mediated in part by divergent effects on FHV RNA polymerase synthesis. These results identify hsp70 chaperones as critical regulators of FHV RNA replication and indicate that cellular chaperones can have both positive and negative regulatory effects on virus replication.The compact genomes of viruses relative to those of other infectious agents restrict their ability to encode all proteins required to complete their replication cycles. To circumvent this limitation, viruses often utilize cellular factors or processes to complete essential steps in replication. One group of cellular proteins frequently targeted by viruses are cellular chaperones, which include a diverse set of heat shock proteins (hsps) that normally facilitate cellular protein translation, folding, trafficking, and degradation (18, 64). The connection between viruses and cellular chaperones was originally identified in bacteria, where the Escherichia coli hsp40 and hsp70 homologues, encoded by dnaJ and dnaK, respectively, were identified as bacterial genes essential for bacteriophage λ DNA replication (62). Research over the past 30 years has further revealed the importance of cellular chaperones in viral replication, such that the list of virus-hsp connections is now quite extensive and includes viruses from numerous families with diverse genome structures (4, 6, 7, 16, 19, 20, 23, 25, 40, 41, 44, 51, 54, 60). These studies have demonstrated the importance of cellular chaperones in multiple steps of the viral life cycle, including entry, viral protein translation, genome replication, encapsidation, and virion release. However, the list of virus-hsp connections is likely incomplete. Further studies to explore this particular host-pathogen interaction will shed light on virus replication mechanisms and pathogenesis, and potentially highlight targets for novel antiviral agents.To study the role of cellular chaperones in the genome replication of positive-sense RNA viruses, we use flock house virus (FHV), a natural insect pathogen and well-studied member of the Nodaviridae family. The FHV life cycle shares many common features with other positive-sense RNA viruses, including the membrane-specific targeting and assembly of functional RNA replication complexes (37, 38), the exploitation of various cellular processes and host factors for viral replication (5, 23, 60), and the induction of large-scale membrane rearrangements (24, 28, 38, 39). FHV virions contain a copackaged bipartite genome consisting of RNA1 (3.1 kb) and RNA2 (1.4 kb), which encode protein A, the viral RNA-dependent RNA polymerase, and the structural capsid protein precursor, respectively (1). During active genome replication, FHV produces a subgenomic RNA3 (0.4 kb), which encodes the RNA interference inhibitor protein B2 (12, 29, 32). These viral characteristics make FHV an excellent model system to study many aspects of positive-sense RNA virus biology.In addition to the benefits of a simple genome, FHV is able to establish robust RNA replication in a wide variety of genetically tractable eukaryotic hosts, including Drosophila melanogaster (38), Caenorhabditis elegans (32), and Saccharomyces cerevisiae (46). The budding yeast S. cerevisiae has been an exceptionally useful model host to study the mechanisms of viral RNA replication complex assembly and function with FHV (31, 37, 39, 45, 53, 55, 56, 60) as well as other positive-sense RNA viruses (11). The facile genetics of S. cerevisiae, along with the vast array of well-defined cellular and molecular tools and techniques, make it an ideal eukaryotic host for the identification of cellular factors required for positive-sense RNA virus replication. Furthermore, readily available yeast libraries with deletions and regulated expression of individual proteins have led to the completion of several high-throughput screens to provide a global survey of host factors that impact virus replication (26, 42, 52). An alternative approach with these yeast libraries that reduces the inherently high false-negative rates associated with high-throughput screens is to focus on a select set of host genes associated with a particular cellular pathway, process, or location previously implicated in virus replication.We have utilized such a targeted approach and focused on examining the impact of cytosolic chaperones on FHV RNA replication. Previously, we have shown that the cellular chaperone hsp90 facilitates protein A synthesis in Drosophila cells (5, 23), and the hsp40 encoded by the yeast YDJ1 gene facilitates FHV RNA replication in yeast, in part through effects on both protein A accumulation and function (60). In this report, we further extend these observations by examining FHV RNA accumulation in a panel of yeast strains with deletions of known or hypothesized cytosolic chaperones. We demonstrate that cytosolic chaperones can have either suppressive or enhancing effects on FHV RNA accumulation. In particular, related hsp70 members encoded by the SSA and SSB yeast chaperone families have marked and dramatically divergent effects on both genomic and subgenomic RNA accumulation and viral polymerase synthesis. These results highlight the complexities of the host-pathogen interactions that influence positive-sense RNA virus replication and identify the hsp70 family of cytosolic chaperones as key regulators of FHV replication.     

Mumps Virus Matrix,Fusion, and Nucleocapsid Proteins Cooperate for Efficient Production of Virus-Like Particles

Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells. To define mumps virus (MuV) proteins important for this process, viral proteins were expressed either singly or in combination in mammalian cells to produce virus-like particles (VLPs). Only the MuV matrix (M) protein when expressed by itself was capable of inducing particle release, but the quantity of these M-alone particles was very small. Efficient production of mumps VLPs occurred only when the M protein was coexpressed together with other viral proteins, with maximum production achieved upon coexpression of the viral M, nucleocapsid (NP), and fusion (F) proteins together. Electron microscopy analysis confirmed that VLPs were morphologically similar to MuV virions. The two MuV glycoproteins were not equal contributors to particle formation. The F protein was a major contributor to VLP production, while the hemagglutinin-neuraminidase protein made a smaller contribution. Evidence for the involvement of class E protein machinery in VLP budding was obtained, with mumps VLP production inhibited upon expression of dominant-negative versions of the class E proteins Vps4A and Chmp4b. Disruption of the sequence 24-FPVI-27 within the MuV M protein led to poor VLP production, consistent with findings of earlier studies of a related sequence, FPIV, important for the budding of parainfluenza virus 5. Together, these results demonstrate that different MuV structural proteins cooperate together for efficient particle production and that particle budding likely involves host class E protein machinery.Mumps virus (MuV) is a paramyxovirus from the Rubulavirus genus. Prior to mass vaccination, mumps was a very common childhood illness, with characteristic symptoms including fever, fatigue, and inflammation of the salivary glands. Less frequently, MuV infection results in serious complications including aseptic meningitis and encephalitis (22). Significant outbreaks of mumps have occurred recently in the United Kingdom (6), Canada (40), and the United States (7, 14), highlighting the continued relevance of this disease even in countries where vaccination is widespread. Like other paramyxoviruses, MuV possesses a genome that consists of single-stranded negative-sense RNA, encapsidated by a nucleocapsid (NP) protein and associated with an RNA-dependent RNA polymerase complex composed of large protein and phosphoprotein subunits. This core is linked to the virion membrane by matrix (M) protein. The outer surface of the virion is covered with glycoprotein spikes consisting of the hemagglutinin-neuraminidase (HN) protein, which binds sialic acid to allow virion attachment to cells, and fusion (F) protein, which induces viral and cellular membranes to fuse together during virus entry. Additional components of MuV include the small hydrophobic protein, which prevents infected cells from undergoing apoptosis (67), and V protein, which prevents induction of interferon-induced antiviral responses (29, 30, 62). The late steps of the MuV life cycle that allow for assembly and budding of MuV virions remain for the most part unexplored.Enveloped virus particles are formed by budding from cellular membranes at specific locations at which viral proteins, and often host factors, have assembled together. For the negative-strand RNA viruses, coordination among the different viral components during virus assembly appears to be directed by the viral matrix proteins, which have the potential to interact with the cytoplasmic tails of the viral glycoproteins and with viral ribonucleoproteins (RNPs) in the cytoplasms of infected cells. M proteins likely assemble as layers beneath the plasma membranes of infected cells and induce other viral components to gather at these locations, from which virus budding occurs (reviewed in references 49 and 57).For many viruses, it has been possible to achieve assembly and budding of particles from cells that have been transfected to produce one or more viral proteins in the absence of virus infection. These particles often resemble virions morphologically and have been termed virus-like particles (VLPs). VLP production provides a useful means for determining the individual roles of different virus proteins in particle formation, and in some cases the VLPs themselves have shown promise as vaccines (45). For most negative-strand RNA viruses, VLP formation is critically dependent on the presence of the viral matrix proteins (49). Indeed, in the cases of Newcastle disease virus (NDV) (37) and Nipah virus (11, 38), M protein expression is sufficient for highly efficient VLP production, with no apparent need for assistance from any of the other viral structural components, such as the viral glycoproteins or NP proteins. In the case of NDV, incorporation of glycoproteins and NP proteins into the budding VLPs requires specific interactions involving the M protein, but these interactions do not appear to facilitate the budding process itself (37).Although expression of viral matrix protein is sufficient for robust VLP production in the above cases, it has long been thought that additional viral components are also important for efficient budding of many negative-strand RNA viruses. For example, an important role for viral glycoproteins in virus assembly has been established based on studies with recombinant viruses that contain glycoproteins lacking their cytoplasmic tails (4, 17, 26, 34, 35, 48, 52, 66) and analyses of assembly-defective subacute sclerosing panencephalitis measles virus strains (5, 47). In fact, recent evidence suggests that for influenza virus it is the viral glycoproteins (and not viral matrix protein) that are the main drivers of virus budding (9). For other negative-strand RNA viruses, expression of viral glycoproteins together with matrix proteins in some cases significantly enhances the efficiency of VLP release. Ebola VLPs (31), Sendai VLPs (55, 56), and parainfluenza virus 5 (PIV5)-like particles (51) are all produced more efficiently in the presence of viral glycoprotein expression. Ebola virus glycoprotein in some cell types functions during virus release to inhibit the action of tetherin, a cellular protein which functions to prevent the release of enveloped virus particles from infected cells (28). In addition to the viral glycoproteins, other viral components can also enhance the production of VLPs. Production of Ebola VLPs and PIV5-like particles can be further enhanced through expression of the corresponding NP proteins (31, 51), and Sendai VLP production is enhanced through expression of Sendai virus C protein (55). Hence, for these viruses, multiple proteins cooperate with one another to achieve maximum VLP production. The extent to which particle formation actually requires this cooperation differs, however. In the case of PIV5, it is absolutely essential; expression of the M protein alone does not lead to VLP production (51). On the other hand, cooperation among viral proteins is beneficial but not strictly required for the production of Sendai or Ebola VLPs, since expression of the matrix proteins of these viruses is sufficient for VLP production (20, 55, 56, 61).The late steps of negative-strand RNA virus budding may occur in a way that is analogous to the budding of retroviruses, which employ protein-protein interaction domains called late domains to manipulate host machinery and allow release of virus particles (reviewed in references 1 and 3). Cellular factors recruited by late domains in many cases are class E proteins that are part of the vacuolar protein sorting (Vps) pathway of the cell. Indeed, disruption of the Vps pathway through expression of dominant-negative (DN) versions of the Vps4 ATPase protein blocks the budding of many retroviruses (reviewed in reference 1), as well as the budding of Ebola virus (32), Lassa fever virus (63), and PIV5 (50). However, other negative-strand RNA viruses, such as influenza virus, bud particles in ways that are not substantially affected by disruption of the cellular Vps pathway (reviewed in reference 8).Here, experiments are described which define MuV proteins important for the assembly and budding of VLPs. Using proteins derived from the 88-1961 wild-type (wt) strain of MuV, optimal production of mumps VLPs is shown to occur upon coexpression of the MuV M, F, and NP proteins together in transiently transfected mammalian cells. Evidence is also provided that supports a role for cellular class E protein machinery in the budding of mumps VLPs.     

Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, with approximately 170 million humans chronically infected. Persistent HCV infection often leads to fibrosis, cirrhosis, and hepatocellular carcinoma (27). There is no vaccine against HCV, and the most widely used therapy involves the administration of type I interferon (IFN-α2Α) combined with ribavirin. However, this treatment is often associated with severe adverse effects and is often ineffective (53).HCV is a member of the Flaviviridae family and is the sole member of the genus Hepacivirus (43). HCV is an enveloped virus with a single-strand positive RNA genome that encodes a unique polyprotein of ∼3,000 amino acids (14, 15). A single open reading frame is flanked by untranslated regions (UTRs), the 5′ UTR and 3′ UTR, that contain RNA sequences essential for RNA translation and replication, respectively (17, 18, 26). Translation of the single open reading frame is driven by an internal ribosomal entry site (IRES) sequence residing within the 5′ UTR (26). The resulting polyprotein is processed by cellular and viral proteases into its individual components (reviewed in reference 55). The E1, E2, and core structural proteins are required for particle formation (5, 6) but not for viral RNA replication or translation (7, 40). These processes are mediated by the nonstructural (NS) proteins NS3, NS4A, NS4B, NS5A, and NS5B, which constitute the minimal viral components necessary for efficient viral RNA replication (7, 40).Expression of the viral polyprotein leads to the formation of virus-like particles (VLPs) in HeLa (48) and Huh-7 cells (23). Furthermore, overexpression of core, E1, and E2 is sufficient for the formation of VLPs in insect cells (3, 4). In the context of a viral infection, the viral structural proteins (65), p7 (31, 49, 61), and all of the nonstructural proteins (2, 29, 32, 41, 44, 63, 67) are required for the production of infectious particles, independent of their role in HCV RNA replication. It is not known whether the nonstructural proteins are incorporated into infectious virions.The current model for HCV morphogenesis proposes that the core protein encapsidates the viral genome in areas where endoplasmic reticulum (ER) cisternae are in contact with lipid droplets (47), forming HCV RNA-containing particles that acquire the viral envelope by budding through the ER membrane (59). We along with others showed recently that infectious particle assembly requires microsomal transfer protein (MTP) activity and apolipoprotein B (apoB) (19, 28, 50), suggesting that these two components of the very-low-density lipoprotein (VLDL) biosynthetic machinery are essential for the formation of infectious HCV particles. This idea is supported by the reduced production of infectious HCV particles in cells that express short hairpin RNAs (shRNAs) targeting apolipoprotein E (apoE) (12, 30).HCV RNA displays various density profiles, depending on the stage of the infection at which the sample is obtained (11, 58). The differences in densities and infectivities have been attributed to the presence of host lipoproteins and antibodies bound to the circulating viral particles (24, 58). In patients, HCV immune complexes that have been purified by protein A affinity chromatography contain HCV RNA, core protein, triglycerides, apoB (1), and apoE (51), suggesting that these host factors are components of circulating HCV particles in vivo.Recent studies using infectious molecular clones showed that both host and viral factors can influence the density profile of infectious HCV particles. For example, the mean particle density is reduced by passage of cell culture-grown virus through chimpanzees and chimeric mice whose livers contain human hepatocytes (39). It has also been shown that a point mutation in the viral envelope protein E2 (G451R) increases the mean density and specific infectivity of JFH-1 mutants (70).HCV particles exist as a mixture of infectious and noninfectious particles in ratios ranging from 1:100 to 1:1,000, both in vivo (10) and in cell culture (38, 69). Extracellular infectious HCV particles have a lower average density than their noninfectious counterparts (20, 24, 38). Equilibrium sedimentation analysis indicates that particles with a buoyant density of ∼1.10 to 1.14 g/ml display the highest ratio of infectivity per genome equivalent (GE) both in cell culture (20, 21, 38) and in vivo (8). These results indicate that these samples contain relatively more infectious particles than any other particle population. Interestingly, mutant viruses bearing the G451R E2 mutation display an increased infectivity-HCV RNA ratio only in fractions with a density of ∼1.1 g/ml (21), reinforcing the notion that this population is selectively enriched in infectious particles.The size of infectious HCV particles has been estimated in vivo by filtration (50 to 80 nm) (9, 22) and by rate-zonal centrifugation (54 nm) (51) and in cell culture by calculation of the Stokes radius inferred from the sedimentation velocity of infectious JFH-1 particles (65 to 70 nm) (20). Previous ultrastructural studies using patient-derived material report particles with heterogeneous diameters ranging from 35 to 100 nm (33, 37, 42, 57, 64). Cell culture-derived particles appear to display a diameter within that range (∼55 nm) (65, 68).In this study we exploited the increased growth capacity of a cell culture-adapted virus bearing the G451R mutation in E2 (70) and the enhanced particle production of the hyperpermissive Huh-7 cell subclone Huh-7.5.1 clone 2 (Huh-7.5.1c2) (54) to produce quantities of infectious HCV particles that were sufficient for electron cryomicroscopy (cryoEM) analyses. These studies revealed two major particle populations with diameters of ∼60 and ∼45 nm. The larger-diameter particles were distinguished by the presence of a membrane bilayer, characterized by electron density attributed to the lipid headgroups in its leaflets. Isopycnic ultracentrifugation showed that the ∼60-nm particles are enriched in fractions with a density of ∼1.1 g/ml, where optimal infectivity-HCV RNA ratios are observed. These results indicate that the predominant morphology of the infectious HCV particle is spherical and pleomorphic and surrounded by a membrane envelope.     

Disulfide Bond Formation at the C Termini of Vaccinia Virus A26 and A27 Proteins Does Not Require Viral Redox Enzymes and Suppresses Glycosaminoglycan-Mediated Cell Fusion

Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L. Chiu, C. L. Lin, M. H. Yang, D. L. Tzou, and W. Chang, J. Virol 81:2149-2157, 2007). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfide-linked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.Vaccinia virus, the prototype of the Orthopoxvirus genus of the family Poxviridae, infects many cell lines and animals (13) and produces several forms of infectious particles, among which the intracellular mature virus (IMV) is the most abundant form inside cells. The IMV can be wrapped with additional Golgi membrane, transported through microtubules, and released from cells as extracellular enveloped viruses (10). The IMV has evolved to enter host cells through plasma membrane fusion (1, 3, 12, 29, 47) or endocytosis (11, 48). Recently, Mercer et al. reported that IMV entered HeLa cells through apoptotic mimicry and macropinocytosis (32), and Huang et al. reported that IMV enters into HeLa cells through a dynamin-dependent fluid-phase endocytosis that required the cellular protein VPEF (22).The IMV contains more than 75 viral proteins. Of these, more than 10 viral envelope proteins are known to be involved in vaccinia virus entry into cells (6, 34, 55). Vaccinia virus contains at least five attachment proteins, with H3, A27, and D8 binding to cell surface glycosaminoglycans (GAGs) (7, 21, 28), A26 protein binding to the extracellular matrix protein laminin (5), and L1 protein binding to unidentified cell surface molecules (14). A27 protein also binds to the viral A17 protein through its C-terminal region (35, 50), and it was recently shown that the coexpression of A17 and A27 proteins resulted in cell fusion in transiently transfected 293T cells (27). In this study, we demonstrate the formation through disulfide bonds of complexes between two viral attachment proteins, A26 and A27, and we determine the cysteine residues that are critical for these disulfide bonds. We also address the biological role of the A26-A27 protein complex formation in cell fusion regulation.