Generation of filamentous instead of icosahedral particles by repression of African swine fever virus structural protein pB438L

The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.     

African swine fever virus polyproteins pp220 and pp62 assemble into the core shell

African swine fever virus (ASFV), a complex enveloped DNA virus, expresses two polyprotein precursors, pp220 and pp62, which after proteolytic processing give rise to several major components of the virus particle. We have analyzed the structural role of polyprotein pp62, the precursor form of mature products p35 and p15, in virus morphogenesis. Densitometric analysis of one- and two-dimensional gels of purified virions showed that proteins p35 and p15, as well as the pp220-derived products, are present in equimolecular amounts in the virus particle. Immunoelectron microscopy revealed that the pp62-derived products localize at the core shell, a matrix-like domain placed between the DNA-containing nucleoid and the inner envelope, where the pp220-derived products are also localized. Pulse-chase experiments indicated that the processing of both polyprotein precursors is concomitant with virus assembly. Furthermore, using inducible ASFV recombinants, we show that pp62 processing requires the expression of the pp220 core precursor, whereas the processing of both precursors pp220 and pp62 is dependent on expression of the major capsid protein p72. Interestingly, when p72 expression is blocked, unprocessed pp220 and pp62 polyproteins assemble into aberrant zipper-like elements consisting of an elongated membrane-bound protein structure reminiscent of the core shell. Moreover, the two polyproteins, when coexpressed in COS cells, interact with each other to form zipper-like structures. Together, these findings indicate that the mature products derived from both polyproteins, which collectively account for about 30% of the virion protein mass, are the basic components of the core shell and that polyprotein processing represents a maturational process related to ASFV morphogenesis.     

The African Swine Fever Virus Virion Membrane Protein pE248R Is Required for Virus Infectivity and an Early Postentry Event

The African swine fever virus (ASFV) protein pE248R, encoded by the gene E248R, is a late structural component of the virus particle. The protein contains intramolecular disulfide bonds and has been previously identified as a substrate of the ASFV-encoded redox system. Its amino acid sequence contains a putative myristoylation site and a hydrophobic transmembrane region near its carboxy terminus. We show here that the protein pE248R is myristoylated during infection and associates with the membrane fraction in infected cells, behaving as an integral membrane protein. Furthermore, the protein localizes at the inner envelope of the virus particles in the cytoplasmic factories. The function of the protein pE248R in ASFV replication was investigated by using a recombinant virus that inducibly expresses the gene E248R. Under repressive conditions, the ASFV polyproteins pp220 and pp62 are normally processed and virus particles with morphology indistinguishable from that of those produced in a wild-type infection or under permissive conditions are generated. Moreover, the mutant virus particles can exit the cell as does the parental virus. However, the infectivity of the pE248R-deficient virions was reduced at least 100-fold. An investigation of the defect of the mutant virus indicated that neither virus binding nor internalization was affected by the absence of the protein pE248R, but a cytopathic effect was not induced and early and late gene expression was impaired, indicating that the protein is required for some early postentry event.African swine fever virus (ASFV) is a large enveloped deoxyvirus that causes a severe hemorrhagic disease in domestic pigs (38). The ASFV genome is a double-stranded DNA molecule of 170 to 190 kbp that encodes more than 150 polypeptides (47). The icosahedral virus particle contains more than 50 polypeptides and is composed of several concentric domains, including an internal DNA-containing nucleoid surrounded by a protein layer designated the core shell, an inner envelope, and an outer icosahedral capsid (8, 10, 20). An additional membrane acquired by budding through the plasma membrane envelops the extracellular virion (14).The complex process of virus assembly occurs at specialized cytoplasmic sites, designated viral factories, and is initiated by the recruitment and modification of endoplasmic reticulum (ER) cisternae, which collapse to form the virus inner envelope, where the viral membrane proteins p54 and p17 are localized (8, 16, 21, 32, 37). This model, however, has been recently questioned, and based on data obtained using samples prepared by high-pressure freezing, it has been suggested that the inner envelope of ASFV consists of a single lipid bilayer (28). The icosahedral capsid layer, formed by protein p72, is then progressively assembled on one side of this envelope, while on the other side, the core shell domain, mainly constituted by the processing products of the polyproteins pp220 and pp62, is simultaneously constructed (6, 7, 20, 26). Finally, the viral DNA and nucleoproteins are packaged and condensed to form the nucleoid (15).The functions of several virus proteins in the formation of the different domains of the virus particle have been investigated in recent years. Thus, the structural proteins p72 and pB438L and the nonstructural pB602L protein, described as a chaperone of p72 (22), have been shown to be required for the construction of the icosahedral capsid (24, 25, 26), while the polyprotein pp220 is essential for the formation of the inner core, constituted by the nucleoid and core shell domains (7). It has also been demonstrated that the processing of the polyproteins pp220 and pp62 by the virus-encoded protease is necessary for the assembly of a proper core (5). In addition, it is known that the transmembrane protein p54 is critical for the recruitment of envelope precursors to assembly sites (35), although the mechanisms underlying the conversion of ER cisternae into functional viral envelopes are mostly unknown. Studies of other transmembrane proteins detected as structural components of the virus particle could shed light on this matter. Some of the virion membrane proteins could also play a role in virus entry, as has been described for the proteins p12, identified as a viral attachment protein (11, 19), and p54, also involved in binding of virus to target cells (27).The ASFV protein pE248R is a late structural component of the virus particle (33) that belongs to a class of myristoylated membrane proteins related to vaccinia virus L1 (30), one of the substrates of the pathway for the formation of disulfide bonds encoded by this virus (41). The protein pE248R also contains intramolecular disulfide bridges and has been recently identified as a possible final substrate of the ASFV-encoded redox system (33). In the present study, we investigated the membrane association, the localization in the virion, and the role of the protein pE248R in ASFV replication. Our results indicate that pE248R is a myristoylated integral membrane protein localized at the inner envelope of the virus particle. By using a conditional lethal ASFV mutant, vE248Ri, with an inducible copy of the gene E248R, we showed that the protein pE248R is required for virus infectivity and an early postentry event but not virus assembly.     

African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity

This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lac repressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.     

African swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites

The assembly of African swine fever virus (ASFV) at the cytoplasmic virus factories commences with the formation of precursor membranous structures, which are thought to be collapsed cisternal domains recruited from the surrounding endoplasmic reticulum (ER). This report analyzes the role in virus morphogenesis of the structural protein p54, a 25-kDa polypeptide encoded by the E183L gene that contains a putative transmembrane domain and localizes at the ER-derived envelope precursors. We show that protein p54 behaves in vitro and in infected cells as a type I membrane-anchored protein that forms disulfide-linked homodimers through its unique luminal cysteine. Moreover, p54 is targeted to the ER membranes when it is transiently expressed in transfected cells. Using a lethal conditional recombinant, vE183Li, we also demonstrate that the repression of p54 synthesis arrests virus morphogenesis at a very early stage, even prior to the formation of the precursor membranes. Under restrictive conditions, the virus factories appeared as discrete electron-lucent areas essentially free of viral structures. In contrast, outside the assembly sites, large amounts of aberrant zipper-like structures formed by the unprocessed core polyproteins pp220 and pp62 were produced in close association to ER cisternae. Altogether, these results indicate that the transmembrane structural protein p54 is critical for the recruitment and transformation of the ER membranes into the precursors of the viral envelope.     

African Swine Fever virus proteinase is essential for core maturation and infectivity

African swine fever virus (ASFV) encodes two polyprotein precursors named pp220 and pp62 that are sequentially processed during viral infection, giving rise to six major structural proteins. These reside at the core shell, a matrix domain located between the endoplasmic reticulum-derived inner envelope and the DNA-containing nucleoid. Proteolytic processing of the polyprotein precursors is catalyzed by the viral proteinase pS273R, a cysteine proteinase that shares sequence similarity with the SUMO1-processing peptidases. We describe here the construction and characterization of an ASFV recombinant, vS273Ri, that inducibly expresses the ASFV proteinase. Using vS273Ri, we show that repression of proteinase expression inhibits polyprotein processing and strongly impairs infective virus production. Electron microscopic examination of vS273Ri-infected cells showed that inhibition of proteolytic processing leads to the assembly of defective icosahedral particles containing a noncentered electron-dense nucleoid surrounded by an abnormal core shell of irregular thickness. The analysis of purified extracellular defective particles revealed that they contain the unprocessed pp220 and pp62 precursors, as well as the major DNA-binding nucleoid proteins p10 and pA104R. Altogether, these results indicate that the proteolytic processing of the polyproteins is not required for their incorporation into the assembling particles nor for the incorporation of the DNA-containing nucleoid. Instead, the ASFV proteinase is involved in a late maturational step that is essential for proper core assembly and infectivity.     

African Swine Fever Virus Is Enveloped by a Two-Membraned Collapsed Cisterna Derived from the Endoplasmic Reticulum

During the cytoplasmic maturation of African swine fever virus (ASFV) within the viral factories, the DNA-containing core becomes wrapped by two shells, an inner lipid envelope and an outer icosahedral capsid. We have previously shown that the inner envelope is derived from precursor membrane-like structures on which the capsid layer is progressively assembled. In the present work, we analyzed the origin of these viral membranes and the mechanism of envelopment of ASFV. Electron microscopy studies on permeabilized infected cells revealed the presence of two tightly apposed membranes within the precursor membranous structures as well as polyhedral assembling particles. Both membranes could be detached after digestion of intracellular virions with proteinase K. Importantly, membrane loop structures were observed at the ends of open intermediates, which suggests that the inner envelope is derived from a membrane cisterna. Ultraestructural and immunocytochemical analyses showed a close association and even direct continuities between the endoplasmic reticulum (ER) and assembling virus particles at the bordering areas of the viral factories. Such interactions become evident with an ASFV recombinant that inducibly expresses the major capsid protein p72. In the absence of the inducer, viral morphogenesis was arrested at a stage at which partially and fully collapsed ER cisternae enwrapped the core material. Together, these results indicate that ASFV, like the poxviruses, becomes engulfed by a two-membraned collapsed cisterna derived from the ER.     

African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.     

Repression of African swine fever virus polyprotein pp220-encoding gene leads to the assembly of icosahedral core-less particles

African swine fever virus (ASFV) polyprotein pp220, encoded by the CP2475L gene, is an N-myristoylated precursor polypeptide that, after proteolytic processing, gives rise to the major structural proteins p150, p37, p34, and p14. These proteins localize at the core shell, a matrix-like virus domain placed between the DNA-containing nucleoid and the inner envelope. In this study, we have examined the role of polyprotein pp220 in virus morphogenesis by means of an ASFV recombinant, v220i, containing an inducible copy of the CP2475L gene regulated by the Escherichia coli repressor-operator system. Under conditions that repress pp220 expression, the virus yield of v220i was about 2.6 log units lower than that of the parental virus or of the recombinant grown under permissive conditions. Electron microscopy revealed that pp220 repression leads to the assembly of icosahedral particles virtually devoid of the core structure. Analysis of recombinant v220i by immunoelectron microscopy, immunoblotting, and DNA hybridization showed that mutant particles essentially lack, besides the pp220-derived products, a number of major core proteins as well as the viral DNA. On the other hand, transient expression of the CP2475L gene in COS cells showed that polyprotein pp220 assembles into electron-dense membrane-bound coats, whereas a mutant nonmyristoylated version of pp220 does not associate with cellular membranes but forms large cytoplasmic aggregates. Together, these findings indicate that polyprotein pp220 is essential for the core assembly and suggest that its myristoyl moiety may function as a membrane-anchoring signal to bind the developing core shell to the inner viral envelope.     

African swine fever virus protease, a new viral member of the SUMO-1-specific protease family

African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.     

Assembly of African swine fever virus: role of polyprotein pp220.

Polyprotein processing is a common strategy of gene expression in many positive-strand RNA viruses and retroviruses but not in DNA viruses. African swine fever virus (ASFV) is an exception because it encodes a polyprotein, named pp220, to produce several major components of the virus particle, proteins p150, p37, p34, and p14. In this study, we analyzed the assembly pathway of ASFV and the contribution of the polyprotein products to the virus structure. Electron microscopic studies revealed that virions assemble from membranous structures present in the viral factories. Viral membranes became polyhedral immature virions after capsid formation on their convex surface. Beneath the lipid envelope, two distinct domains appeared to assemble consecutively: first a thick protein layer that we refer to as core shell and then an electron-dense nucleoid, which was identified as the DNA-containing domain. Immunofluorescence studies showed that polyprotein pp220 is localized in the viral factories. At the electron microscopic level, antibodies to pp220 labeled all identifiable forms of the virus from the precursor viral membranes onward, thus indicating an early role of the polyprotein pp220 in ASFV assembly. The subviral localization of the polyprotein products, examined on purified virions, was found to be the core shell. In addition, quantitative studies showed that the polyprotein products are present in equimolar amounts in the virus particle and account for about one-fourth of its total protein content. Taken together, these results suggest that polyprotein pp220 may function as an internal protein scaffold which would mediate the interaction between the nucleoid and the outer layers similarly to the matrix proteins of other viruses.     

The Major Structural Protein of African Swine Fever Virus, p73, Is Packaged into Large Structures, Indicative of Viral Capsid or Matrix Precursors, on the Endoplasmic Reticulum

African swine fever virus (ASFV) is a large enveloped DNA virus that shares the striking icosahedral symmetry of iridoviruses. To understand the mechanism of assembly of ASFV, we have been studying the biosynthesis and subcellular distribution of p73, the major structural protein of ASFV. Sucrose density sedimentation of lysates prepared from infected cells showed that newly synthesized p73 was incorporated into a complex with a size of 150 to 250 kDa. p73 synthesized by in vitro translation migrated at 70 kDa, suggesting that cellular and/or viral proteins are required for the formation of the 150- to 250-kDa complex. During a 2-h chase, approximately 50% of the newly synthesized pool of p73 bound to the endoplasmic reticulum (ER). During this period, the membrane-bound pool of p73, but not the cytosolic pool, formed large complexes of approximately 50,000 kDa. The complexes were formed via assembly intermediates, and the entire membrane-associated pool of p73 was incorporated into the 50,000-kDa complex within 2 h. The 50,000-kDa complexes containing p73 were also detected in virions secreted from cells. Immunoprecipitation of sucrose gradients with sera taken from hyperimmune pigs suggested that p73 was the major component of the 50,000-kDa complex. It is possible, therefore, that the complex contains between 600 and 700 copies of p73. The kinetics of complex formation and envelopment of p73 were similar, and complex formation and envelopment were both reversibly inhibited by cycloheximide, suggesting a functional link between complex assembly and ASFV envelopment. A protease protection assay detected 50,000-kDa complexes on the inside and outside of the membranes forming the viral envelope. The identification of a complex containing p73 beneath the envelope of ASFV suggests that p73 may be a component of the inner core shell or matrix of ASFV. The outer pool may represent p73 within the outer capsid layer of the virus. In summary, the data suggest that the assembly of the inner core matrix and outer capsid of ASFV takes place on the ER membrane during envelopment and that these structures are not preassembled in the cytosol.     

Rubella virus capsid associates with host cell protein p32 and localizes to mitochondria

Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.     

African swine fever virus protein pE296R is a DNA repair apurinic/apyrimidinic endonuclease required for virus growth in swine macrophages

We show here that the African swine fever virus (ASFV) protein pE296R, predicted to be a class II apurinic/apyrimidinic (AP) endonuclease, possesses endonucleolytic activity specific for AP sites. Biochemical characterization of the purified recombinant enzyme indicated that the K(m) and catalytic efficiency values for the endonucleolytic reaction are in the range of those reported for Escherichia coli endonuclease IV (endo IV) and human Ape1. In addition to endonuclease activity, the ASFV enzyme has a proofreading 3'-->5' exonuclease activity that is considerably more efficient in the elimination of a mismatch than in that of a correctly paired base. The three-dimensional structure predicted for the pE296R protein underscores the structural similarities between endo IV and the viral protein, supporting a common mechanism for the cleavage reaction. During infection, the protein is expressed at early times and accumulates at later times. The early enzyme is localized in the nucleus and the cytoplasm, while the late protein is found only in the cytoplasm. ASFV carries two other proteins, DNA polymerase X and ligase, that, together with the viral AP endonuclease, could act as a viral base excision repair system to protect the virus genome in the highly oxidative environment of the swine macrophage, the virus host cell. Using an ASFV deletion mutant lacking the E296R gene, we have determined that the viral endonuclease is required for virus growth in macrophages but not in Vero cells. This finding supports the existence of a viral reparative system to maintain virus viability in the infected macrophage.     

Polymorphism in the assembly of polyomavirus capsid protein VP1.

Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid.     

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.     

Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed.     

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.     

Dimer Interface Migration in a Viral Sulfhydryl Oxidase

Large double-stranded DNA viruses, including poxviruses and mimiviruses, encode enzymes to catalyze the formation of disulfide bonds in viral proteins produced in the cell cytosol, an atypical location for oxidative protein folding. These viral disulfide catalysts belong to a family of sulfhydryl oxidases that are dimers of a small five-helix fold containing a Cys-X-X-Cys motif juxtaposed to a flavin adenine dinucleotide cofactor. We report that the sulfhydryl oxidase pB119L from African swine fever virus (ASFV) uses for self-assembly surface different from that observed in homologs from mammals, plants, and fungi. Within a protein family, different packing interfaces for the same oligomerization state are extremely rare. We find that the alternate dimerization mode seen in ASFV pB119L is not characteristic of all viral sulfhydryl oxidases, as the flavin-binding domain from a mimivirus sulfhydryl oxidase assumes the same dimer structure as the known eukaryotic enzymes. ASFV pB119L demonstrates the potential of large double-stranded DNA viruses, which have faster mutation rates than their hosts and the tendency to incorporate host genes, to pioneer new protein folds and self-assembly modes.