Douglas P. Gladue

Foot-and-Mouth Disease Virus Nonstructural Protein 2C Interacts with Beclin1, Modulating Virus Replication

ABSTRACT Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the process of virus replication, we used a yeast two-hybrid approach to identify host proteins that interact with 2C. We report here that cellular Beclin1 is a specific host binding partner for 2C. Beclin1 is a regulator of the autophagy pathway, a metabolic pathway required for efficient FMDV replication. The 2C-Beclin1 interaction was further confirmed by coimmunoprecipitation and confocal microscopy to actually occur in FMDV-infected cells. Overexpression of either Beclin1 or Bcl-2, another important autophagy factor, strongly affects virus yield in cell culture. The fusion of lysosomes to autophagosomes containing viral proteins is not seen during FMDV infection, a process that is stimulated by Beclin1; however, in FMDV-infected cells overexpressing Beclin1 this fusion occurs, suggesting that 2C would bind to Beclin1 to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival. Using reverse genetics, we demonstrate here that modifications to the amino acids in 2C that are critical for interaction with Beclin1 are also critical for virus growth. These results suggest that interaction between FMDV 2C and host protein Beclin1 could be essential for virus replication.

Classical Swine Fever Virus p7 Protein Is a Viroporin Involved in Virulence in Swine

ABSTRACT The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.

A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle.

The role of non-structural protein 3A of foot-and-mouth disease virus (FMDV) on the virulence in cattle has received significant attention. Particularly, a characteristic 10-20 amino acid deletion has been implicated as responsible for virus attenuation in cattle: a 10 amino acid deletion in the naturally occurring, porcinophilic FMDV O1 Taiwanese strain, and an approximately 20 amino acid deletion found in egg-adapted derivatives of FMDV serotypes O1 and C3. Previous reports using chimeric viruses linked the presence of these deletions to an attenuated phenotype in cattle although results were not conclusive. We report here the construction of a FMDV O1Campos variant differing exclusively from the highly virulent parental virus in a 20 amino acid deletion between 3A residues 87-106, and its characterization in vitro and in vivo. We describe a direct link between a deletion in the FMDV 3A protein and disease attenuation in cattle.

African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus

ABSTRACT African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 102 or 104 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. IMPORTANCE The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-ΔMGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-ΔMGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G.

Interaction of Foot-and-Mouth Disease Virus Nonstructural Protein 3A with Host Protein DCTN3 Is Important for Viral Virulence in Cattle

ABSTRACT Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular protein DCTN3 as a specific host binding partner for 3A. DCTN3 is a subunit of the dynactin complex, a cofactor for dynein, a motor protein. The dynactin-dynein duplex has been implicated in several subcellular functions involving intracellular organelle transport. The 3A-DCTN3 interaction identified by the yeast two-hybrid approach was further confirmed in mammalian cells. Overexpression of DCTN3 or proteins known to disrupt dynein, p150/Glued and 50/dynamitin, resulted in decreased FMDV replication in infected cells. We mapped the critical amino acid residues in the 3A protein that mediate the protein interaction with DCTN3 by mutational analysis and, based on that information, we developed a mutant harboring the same mutations in O1 Campos FMDV (O1C3A-PLDGv). Although O1C3A-PLDGv FMDV and its parental virus (O1Cv) grew equally well in LFBK-αvβ6, O1C3A-PLDGv virus exhibited a decreased ability to replicate in primary bovine cell cultures. Importantly, O1C3A-PLDGv virus exhibited a delayed disease in cattle compared to the virulent parental O1Campus (O1Cv). Virus isolated from lesions of animals inoculated with O1C3A-PLDGv virus contained amino acid substitutions in the area of 3A mediating binding to DCTN3. Importantly, 3A protein harboring similar amino acid substitutions regained interaction with DCTN3, supporting the hypothesis that DCTN3 interaction likely contributes to virulence in cattle. IMPORTANCE The objective of this study was to understand the possible role of a FMD virus protein 3A, in causing disease in cattle. We have found that the cellular protein, DCTN3, is a specific binding partner for 3A. It was shown that manipulation of DCTN3 has a profound effect in virus replication. We developed a FMDV mutant virus that could not bind DCTN3. This mutant virus exhibited a delayed disease in cattle compared to the parental strain highlighting the role of the 3A-DCTN3 interaction in virulence in cattle. Interestingly, virus isolated from lesions of animals inoculated with mutant virus contained mutations in the area of 3A that allowed binding to DCTN3. This highlights the importance of the 3A-DCTN3 interaction in FMD virus virulence and provides possible mechanisms of virus attenuation for the development of improved FMD vaccines.

Interaction between Core protein of classical swine fever virus with cellular IQGAP1 protein appears essential for virulence in swine

Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified residues within CSFV Core protein (designated as areas I, II, III and IV) that maintain homology to regions within the matrix protein of Moloney Murine Leukemia Virus (MMLV) that mediate binding to IQGAP1 [EMBO J, 2006 25:2155]. Alanine-substitution within Core regions I, II, III and IV identified residues that specifically mediate the Core-IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues (207)ATI(209) (I), (210)VVE(212) (II), (213)GVK(215) (III), or (232)GLYHN(236) (IV) have defective growth in primary swine macrophage cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in swine. These data shows that the interaction of Core with an integral component of cytoskeletal regulation plays a role in the CSFV cycle.

Patterns of gene expression in swine macrophages infected with classical swine fever virus detected by microarray

Infection of domestic swine with highly virulent, classical swine fever virus (CSFV) strain Brescia, causes lethal disease in all infected animals. However, the molecular mechanisms involved in modulating the host cellular processes and evasion of the immune response have not been clearly established. To gain insight into, the early host response to CSFV, we analyzed the pattern of gene expression in infected swine macrophages, using custom designed swine microarrays. Macrophages, the target cell for CSFV infection, were isolated from primary cultures of peripheral blood mononuclear cells, allowing us to utilize identical uninfected macrophages at the same time points as CSFV-infected macrophages, allowing only genes induced by CSFV to be identified. First, microarray probes were optimized by screening 244,000 probes for hybridization with RNA from infected and uninfected macrophages. Probes that hybridized and passed quality control standards were used to design a 44,000 probe microarray for this study. Changes in expression levels of 79 genes (48 up- and 31 down-regulated) during the first 48h post-infection were observed. As expected many of the genes with an altered pattern of expression are involved in the development of an innate immune response. Several of these genes had differential expression in an attenuated strain NS4B.VGIv, suggesting that some of these differences are responsible for virulence. The observed gene expression profile might help to explain the immunological and pathological changes associated with infection of pigs with CSFV Brescia.

African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge

ABSTRACT African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G), has caused an epizootic that spread rapidly into Eastern European countries. Currently no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine, inducing protective immunity against challenge with homologous parental viruses. Deletion of the gene 9GL (open reading frame [ORF] B119L) in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at 106 50% hemadsorption doses (HAD50). Here we report the construction of a genetically modified ASFV-G strain (ASFV-G-Δ9GLv) harboring a deletion of the 9GL (B119L) gene. Like Malawi-Lil-20/1-Δ9GL, ASFV-G-Δ9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 104 HAD50 of ASFV-G-Δ9GL produced a virulent phenotype that, unlike Malawi-Lil-20/1-Δ9GL, induced a lethal disease in swine like parental ASFV-G. Interestingly, lower doses (102 to 103 HAD50) of ASFV-G-Δ9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 102 HAD50 of ASFV-G-Δ9GLv conferred partial protection when pigs were challenged at either 21 or 28 days postinfection (dpi). An ASFV-G-Δ9GL HAD50 of 103 conferred partial and complete protection at 21 and 28 dpi, respectively. The information provided here adds to our recent report on the first attempts toward experimental vaccines against ASFV-G. IMPORTANCE The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce protection in pigs but only against homologous virus challenges. Here we produced a recombinant ASFV lacking virulence-associated gene 9GL in an attempt to produce a vaccine against virulent ASFV-G, a highly virulent virus isolate detected in the Caucasus region in 2007 and now spreading though the Caucasus region and Eastern Europe. Deletion of 9GL, unlike with other ASFV isolates, did not attenuate completely ASFV-G. However, when delivered once at low dosages, recombinant ASFV-G-Δ9GL induces protection in swine against parental ASFV-G. The protection against ASFV-G is highly effective after 28 days postvaccination, whereas at 21 days postvaccination, animals survived the lethal challenge but showed signs of ASF. Here we report the design and development of an experimental vaccine that induces protection against virulent ASFV-G.

Effects of the interactions of classical swine fever virus Core protein with proteins of the SUMOylation pathway on virulence in swine.

Here we have identified host cell proteins involved with the cellular SUMOylation pathway, SUMO-1 (small ubiquitin-like modifier) and UBC9, a SUMO-1 conjugating enzyme that interact with classical swine fever virus (CSFV) Core protein. Five highly conserved lysine residues (K179, K180, K220, K221, and K246) within the CSFV Core were identified as putative SUMOylation sites. Analysis of these interactions showed that K179A, K180A, and K221A substitutions disrupt Core-SUMO-1 binding, while K220A substitution precludes Core-UBC9 binding. In vivo, Core mutant viruses (K179A, K180A, K220A, K221A) and (K220A, K221A) harboring those substitutions were attenuated in swine. These data shows a clear correlation between the disruption of Core protein binding to SUMO-1 and UBC9 and CSFV attenuation. Overall, these data suggest that the interaction of Core with the cellular SUMOylation pathway plays a significant role in the CSFV growth cycle in vivo.

The progressive adaptation of a Georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome.

ABSTRACT African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed. IMPORTANCE The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection.