A lipid modified ubiquitin is packaged into particles of several enveloped viruses.
An anti-ubiquitin cross-reactive protein which migrates more slowly (6.5 kDa) by SDS-PAGE than ubiquitin was identified in African swine fever virus particles. This protein was extracted into the detergent phase in Triton X-114 phase separations, showing that it is hydrophobic, and was radiolabelled with both [3H]palmitic acid and [32P]orthophosphate. This indicates that the protein has a similar structure to the membrane associated phosphatidyl ubiquitin described in baculovirus particles. A similar molecule was found in vaccinia virus and herpes simplex virus particles, suggesting that it may be a component of uninfected cell membranes, which is incorporated into membrane layers in virions during morphogenesis. (+info)
Nuclear and nucleolar localization of an African swine fever virus protein, I14L, that is similar to the herpes simplex virus-encoded virulence factor ICP34.5.
PCR analysis of the genomes of 18 different African swine fever virus (ASFV) isolates showed that the I14L open reading frame (ORF) was present as either a long form or short form in all of the isolates. Sequencing of the ORF from eight isolates confirmed that both forms of the ORF were well conserved. Antisera raised against the I14L protein identified the long form of the protein as a 21 kDa protein expressed late during ASFV infection. Immunofluorescent analysis of transiently expressed haemagglutinin-tagged forms of the I14L protein showed that the long form of the protein localized predominantly to the nucleus and within the nucleoli. In contrast, although the short form of the protein was also present predominantly in the nucleus, it did not localize to the nucleoli. Deletion of the N-terminal 14 amino acids from the long form of the I14L protein, which includes a high proportion of basic Arg/Lys residues, abolished the specific nucleolar localization of the protein, although the protein was still present in the nucleus. Addition of this 14 amino acid sequence to beta-galactosidase or replacement of the N-terminal 14 amino acids of the I14L short form with those from the long form directed both of these modified proteins to the nucleolus. This indicates that this 14 amino acid sequence contains all the signals required for nucleolar localization. (+info)
Modulation of monocytic cell activity and virus susceptibility during differentiation into macrophages.
A major component of innate immune responses relies on monocytes and macrophages, virus infection of which will pose a particular problem for immunological defense. Consequently, the monocytic cell differentiation pathway was analyzed in terms of cellular modulations therein and their relation to monocytotropic virus infection. Differentiation was characterized by down-regulation of CD14, MHC Ags, the monocytic SWC1 marker, and p53; concomitant up-regulation of the SWC9 macrophage marker, a putative porcine CD80 (detected with anti-human CD80 Ab), and acid phosphatase secretion were also characteristic. Elevated phagocytic and endocytic activities as well as endosomal/lysosomal acidification were identified as being important to the macrophage. In contrast, monocytes possessed high accessory activity. This was multifactorial, concomitantly requiring 1) high MHC Ag expression; 2) enzyme activity of esterase, peroxidase, myeloperoxidase, and 5' nucleotidase in preference to glucosidase, galactosidase, and glucuronidase; and 3) elevated capacity for spontaneous IL-1 production. Only with all parameters was efficient stimulation of Ag-specific lymphocytes possible. These results point to a continuous process during differentiation, involving inter-related characteristics linking the more accessory monocyte to the scavenger macrophage, both in vitro and in vivo. Of particular interest was how these characteristics related to monocytotropic virus infection, and how a particular virus could show a clear preference for the differentiating macrophages. Such results not only further our understanding of porcine immunology, but also provide evidence and a potential model for the determination and characterization of monocytotropic virus-host cell interactions. (+info)
The biological effects induced in mice by p36, a proteinaceous factor of virulence produced by African swine fever virus, are mediated by interleukin-4 and also to a lesser extent by interleukin-10.
We have previously presented indirect evidence that both specific immunosuppression and lymphocyte mitogenicity induced in mice by p36, a proteinaceous factor of virulence produced by porcine monocytes infected by African swine fever virus, were consistent with a Th2-driven response. Here we show: (1) Interleukin-4 (IL-4) and interleukin-10 (IL-10) mRNA expression in the spleen and thymus of C57BL/6 mice were displayed early after p36 inoculation. The expression of thymic IL-10 mRNA occurred, however, later than that of IL-4 mRNA. (2) Increased serum levels of these two cytokines were also soon detected after the protein inoculation. (3) Both immunosuppressive and mitogenic effects of p36 were absent in IL-4 gene-targeted mice and partially abrogated in mice depleted of IL-4 by neutralizing monoclonal antibodies. (4) IL-10 depletion abrogated the immunosuppressive but not the p36 lymphocyte mitogenic biological effects. (5) The increase in the serum concentrations of both IL-4 and IL-10 were lower in thymectomized than in non-thymectomized mice. (6) The expression of interferon-gamma (IFN-gamma) mRNA was weakly or not at all induced in p36-treated mice. Taken together, these results are in agreement with the promotion of a Th2 immune response induced by p36. (+info)
Replication of African swine fever virus DNA in infected cells.
We have examined the ultrastructural localization of African swine fever virus DNA in thin-sections of infected cells by in situ hybridization and autoradiography. Virus-specific DNA sequences were found in the nucleus of infected Vero cells at early times in the synthesis of the viral DNA, forming dense foci localized in proximity to the nuclear membrane. At later times, the viral DNA was found exclusively in the cytoplasm. Electron microscopic autoradiography of African swine fever virus-infected macrophages showed that the nucleus is also a site of viral DNA replication at early times. These results provide further evidence of the existence of nuclear and cytoplasmic stages in the synthesis of African swine fever virus DNA. On the other hand, alkaline sucrose sedimentation analysis of the replicative intermediates synthesized in the nucleus and cytoplasm of infected macrophages showed that small DNA fragments ( approximately 6-12S) were synthesized in the nucleus at an early time, whereas at later times, larger fragments of approximately 37-49S were labeled in the cytoplasm. Pulse-chase experiments demonstrated that these fragments are precursors of the mature cross-linked viral DNA. The formation of dimeric concatemers, which are predominantly head-to-head linked, was observed by pulsed-field electrophoresis and restriction enzyme analysis at intermediate and late times in the replication of African swine fever virus DNA. Our findings suggest that the replication of African swine fever virus DNA proceeds by a de novo start mechanism with the synthesis of small DNA fragments, which are then converted into larger size molecules. Ligation or further elongation of these molecules would originate a two-unit concatemer with dimeric ends that could be resolved to generate the genomic DNA by site-specific nicking, rearrangement, and ligation as has been proposed in the de novo start model of Baroudy et al. (B. M. Baroudy, S. Venkatesam, and B. Moss, 1982, Cold Spring Harbor Symp. Quant. Biol. 47, 723-729) for the replication of vaccinia virus DNA. (+info)
The African swine fever virus prenyltransferase is an integral membrane trans-geranylgeranyl-diphosphate synthase.
In a previous study, it was shown that the protein encoded by the gene B318L of African swine fever virus (ASFV) is a trans-prenyltransferase that catalyzes in vitro the condensation of farnesyl diphosphate and isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and longer chain prenyl diphosphates (Alejo, A., Yanez, R. J., Rodriguez, J. M., Vinuela, E., and Salas, M. L. (1997) J. Biol. Chem. 272, 9417-9423). To investigate the in vivo function of the viral enzyme, we have determined, in this work, its subcellular localization and activity in cell extracts. Two systems were used in these studies: cells infected with ASFV and cells infected with a recombinant pseudo-Sindbis virus carrying the complete B318L gene. In this latter system, the trans-prenyltransferase was found to colocalize with the endoplasmic reticulum marker protein-disulfide isomerase, whereas in cells infected with ASFV, the viral enzyme was present in cytoplasmic viral assembly sites, associated with precursor viral membranes derived from the endoplasmic reticulum. In addition, after subcellular fractionation, the viral enzyme partitioned into the membrane fraction. Extraction of membrane proteins with alkaline carbonate and Triton X-114 indicated that the ASFV enzyme behaved as an integral membrane protein. The membrane enzyme synthesized predominantly all-trans-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate. These results indicate that the viral B318L protein is a trans-geranylgeranyl-diphosphate synthase, being the only enzyme of this type that is known to have a membrane localization. (+info)
African swine fever virus: a B cell-mitogenic virus in vivo and in vitro.
The two major characteristics of pathogenesis in African swine fever virus (ASFV) infections of domestic pigs are massive B-cell apoptosis and haemorrhage. The effects of ASFV on porcine B cells have therefore been systematically examined in vivo, by using virus-infected pigs and SCID-Beige mice reconstituted with porcine bone marrow, and in vitro, by using porcine B-cell lines and B cells from normal and ASFV-infected pigs. Secretion of porcine Ig was stimulated by ASFV both in vivo and in bone marrow cultures in vitro, with the virulent Malawi isolate of ASFV being the most effective. Stimulation of Ig secretion in vitro depended on the presence of ASFV-infected macrophages and did not occur with supernatants from ASFV-infected macrophages. Although the virus alone did not stimulate proliferation of purified B cells in vitro, it was co-stimulatory with CD154 (CD40 ligand). The B cells recovered from ASFV-infected porcine lymphoid tissue were of activated surface marker phenotypes and, interestingly, expressed diminished levels of the B-cell co-stimulatory surface molecule CD21. In addition, they were highly sensitive to IL-4 and CD154. These results may be integrated into a model of pathogenesis in which those B cells activated indirectly as a result of virulent ASFV infection of macrophages are not rescued from apoptosis through interaction with CD154, due to the drastic depletion of T cells that occurs early in infection. The consequently diminished specific anti-ASFV antibody response would favour survival of the virus, with the non-specific hypergammaglobulinaemia being perhaps another example of pathogen-mediated immune deviation. (+info)
African swine fever virus replication in the midgut epithelium is required for infection of Ornithodoros ticks.
Although the Malawi Lil20/1 (MAL) strain of African swine fever virus (ASFV) was isolated from Ornithodoros sp. ticks, our attempts to experimentally infect ticks by feeding them this strain failed. Ten different collections of Ornithodorus porcinus porcinus ticks and one collection of O. porcinus domesticus ticks were orally exposed to a high titer of MAL. At 3 weeks postinoculation (p.i.), <25% of the ticks contained detectable virus, with viral titers of <4 log(10) 50% hemadsorbing doses/ml. Viral titers declined to undetectability in >90% of the ticks by 5 weeks p.i. To further study the growth defect, O. porcinus porcinus ticks were orally exposed to MAL and assayed at regular intervals p.i. Whole-tick viral titers dramatically declined (>1,000-fold) between 2 and 6 days p.i., and by 18 days p.i., viral titers were below the detection limit. In contrast, viral titers of ticks orally exposed to a tick-competent ASFV isolate, Pretoriuskop/96/4/1 (Pr4), increased 10-fold by 10 days p.i. and 50-fold by 14 days p.i. Early viral gene expression, but not extensive late gene expression or viral DNA synthesis, was detected in the midguts of ticks orally exposed to MAL. Ultrastructural analysis demonstrated that progeny virus was rarely present in ticks orally exposed to MAL and, when present, was associated with extensive cytopathology of phagocytic midgut epithelial cells. To determine if viral replication was restricted only in the midgut epithelium, parenteral inoculations into the hemocoel were performed. With inoculation by this route, a persistent infection was established although a delay in generalization of MAL was detected and viral titers in most tissues were typically 10- to 1,000-fold lower than those of ticks injected with Pr4. MAL was detected in both the salivary secretion and coxal fluid following feeding but less frequently and at a lower titer compared to Pr4. Transovarial transmission of MAL was not detected after two gonotrophic cycles. Ultrastructural analysis demonstrated that, when injected, MAL replicated in a number of cell types but failed to replicate in midgut epithelial cells. In contrast, ticks injected with Pr4 had replicating virus in midgut epithelial cells. Together, these results indicate that MAL replication is restricted in midgut epithelial cells. This finding demonstrates the importance of viral replication in the midgut for successful ASFV infection of the arthropod host. (+info)