Human MxA protein inhibits the replication of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Nairovirus within the family Bunyaviridae and is the causative agent of severe hemorrhagic fever. Despite increasing knowledge about hemorrhagic fever viruses, the factors determining their pathogenicity are still poorly understood. The interferon-induced MxA protein has been shown to have an inhibitory effect on several members of the Bunyaviridae family, but the effect of MxA against CCHFV has not previously been studied. Here, we report that human MxA has antiviral activity against CCHFV. The yield of progeny virus in cells constitutively expressing MxA was reduced up to 1,000-fold compared with control cells, and accumulation of viral genomes was blocked. Confocal microscopy revealed that MxA colocalizes with the nucleocapsid protein (NP) of CCHFV in the perinuclear regions of infected cells. Furthermore, we found that MxA interacted with NP by using a coimmunoprecipitation assay. We also found that an amino acid substitution (E645R) within the C-terminal domain of MxA resulted in a loss of MxA antiviral activity and, concomitantly, in the capacity to interact with CCHFV NP. These results suggest that MxA, by interacting with a component of the nucleocapsid, prevents replication of CCHFV viral RNA and thereby inhibits the production of new infectious virus particles.     

MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes

Human MxA protein accumulates in the cytoplasm of interferon-treated cells and inhibits the multiplication of several RNA viruses, including Thogoto virus (THOV), a tick-borne orthomyxovirus that transcribes and replicates its genome in the cell nucleus. The antiviral mechanism of MxA was investigated by using two alternative minireplicon systems in which recombinant viral ribonucleoprotein complexes (vRNPs) of THOV were reconstituted from cloned cDNAs. A chloramphenicol acetyltransferase reporter minigenome RNA was expressed either by T7 RNA polymerase in the cytoplasm of transfected cells or, alternatively, by RNA polymerase I in the nucleus. The inhibitory effect of MxA was studied in both cellular compartments by coexpressing wild-type MxA or TMxA, an artificial nuclear form of MxA. Our results indicate that both MxA proteins recognize the assembled vRNP rather than the newly synthesized unassembled components. The present findings are consistent with previous data which indicated that cytoplasmic MxA prevents transport of vRNPs into the nucleus, whereas nuclear MxA directly inhibits the viral polymerase activity in the nucleus.     

Thogoto virus lacking interferon-antagonistic protein ML is strongly attenuated in newborn Mx1-positive but not Mx1-negative mice

The Thogoto virus ML protein suppresses interferon synthesis in infected cells. Nevertheless, a virus mutant lacking ML remained highly pathogenic in standard laboratory mice. It was strongly attenuated, however, in mice carrying the interferon-responsive Mx1 gene found in wild mice, demonstrating that enhanced interferon synthesis is protective only if appropriate antiviral effector molecules are present. Our study shows that the virulence-enhancing effects of some viral interferon antagonists may escape detection in conventional animal models.     

Structural Requirements for the Antiviral Activity of the Human MxA Protein against Thogoto and Influenza A Virus

The interferon-induced dynamin-like MxA protein has broad antiviral activity against many viruses, including orthomyxoviruses such as influenza A and Thogoto virus and bunyaviruses such as La Crosse virus. MxA consists of an N-terminal globular GTPase domain, a connecting bundle signaling element, and the C-terminal stalk that mediates oligomerization and antiviral specificity. We previously reported that the disordered loop L4 that protrudes from the compact stalk is a key determinant of antiviral specificity against influenza A and Thogoto virus. However, the role of individual amino acids for viral target recognition remained largely undefined. By mutational analyses, we identified two regions in the C-terminal part of L4 that contribute to an antiviral interface. Mutations in the proximal motif, at positions 561 and 562, abolished antiviral activity against orthomyxoviruses but not bunyaviruses. In contrast, mutations in the distal motif, around position 577, abolished antiviral activity against both viruses. These results indicate that at least two structural elements in L4 are responsible for antiviral activity and that the proximal motif determines specificity for orthomyxoviruses, whereas the distal sequence serves a conserved structural function.     

Enhanced virus resistance of transgenic mice expressing the human MxA protein.

MxA is a GTPase that accumulates to high levels in the cytoplasm of interferon-treated human cells. Expression of MxA cDNA confers to transfected cell lines a high degree of resistance against several RNA viruses, including influenza, measles, vesicular stomatitis, and Thogoto viruses. We have now generated transgenic mice that express MxA cDNA in the brain and other organs under the control of a constitutive promoter. Embryonic fibroblasts derived from the transgenic mice were nonpermissive for Thogoto virus and showed reduced susceptibility for influenza A and vesicular stomatitis viruses. The transgenic animals survived challenges with high doses of Thogoto virus by the intracerebral or intraperitoneal route. Furthermore, the transgenic mice were more resistant than their nontransgenic littermates to intracerebral infections with influenza A and vesicular stomatitis viruses. These results demonstrate that MxA is a powerful antiviral agent in vivo, indicating that it may protect humans from the deleterious effects of infections with certain viral pathogens.     

Measles virus inhibits acquisition of lymphocyte functions but not established effector functions

The effect of measles-virus infection on effector activities of human lymphocytes and on the generation of certain effector activities was studied in vitro. Addition of measles virus to allogeneic mixed lymphocyte cultures resulted in a strongly depressed cytolytic activity in a subsequent cell-mediated lympholysis assay. Late addition of measles virus did not inhibit cytotoxic effector function, although effector cells were probably infected. Similarly, measles-virus infection did not affect the ability of lymphocytes to mediate antibody-dependent cellular cytotoxicity. Addition of measles virus to lymphocytes with, or shortly after, exposure of the cells to the polyclonal activator pokeweed mitogen resulted in abolition of the synthesis of immunoglobulins in vitro. When the virus was added late, the rate of Ig secretion was only partially inhibited. Finally, when lymphocytes were cultured without stimulus in medium supplemented with fetal bovine serum, a population of inhibitory cells was generated. Measles virus was able to prevent the generation of such inhibitory cells. In conclusion, measles virus inhibited acquisition of various effector functions, but the activities of committed lymphocytes were generally not affected.     

Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein.

MxA and MxB are interferon-induced proteins of human cells and are related to the murine protein Mx1, which confers selective resistance to influenza virus. In contrast to the nuclear murine protein Mx1, MxA and MxB are located in the cytoplasm, and their role in the interferon-induced antiviral state was unknown. In this report we show that transfected cell lines expressing MxA acquired a high degree of resistance to influenza A virus. Surprisingly, MxA also conferred resistance to vesicular stomatitis virus. Expression of MxA in transfected 3T3 cells had no effect on the multiplication of two picornaviruses, a togavirus, or herpes simplex virus type 1. Treatment of MxA-expressing cells with antibodies to mouse alpha-beta interferon did not abolish the resistance phenotype. The conclusion that resistance to influenza virus and vesicular stomatitis virus was due to the specific action of MxA is further supported by the observation that transfected 3T3 cell lines expressing the related MxB failed to acquire virus resistance.     

A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus.

Epstein-Barr virus (EBV) codes for at least three glycoproteins, gp350, gp220, and gp85. The two largest glycoproteins are thought to be involved in the attachment of the virus to its receptor on B cells, but despite the fact that gp85 induces neutralizing antibody, no function has been attributed to it. As an indirect approach to understanding the role of gp85 in the initiation of infection, we determined the point at which a neutralizing, monoclonal antibody that reacted with the glycoprotein interfered with virus replication. The antibody had no effect on virus binding. To examine the effect of the antibody on later stages of infection, the fusion assay of Hoekstra and colleagues (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilshaut, Biochemistry 23:5675-5681, 1984) was adapted for use with EBV. The virus was labeled with a fluorescent amphiphile that was self-quenched at the high concentration obtained in the virus membrane. When the virus and cell membrane fused, there was a measurable relief of self-quenching that could be monitored kinetically. Labeling had no effect on virus binding or infectivity. The assay could be used to monitor virus fusion with lymphoblastoid lines or normal B cells, and its validity was confirmed by the use of fixed cells and the Molt 4 cell line, which binds but does not internalize the virus. The monoclonal antibody to gp85 that neutralized virus infectivity, but not a second nonneutralizing antibody to the same molecule, inhibited the relief of self-quenching in a dose-dependent manner. This finding suggests that gp85 may play an active role in the fusion of EBV with B-cell membranes.     

IHF protein inhibits cleavage but not assembly of plasmid R388 relaxosomes

Relaxosomes are specific nucleoprotein structures involved in DNA-processing reactions during bacterial conjugation. In this work, we present evidence indicating that plasmid R388 relaxosomes are composed of origin of transfer (oriT) DNA plus three proteins TrwC relaxase, TrwA nic-cleavage accessory protein and integration host factor (IHF), which acts as a regulatory protein. Protein IHF bound to two sites (ihfA and ihfB) in R388 oriT, as shown by gel retardation and DNase I footprinting analysis. IHF binding in vitro was found to inhibit nic-cleavage, but not TrwC binding to supercoiled DNA. However, no differences in the frequency of R388 conjugation were found between IHF- and IHF+ donor strains. In contrast, examination of plasmid DNA obtained from IHF- strains revealed that R388 was obtained mostly in relaxed form from these strains, whereas it was mostly supercoiled in IHF+ strains. Thus, IHF could have an inhibitory role in the nic-cleavage reaction in vivo. It can be speculated that triggering of conjugative DNA processing during R388 conjugation can be mediated by IHF release from oriT.     

Human immunodeficiency virus envelope protein Gp120 induces proliferation but not apoptosis in osteoblasts at physiologic concentrations

Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05), which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism.     

Tobacco mosaic virus inoculation inhibits wound-induced jasmonic acid-mediated responses within but not between plants

Jasmonic acid (JA) and salicylic acid (SA) have both been implicated as important signal molecules mediating induced defenses of Nicotiana tabacum L. against herbivores and pathogens. Since the application of SA to a wound site can inhibit both wound-induced JA and a defense response that it elicits, namely nicotine production, we determined if tobacco mosaic virus (TMV) inoculation, with its associated endogenous systemic increase in SA, reduces a plant's ability to increase JA and nicotine levels in response to mechanical damage, and evaluated the consequences of these interactions for the amount of tissue removed by a nicotine-tolerant herbivore, Manduca sexta. Additionally, we determined whether the release of volatile methyl salicylic acid (MeSA) from inoculated plants can reduce wound-induced JA and nicotine responses in uninoculated plants sharing the same chamber. The TMV-inoculated plants, though capable of inducing nicotine normally in response to methyl jasmonate applications, had attenuated wound-induced JA and nicotine responses. Moreover, larvae consumed 1.7- to 2.7-times more leaf tissue from TMV-inoculated plants than from mock-inoculated plants. Uninoculated plants growing in chambers downwind of either TMV-inoculated plants or vials releasing MeSA at 83- to 643-times the amount TMV-inoculated plants release, exhibited normal wound-induced responses. We conclude that tobacco plants, when inoculated with TMV, are unable to elicit normal wound responses, due likely to the inhibition of JA production by the systemic increase in SA induced by virus-inoculation. The release of volatile MeSA from inoculated plants is not sufficient to influence the wound-induced responses of neighboring plants. Received: 6 January 1999 / Accepted: 11 January 1999     

Vitamin A inhibits chondrogenesis but not myogenesis

Limb bud cells were isolated from HH stage 22–23 chick embryos and were grown as a ‘spot culture’ in in vitro conditions which support their differentiation into chondrocytes and myotubes. By day 4 of culture, numerous chondrocyte nodules developed and were scattered mainly in the very centre of the cell spot. In contrast, multinucleated myotubes formed at both the centre and the periphery of the cell spot. Treatment with vitamin A starting on day 1, inhibited chondrogenesis in these cultures, and by day 4–6 chondrocyte nodules could not be detected histologically. In contrast, no dose of vitamin A tested was effective in suppressing the development of multi-nucleated myotubes. These data show that vitamin A selectively inhibits chondrogenesis but not myogenesis in limb bud cell cultures.     

A short form of the tick-borne encephalitis virus NS3 protein.

Using monoclonal antibodies to the tick-borne encephalitis virus (TBE) nonstructural protein NS3 two forms of this protein were revealed in TBE-infected mammalian cells: a full-length form (69 kDa) and a short form (49 kDa) which has not been observed before and was called NS3'. Recombinant plasmids were constructed and various fragments of the TBE NS3 gene were expressed in rabbit reticulocyte lysate. By analyzing immune precipitates of 35S-labeled translation products, we could monitor and localize internal cleavage of NS3, due to which the NS3' protein was generated.