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O Haller

Publications and source records attributed to O Haller.

At least 37 records · Page 2Linked to original sources
PubMed1998

A classical bipartite nuclear localization signal on Thogoto and influenza A virus nucleoproteins.

We have previously shown that the nucleoprotein (NP) of Thogoto virus (THOV), a tick-borne member of the Orthomyxoviridae family, accumulates in the cell nucleus. Here we demonstrate that THOV NP contains a motif (KRxxxxxxxxxKTKK) at amino acid positions 179-193 that represents a classical bipartite nuclear localization signal (NLS). This sequence motif (named cNLS) was able to translocate a cytoplasmic 80-kDa reporter protein into the nucleus. Targeted mutations substituting lysines for alanines in the downstream cluster of the bipartite motif abolished the capacity of cNLS to mediate nuclear import. In contrast, identical mutations had no effect on nuclear localization when introduced into THOV NP, indicating that additional transport signals are present in NP. Amino-acid sequence comparisons revealed that THOV NP lacks the N-terminal nonconvential NLS (named here nNLS), which has been implicated in nuclear import of influenza A virus NP. Accordingly, THOV NP failed to interact in coprecipitation assays with the cellular NPI-1/3 transport factors of the karyopherin alpha family. A highly conserved motif identified in THOV NP was the so-called nuclear accumulation sequence (NAS). Mutating NAS alone, or in combination with cNLS, had no gross effect on the intracellular distribution of the protein, indicating that a functional NAS is not required for nuclear accumulation of THOV NP in mammalian cells. We also studied nuclear transport of influenza A/PR/8/34 virus NP. Interestingly, we found a cNLS motif at amino acid positions 198-216 in addition to the previously described nonconventional nNLS. To further assess the functional role of cNLS, nNLS, and NAS, we analyzed single, double, and triple mutants of influenza A virus NP. When nNLS was destroyed, the protein stayed in the cytoplasm as expected. When NAS was disrupted in addition to nNLS, the double mutant accumulated in the nucleus, suggesting that cNLS was active. Indeed, when cNLS was also inactivated, the triple mutant protein localized again predominantly to the cytoplasm. These findings suggest that NP of orthomyxoviruses have two independent NLSs, namely cNLS and nNLS. They further suggest that NAS and NLSs may assume opposing roles in nucleocytoplasmic transport of NP.

Amino Acid Sequence↗
PubMed1998

MxA GTPase: oligomerization and GTP-dependent interaction with viral RNP target structures.

MxA protein is an interferon-induced GTPase of human cells that inhibits the multiplication of several RNA viruses, including influenza viruses and bunyaviruses. Studies on MxA transgenic mice have shown that MxA is a powerful antiviral agent in vivo. It has been suggested that this cellular protein also protects humans from viral disease, but the mechanism(s) by which MxA exerts its antiviral action is still poorly understood. Using an in vitro cosedimentation assay, we now demonstrate that MxA tightly interacts with components of the ribonucleoprotein complex of Thogoto virus, an influenza-like virus transmitted by ticks. This assay demonstrates for the first time a physical interaction between MxA GTPase and a viral target structure. It is based on three elements, namely, highly active MxA GTPases as effector molecules, viral ribonucleoprotein particles as viral targets, and GTPgammaS as a stabilizing factor. Furthermore, using a simple nuclear translocation assay, we show that human MxA protein forms oligomers in vivo. This assay provides a stringent test for tight association of partner molecules in intact mammalian cells. It not only will be useful for studying physical interactions of MxA with partner molecules, but may also be applicable to other studies on protein-protein interactions in living cells.

Animals↗
PubMed1998

In vivo reconstitution of active Thogoto virus polymerase: assays for the compatibility with other orthomyxovirus core proteins and template RNAs.

Tick-borne Thogoto virus (THOV), the prototype of a new genus in the Orthomyxoviridae family, contains six single-stranded RNA segments of negative polarity. Four of them encode gene products that correspond to the influenza virus PB1, PB2, PA and NP core proteins. Here we describe an in vivo system in which the expression of a THOV model RNA is driven by THOV core proteins synthesized from cloned cDNAs. Our results demonstrated the biological activity of our cloned genes and showed that the three polymerase subunits and the NP are required for gene expression. For comparison, we also used the in vivo reconstituted systems of the influenza A and B viruses. None of the polymerase or NP proteins was active in a heterologous orthomyxovirus core, indicating a high specificity in core assembly and/or function. Interestingly, the THOV polymerase did not recognize the influenza A virus promoter and vice versa.

Cloning, Molecular↗
PubMed1998

Characterization and expression of the Mx1 gene in wild mouse species.

The mouse Mx1 gene encodes an interferon (IFN)-inducible nuclear protein and confers resistance to influenza virus infection. The standard laboratory mouse strains all carry the Mx1- allele and are susceptible to influenza virus. In this study, several mouse strains established from wild mice were tested to determine their Mx1+ or Mx1- allele status with polymerase chain reaction-restriction fragment length variation (PCR-RFLV), sequence analysis, reverse transcription (RT)-PCR, and immunofluorescence staining. All of the mouse strains originating from wild mice were found uniformly to carry the Mx1+ allele. Therefore, it is conceivable that the Mx1+ allele in wild populations serves a function against some pathogens related to orthomyxoviruses. The PCR-RFLV and sequence analysis allowed us to classify the Mx1+ alleles of the laboratory and wild-origin mouse strains into distinct classes. RT-PCR and immunofluorescence staining demonstrated that the Mx1 transcripts and proteins were induced by IFN-alpha/beta in macrophages from wild mouse species.

Animals↗
PubMed1998

Intragenic variability of human cytomegalovirus glycoprotein B in clinical strains.

Human cytomegalovirus (HCMV) strains can be classified into four glycoprotein B (gB) genotypes, and there has been evidence of differences in viral virulence. In this study, intragenic variability of HCMV gB strains was analyzed. The gB gene was amplified by nested polymerase chain reaction using samples from immunosuppressed patients. The genotype of fragments corresponding to the cleavage site of gB was determined by restriction fragment analysis; fragments corresponding to the N- and C-termini (gBn and gBc) were sequenced and compared with published sequences. At the cleavage site, the four known genotypes were found. Typing revealed four major genotypes at the N-terminus and two at the C-terminus. In 22 of 44 strains, the gB type determined at the cleavage site was different from the gBn or gBc type (or either), indicating that intragenic variability within the gB gene occurs frequently.

Base Sequence↗
PubMed1998

Mx proteins: mediators of innate resistance to RNA viruses.

Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted attention because some display antiviral activity against pathogenic RNA viruses, for example against members of the orthomyxovirus (influenzavirus) family or the bunyavirus family. Transfected cells and transgenic mice expressing Mx proteins are highly resistant to Mx-sensitive viruses, demonstrating that Mx proteins are powerful antiviral agents. In humans, synthesis of MxA is observed during self-limiting viral infections and may thus promote recovery from disease.

Animals↗
PubMed1997

Conserved vRNA end sequences of Thogoto-orthomyxovirus suggest a new panhandle structure.

Panhandles are dsRNA structures formed by conserved sequences at the 5' and 3' ends of the influenza virus genomic RNA. They consist of two stems separated by a flexible bulge and serve as promoter for the viral polymerase. In the outer stem, melting of base pairs is a prerequisite for initiation of transcription. We compared the terminal sequences of Thogoto virus (THOV), a tick-borne orthomyxovirus, with those of influenza virus. Despite their overall similarity, the first U downstream of the 5' end of the panhandle (position 3 in influenza virus) is found at position 8 in the THOV sequence. This shift from position 3 to position 8 results in a radical change of the predicted secondary structure. In the outer stem, intra-strand base pairings are clearly favoured above inter-strand hybridizations. As this secondary structure can explain the functioning of a mutant promoter of influenza virus with twice the activity of the wild-type, we propose a general validity of our "hook-like" panhandle structure.

Base Sequence↗
PubMed1997

Germinal centre CD4+ T cells are an important site of HIV replication in vivo.

OBJECTIVE: CD4+ T cells are the main target for HIV. However, the highest HIV antigen concentration in infected subjects accumulates on the cell surface of follicular dendritic cells in the germinal centres of the lymphoid tissue. Germinal centres contain a T-helper cell subset which expresses CD57 molecules. Here we analysed virus replication and viral load in CD57+CD4+ germinal centre T cells and in the CD4+ T cells found mostly outside germinal centres (CD57-CD4+). METHODS: Peripheral blood mononuclear cells and lymph-node cells were prepared, stained for CD4 and CD57 and purified by FACS. Defined cell numbers of CD4+CD57+ cells and CD4+CD57- cells were sorted directly into polymerase chain reaction (PCR) tubes by FACS, equipped with an automated cell deposition unit and analysed by PCR to detect proviral DNA. Based on Poisson distribution, the expected level of infection was calculated. Viral replication was determined by amplifying double-spliced, single-spliced, and full-length transcripts of HIV using serially diluted cDNA of the FACS-sorted cells. RESULTS: An up to 10-fold higher frequency of infected cells was found in the CD57+CD4+ germinal centre T cells compared with CD57-CD4+ T cells. Furthermore, active viral replication was detected almost exclusively in the CD57+CD4+ T cells. CONCLUSIONS: The CD57+CD4+ germinal centre T cells are one of the sites of HIV infection and replication that may play a pivotal role in the pathogenesis of HIV infection.

Base Sequence↗
PubMed1997

Mx1 sensitivity: Batken virus is an orthomyxovirus closely related to Dhori virus.

Batken virus, isolated from mosquitoes and ticks, was tentatively classified as a member of the family Bunyaviridae. Here we show that Batken virus is inhibited by the interferon-induced Mx1 protein of mice which selectively blocks the growth of orthomyxoviruses, including Thogoto and Dhori viruses. Furthermore, we show that Batken virus multiplication is characterized by accumulation of viral proteins in the nucleus and by budding of viral particles from the cell surface. Serological cross-reactions between Batken and Dhori viruses revealed a phylogenetic relationship of these viruses, as previously also proposed by D. K. Lvov. Fragments of the Batken virus glycoprotein and nucleoprotein genes were amplified by RT-PCR. The deduced amino acid sequences were similar to the corresponding Dhori virus sequences. Therefore, Batken virus should be classified into the newly established genus Thogotovirus of the family Orthomyxoviridae. Finally, our results demonstrate that Mx1 susceptibility of orthomyxoviruses is a reliable marker in the hunt for new family members.

Amino Acid Sequence↗
PubMed1997

Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.

Human MxA protein is an interferon-induced 76-kDa GTPase that exhibits antiviral activity against several RNA viruses. Wild-type MxA accumulates in the cytoplasm of cells. TMxA, a modified form of wild-type MxA carrying a foreign nuclear localization signal, accumulates in the cell nucleus. Here we show that MxA protein is translocated into the nucleus together with TMxA when both proteins are expressed simultaneously in the same cell, demonstrating that MxA molecules form tight complexes in living cells. To define domains important for MxA-MxA interaction and antiviral function in vivo, we expressed mutant forms of MxA together with wild-type MxA or TMxA in appropriate cells and analyzed subcellular localization and interfering effects. An MxA deletion mutant, MxA(359-572), formed heterooligomers with TMxA and was translocated to the nucleus, indicating that the region between amino acid positions 359 and 572 contains an interaction domain which is critical for oligomerization of MxA proteins. Mutant T103A with threonine at position 103 replaced by alanine had lost both GTPase and antiviral activities. T103A exhibited a dominant-interfering effect on the antiviral activity of wild-type MxA rendering MxA-expressing cells susceptible to infection with influenza A virus, Thogoto virus, and vesicular stomatitis virus. To determine which sequences are critical for the dominant-negative effect of T103A, we expressed truncated forms of T103A together with wild-type protein. A C-terminal deletion mutant lacking the last 90 amino acids had lost interfering capacity, indicating that an intact C terminus was required. Surprisingly, a truncated version of MxA representing only the C-terminal half of the molecule exerted also a dominant-negative effect on wild-type function, demonstrating that sequences in the C-terminal moiety of MxA are necessary and sufficient for interference. However, all MxA mutants formed hetero-oligomers with TMxA and were translocated to the nucleus, indicating that physical interaction alone is not sufficient for disturbing wild-type function. We propose that dominant-negative mutants directly influence wild-type activity within hetero-oligomers or else compete with wild-type MxA for a cellular or viral target.

3T3 Cells↗
PubMed1996

Thogoto and Dhori virus replication is blocked by inhibitors of cellular polymerase II activity but does not cause shutoff of host cell protein synthesis.

Tick-transmitted Thogoto and Dhori viruses share structural and genetic properties with the influenza viruses. Here, we compare different steps of their replication cycle in mammalian cells in comparison with influenza A virus. Viral antigens of both viruses accumulated in the nuclei of infected cells, suggesting a nuclear phase of viral replication. Furthermore, as observed with influenza viruses, transcription of Thogoto and Dhori viruses was inhibited by alpha-amanitin and actinomycin D, suggesting a dependence of viral transcription on cellular RNA polymerase II activity. In contrast to influenza viruses, Thogoto and Dhori virus infection did not lead to down-regulation of cellular protein synthesis indicating marked differences regarding the fate of infected cells.

3T3 Cells↗
PubMed1996

Nucleoprotein viral RNA and mRNA of Thogoto virus: a novel "cap-stealing" mechanism in tick-borne orthomyxoviruses?

Tick-borne Thogoto virus (THOV) represents the prototype virus of a new genus in the Orthomyxoviridae family. Its genome consists of six segments of negative-sense, single-stranded RNA. We have cloned and sequenced the fifth genomic segment, which codes for the viral nucleoprotein (NP). The deduced amino acid sequence shows 43% similarity to the NP of Dhori virus, a related tick-transmitted orthomyxovirus, and about 14% sequence similarity to those of the influenza viruses. To reveal the mechanism by which THOV initiates mRNA synthesis, we characterized the 5' ends of the NP mRNAs. Transcripts were recognized by a cap-specific monoclonal antibody, indicating that THOV mRNAs are capped. Surprisingly, no large heterogeneous extensions were found at the 5' end, as would have been expected if THOV were using a classical "cap-stealing" mechanism. We therefore propose that THOV is stealing only the cap structure with one or two additional nucleotides from cellular mRNAs to generate appropriate primers for initiation of viral mRNA transcription.

Amino Acid Sequence↗
PubMed1996

Inhibition of bunyaviruses, phleboviruses, and hantaviruses by human MxA protein.

Viruses of the Bunyaviridae family cause a variety of diseases ranging from uncomplicated fever to potentially lethal encephalitis and hemorrhagic fever. Little is known about the factors determining pathogenicity in the vertebrate host. Interferons have been reported to be inhibitory, but their mode of action against members of the Bunyaviridae has not yet been elucidated. The interferon-induced MxA protein encoded on human chromosome 21 is a large GTPase with antiviral activity against distinct negative-strand RNA viruses, notably influenza viruses. Here we show that MxA inhibits representative members of the Bunyaviridae family by interacting with an early step of virus replication. When constitutively expressed in stably transfected Vero cells, MxA prevented the accumulation of viral transcripts and proteins of Hantaan virus (genus Hantavirus). Other members of the family such as La Crosse virus (genus Bunyavirus) and Rift Valley fever virus and sandfly fever virus (both genus Phlebovirus) were likewise inhibited, and virus titers were reduced up to 10(4)-fold. Our data indicate that humans have evolved a mechanism of controlling these viruses irrespective of differences in viral coding strategies.

3T3 Cells↗
PubMed1996

Glycoprotein B genotype of human cytomegalovirus: distribution in HIV-infected patients.

Glycoprotein B (gB) is involved in cell to cell transmission of human cytomegalovirus (HCMV) and may be a critical factor in tissue tropism and viral pathogenesis. We analyzed the distribution of the four known gB genotypes of HCMV in 99 HIV-positive patients. 29 patients had HCMV retinitis, and 70 patients had asymptomatic HCMV infection. DNA was isolated from blood, urine, and aqueous humor, and gB genotypes were determined by PCR and restriction analysis. Infections with gB type 1 were less frequent in patients with retinitis than in patients with asymptomatic HCMV infection (17% versus 37%; p = 0.05). Furthermore, the gB type was correlated with dissemination of infection. In patients with HCMV detected in only one compartment (blood or urine) the gB type 1 was found more frequently than in patients with HCMV detected in at least two compartments (p = 0.01). The data show that gB genotypes differ in their association with clinical disease, and indicate that the gB genotype may contribute to the course of HCMV infection.

Cytomegalovirus↗
PubMed1996

The origin of SL family mice.

The origin of SL family mice was studied by analyzing 100 microsatellite loci, the major histocompatibility complex, the Mx gene, murine leukemia provirus, and mammary tumor provirus. From the genetic profile of family members and their history, we assumed the existence of a proto-SL mouse, an ancestor of all SL family members. Many alleles were contributed to the proto-SL by the ancestors related to strains A2G and CF#1, and/or some wild mice. Among four existing family members, SL/Am and SL/Ni mice were almost identical and presumably closest to the proto-SL. The SL/Kh mouse was derived from a cross of the proto-SL and AKR mice, because SL/Kh mice inherited a considerable number of genes from AKR mice, the most outstanding of which were those of the provirus Emv-11 and Thy-1.1. The SL/QDj mice seemed to be a recombinant inbred strain between SL/Am and SL/Kh mice, because their alleles at all 100 microsatellite loci were shared by SL/Am or SL/Kh strains or both. All four SL family members shared the major histocompatibility complex haplotype q.

Animals↗
PubMed1995

Mx1 but not MxA confers resistance against tick-borne Dhori virus in mice.

The interferon-induced nuclear Mx1 protein is responsible for innate resistance of mice to influenza virus. It has been unclear why mice are equipped with a powerful and specific defense mechanism against influenza viruses for which they are not natural hosts. Here, we show that Dhori virus, an influenza-like virus transmitted by ticks and known to infect small mammals, is sensitive to the Mx1 resistance mechanism. Influenza virus-susceptible BALB/c and C57BL/6 mice (lacking a functional Mx1 gene) developed severe disease symptoms and died within a few days after intraperitoneal infection with a lethal dose of Dhori virus. In contrast, Mx1(+)-congenic, influenza virus-resistant BALB.A2G-Mx1 and B6.A2G-Mx1 mice remained healthy and survived. The Mx1 resistance phenotype was expressed in cultured peritoneal macrophages and interferon-treated embryonic cells obtained from these mice. Moreover, stable lines of transfected mouse 3T3 cells constitutively expressing Mx1 protein were protected from Dhori virus infection. The MxA protein of human cells shows a high degree of sequence similarity to Mx1 but, unlike Mx1, inhibits a broad range of RNA viruses. Transgenic mice that permanently express the human MxA protein in various organs became resistant to infection with Thogoto virus but remained fully susceptible to Dhori virus. These in vivo results show that DHO virus is unique in being resistant to human MxA but susceptible to mouse Mx1 protein. They further indicate that the Mx1 system functions as a potent defense mechanism against tick-borne influenza-like viruses in mice.

3T3 Cells↗