defective for catalysis, but remained proficient for bind- ing mRNAs. Such mutants could manifest a dominant- negative phenotype by competing with wild-type RNase E to access the same (limiting) substrates. However, we believe that this is not a likely possibility. Our screen- ing for dominant-negativemutants did not reveal any catalytically inactive missense mutants, even though, on the basis of our current understanding of ribonucleases, such inactive mutants should exist. This suggests that inactive missense mutants do not confer a dominant- negative phenotype. Supporting this notion, we have identified a number of RNase E missense mutants that are functionally defective and that are expressed at high levels in the cell, but do not confer a dominant-negative phenotype (K. B riegel and C. J ain , unpublished data). A second possibility is that the dominant-negativemutants could sequester limiting host factors that are necessary for RNase E function. Although RNase E is known to bind a number of proteins, notably the components of the degradosome, these associations require the carboxy- terminal domain of RNase E. It is unclear how expres- sion of the dominant-negative RNase E variants that lack the carboxy-terminal domain could interfere with the interactions between those proteins and wild-type RNase E.
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-negativemutants directly influ- ence wild-type activity within hetero-oligomers or else compete with wild-type MxA for a cellular or viral target.
FIG. 4. Flag tagging does not affect the inhibitory features of DN s309. (A) Schematic representation of the tagging strategy. Depicted are the wt M53 ORF domains. The N-terminal epitope recognized by the M53-specific serum (ab), the variable region (VR), and the C-terminal four conserved regions (CR1 to -4) are indicated. The N-terminal end of the s309 mutant was replaced by a Flag tag (black circle). This construct (Flag- ⌬ N-s309) is not recognized by the anti-M53 serum. (B) Flag tagging does not affect the inhibitory features of s309. M2-10B4 cells were in- fected at an MOI of 0.1 in either the absence or the presence of 1 g/ml dox with Flag-M53R and Flag-DN-s309R viruses. Cell supernatants were collected on the indicated days postinfection, and the released infectious units were determined by titration on MEFs. The balcony diagram shows the regulation of virus growth in response to dox administration 3 days or 5 days after infection. The titer reduction induced by dox is the ratio between the titer in the absence of dox and the titer in the presence of dox. The results of one representative experiment set up by duplicates of both viruses are shown. (C) NIH 3T3 cells were infected at an MOI of 0.5 with the FlagM53R (lanes 1 and 2) and the Flag- ⌬ N-s309R (lanes 3 and 4) viruses in the absence ( ⫺ ; lanes 1 and 3) and presence ( ⫹ ; lanes 2 and 4) of 1 g/ml dox. Uninfected cells (lane 5, ⫹ ) and wt infection (lane 6, ⫹ ) were used as negative and positive controls, respectively. Protein expres- sion was analyzed 24 h after infection by Western blotting using antibodies against pp89 (immediate-early protein, 89 kDa; IE1), M86 (late protein, 150 kDa; MCP), M53 (38 kDa), and M50 (35 kDa) (NEC proteins); and Flag tag. pp89 and M86 did not show any significant difference after dox was added to the system. The native M53 protein (38 kDa) was detected in the absence of dox, together with Flag-M53 protein ( ⱖ 40 kDa). Accu- mulation of Flag-tagged wt M53 (Flag-M53; ⱖ 40 kDa) and s309 (Flag- ⌬ N-s309; ⬃ 32 to 33 kDa) upon dox induction was associated with a reduction in the native M53 protein levels.
Until recently, the nuclear export pathway exploited by herpesviruses had been thought to be unique to them. However, Speese and colleagues, during their investigation of the Drosophila synaptic development, found ribonucleoprotein particles (RNPs) that complexed with lamin C and the C-terminal end of a cellular signalling receptor, DFz2C, which is transported from the plasma membrane to the nucleus. These large granules do not undergo conformational adaptations to fit the nuclear pore complex export machinery as reported for other RNPs . Instead, they also enter the cytoplasm by a mechanism of transition through the nuclear envelope accompanied by envelopment and de-envelopment  akin to that described for herpesviruses. Both exit strategies share the disassembly of the lamina underlying the nuclear membrane, which is disrupted by multiple phosphorylation events. In herpesvirus infection, viral and cellular kinases are recruited to the INM to dissolve the lamin layer [15, 27, 73, 115, 118, 123, 136, 144]. The NEC proteins pUL31 and pUL34 are thought to induce membrane curvature, thereby facilitating vesicle formation . Speese et al. demonstrated that a cellular kinase, atypical PKC, is required for lamina remodelling and formation of DFz2C granule containing invaginations of the INM . Based on their observations, Speese et al. propose that herpesviruses not only hijacked the lamina disassembly pathway operationally during nuclear export, but a complete nuclear exit mechanism used by endogenous RNPs. The data presented here, however, point towards a more general role of the NEC in herpesvirus morphogenesis. Clearly processes other than the nuclear egress were inhibited by the DN mutants of pM53. For the first time a mutant of an NEC protein was described to induce inhibition of DNA synthesis. This may indicate that the role of the NEC is more fundamental in nuclear viral morphogenesis than it was believed before and that the NEC may be required for efficient execution of all functions which require the reorganisation of chromatin dynamics during infection . Supporting this idea it was reported that the HSV-1 homologues affect the late chromatin reorganisation in infected cells [162, 163].
The interaction between the MCMV NEC proteins M50 and M53 is essential for function. We have mapped the mutual binding sites of M50 and M53, but no further structure-func- tion relations are known (6, 19). A library of M50 mutants was made available from this study which relies on the random insertion of 5 aa or a stop codon into the protein coding sequence. Longer parts of the M50 coding region in which insertions did not lead to a null phenotype were subjected to deletions until the mutant became nonfunctional. Loss-of- function mutants from this library were now placed under the control of the strong HCMV immediate-early promoter-en- hancer and reinserted via an FRT site into the MCMV BAC that still contains the wt M50 gene (Fig. 1A). The resulting recombinants were screened for inhibition of virus reconstitu- tion. To this end the DNAs of mutant MCMV BACs were transfected one by one into primary mouse fibroblasts (MEFs) and viral plaque formation was monitored. For the M50 gene, a total of 32 derivatives, 29 loss-of-function mutants, one non- functional deletion mutant, and two functional mutants for control purposes were analyzed (Fig. 2). Four of the tested FIG. 3. The effect of conditional expression of the inhibitory M50 mutants on the virus growth. The schematic representation of the analyzed mutants is shown on the left. The wt M50 ORF is shown first. (A) The N-terminal region of the M50 (CR), which is conserved in alpha-, beta-, and gammaherpesvirus families, is indicated by a dark gray box, and the C-terminal variable region (VR) is indicated by a light gray box. The open box labeled with TM indicates the transmembrane domain, and “P” indicates the proline-rich sequence. The HA tag is indicated by a black box. The open arrowheads show the positions of 5-aa insertions affecting M53 protein binding (aa 53 to 57 and aa 114) (6). The sequence used to generate the anti-M50 antiserum (aa 201 to 213) is shown by a thick bar (ab). (B to D) The analyzed M50
APOBEC3G (A3G) is a host cytidine deaminase that serves as a potent intrinsic inhibitor of retroviral replication. A3G is packaged into human immunodeficiency virus type 1 virions and deaminates deoxycytidine to deoxyuridine on nascent minus-strand retroviral cDNA, leading to hyper-deoxyguanine-to-deoxyadenine mutations on positive-strand cDNA and inhibition of viral replication. The antiviral activity of A3G is suppressed by Vif, a lentiviral accessory protein that prevents encapsidation of A3G. In this study, we identified dominantnegativemutants of Vif that interfered with the ability of wild-type Vif to inhibit the encapsidation and antiviral activity of A3G. These mutants were nonfunctional due to mutations in the highly conserved HCCH and/or SOCS box motifs, which are required for assembly of a functional Cul5-E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominantnegative phenotype. Expression of dominantnegative Vif counteracted the Vif-induced reduction of intracellular A3G levels, presumably by preventing Vif-induced A3G degradation. Consequently, dominantnegative Vif interfered with wild-type Vif’s ability to exclude A3G from viral particles and reduced viral infectivity despite the presence of wild-type Vif. The identification of dominantnegativemutants of Vif presents exciting possibilities for the design of novel antiviral strategies.
Interleukin-1 (IL-1) activates p38 MAP kinase via the small G protein Ras, and this activity can be down-reg- ulated by another small G protein Rap. Here we have further investigated the role of Ras and Rap in p38 MAPK activation by IL-1. Transient transfection of cells with constitutively active forms of the known IL-1 sig- naling components MyD88, IRAK, and TRAF-6, or the upstream kinases MKK6 and MKK3, activated p38 MAPK. Dominantnegative forms of these were found to inhibit activation of p38 MAPK by IL-1. Dominant nega- tive RasN17 blocked the effect of the active forms of all but MKK3 and MKK6, indicating that Ras lies down- stream of TRAF-6 but upstream of MKK3 and MKK6 on the pathway. Furthermore, the activation of p38 MAPK caused by overexpressing active RasVHa could not be inhibited using dominantnegativemutants of MyD88, IRAK, or IRAK-2, or TRAF6, but could be inhibited by dominantnegative MKK3 or MKK6. In the same manner, the inhibitory effect of Rap on the activation of p38 by IL-1 occurred at a point downstream of MyD88, IRAK, and TRAF6, since the activation of p38 MAPK by these components was inhibited by overexpressing active Rap1AV12, while neither MKK3 nor MKK6 were af- fected. Active RasVHa associated with IRAK, IRAK2, and TRAF6, but not MyD88. In addition we found a role for TAK-1 in the activation of p38 MAPK by IL-1, with TAK-1 also associating with active Ras. Our study sug- gests that upon activation Ras becomes associated with IRAK, Traf-6, and TAK-1, possibly aiding the assembly of this multiprotein signaling complex required for p38 MAPK activation by IL-1.
To test whether dominant-negative Rab1b mutants interfere with Ebolavirus virion formation by affecting GBF1, we cloned the N-terminally FLAG-tagged ORF of GBF1 (NM_004193) into pCAGGS/MCS. A dominant-negative mutant of GBF1 which abolishes the ARF1 nucleotide-exchange activity was created by replacing glutamic acid with lysine at position 794 (FLAG-GBF1_E794K) (5, 21). As reported, expression of dominant-negative GBF1 resulted in the relocalization of GM130 (a Golgi marker) to the cytoplasm in a dotted staining pattern (Fig. 3A, arrows) (5). We then asked whether this mutant inhibited VLP production (Fig. 3B). Expression of FLAG-GBF1 (Fig. 3B, lane 2, “Anti-FLAG”) or its dominant- negative variant (Fig. 3B, lane 3, “Anti-FLAG”) did not affect VP40 expression levels in plasmid-transfected 293T cells. How- ever, the efficiency of Ebolavirus VP40-induced VLP formation was reduced upon coexpression of FLAG-tagged, dominant- negative GBF1 (Fig. 3B, lane 3), but not FLAG-GBF1 (Fig. 3B, lane 2). We next visually evaluated the involvement of GBF1 in Ebolavirus VP40 intracellular transport by using a Venus-VP40 fusion protein (Fig. 3C). The dominant-negative GBF1 mutant (GBF1_E794K) reduced the number of cell protrusions induced by Venus-VP40, similar to that seen with the Rab1b dominant-negativemutants (Fig. 2D). These results suggest that GBF1 is required for efficient Ebolavirus VP40 intracellular transport and VP40-induced virion formation.
Our data point to C91 as being the most reactive cysteine in MAL and available for posttranslational modification. Indeed, we identified that MAL can be glutathionylated on this residue. MAL was basally glutathionylated in murine BMDMs, LPS transiently increased MAL glutathionylation, and the glutathionylation returned to basal levels after 60 min. Mutagenesis of C91 to alanine confirms this residue as the site of glutathionylation. Mutagenesis of the neighboring amino acid H92 also abrogated gluta- thionylation. MAL C91A or MAL H92P were unable to signal to NF-κB when overexpressed and acted as dominant-negativemutants of TLR4 signaling and could not reconstitute MAL-deficient cells. Interestingly, MAL C91A showed only minor dominant-negative ef- fects against TLR2. Previous reports suggest MAL is not required by TLR2 (42), which may explain the failure of MAL C91A to exert a strong dominant-negative effect on TLR2. Finally, immunopre- cipitation showed that the interaction with MyD88 of MAL C91A or MAL H92P was reduced, and both mutants failed to undergo deg- radation in response to IRAK4 overexpression. In the cell, gluta- thionylation of MAL on C91 might therefore be critical for MAL to signal via the Myddosome and IRAK4. As the modified residue is close to the BB loop, the modification could influence the conformation of this functionally important flexible loop.
Consistent with these characteristics, interference with Survivin function or expression in cultured tumor cell lines by expression of dominant-negativemutants or by antisense-mediated reductions in SURVIVIN expression has been associated with supernumer- ary centrosomes, aberrant mitosis, and defective cytokinesis, with cells becom- ing polyploid and multinucleated. Homozygous disruption of both alle- les of the Survivin gene in mouse embryonic stem cells results in embry- onic lethality at day 4–5 due to chro- mosome segregation problems and failed cytokinesis (22). Moreover, knockouts of putative Survivin homo- logues in fission yeast (bir-1) and bud- ding yeast (BIR-1), and knock-downs of a Survivin-like gene (bir-1) in C. ele- gans embryos, cause various defects in centrosome formation, mitotic spin- dle assembly, chromosome segrega- tion, and cytokinesis (reviewed in ref. 5). Thus, loss-of-function mutations or interference with endogenous func- tion or expression of Survivin and its homologues in lower organisms clear- ly results in defects in late steps of cell division. Often these defects in cell division are followed by cell death, but it could be argued that most anything that disrupts chromosome segrega- tion could result secondarily in cell demise. How Survivin and its homo- logues assist in chromosome segrega- tion and cytokinesis is unknown, but evidence has been presented support-
Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence micros- copy, colocalization with specific endocytic markers, dominantnegativemutants, and pharmacological inhib- itors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na ⴙ /H ⴙ ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via ␣ v integrin/CD46 in HeLa cells.
The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5= untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negativemutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5= UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation.
An analysis of the action of dominantnegative Tsg101 and Vps4 proteins on mutant PFV budding revealed discrepancies between the current models of L-domain exploitation of ESCRT machinery and that apparently used by PFV. Current models predict that if both PSAP and PPPI domains act by recruiting components of the class E VPS pathway, then a dominantnegative form of Vps4 should be inhibitory for both. However, for both replication-competent PFV and the PFV- based vector, dominantnegative Vps4 and Vps4B/SKD1 re- duced virion yield only in the presence of an intact PSAP motif. Dominantnegative Tsg101 inhibited both PSAP and PPPI domain function. This might suggest that the PPPI motif is not a true L domain and facilitates PFV egress by some mechanism completely independent of the class E VPS pathway. However, this interpretation is difficult to reconcile with the observation that the presence of the PPPI motif both stimulates PFV egress and confers at least some degree of sensitivity to dominantnegative Tsg101 in the absence of PSAP. Conversely, absence of the PPPI motif apparently renders PSAP-dependent PFV egress hypersensitive to dominantnegative Tsg101. This find- FIG. 4. Effect of the dominantnegative Vps4, Tsg101 or AIP-1/ALIX proteins on release of PFV virus or vector. Hatched bars indicate titers of virus produced from 293T cells transfected with pczHSRV2 (wild type or mutant), solid bars show titers of vector from cells transfected with MH71 (wild type or mutant). All vector constructs were cotransfected with pczHFVenv. In all cases, negative controls were also cotransfected with pCR3.1GFP or pDSRED-C1. (A) Effect of expression of dominantnegative Vps4 (VPS4-223 M-GFP). (B) Expression of dominantnegative Vps4B/SKD1 E228Q. (C) Expression of dominantnegative truncated Tsg101 (pCR3.1-YFP-Tsg101 [1-157]). (D) Expression of dominantnegative truncated AIP-1/ALIX [pCR3.1-YFP-ALIX(d1-176)].
Unsurprisingly, the truncated B30.2 domain of Mamu-7 has no apparent antiretroviral activity when fused to the Mamu-1 RBCC (Fig. 1). Whether Mamu-7 TRIM5 exon 8 is expressed in vivo remains unclear. Similar TRIMCyp-encoding alleles in Macaca nemestrina bear the same splicing mutant at the intron 6-exon 7 boundary that leads to exon skipping to the CypA cDNA (5). In M. nemestrina, at least, this mutation appears to prevent the expression of a functional TRIM5 from the TRIMCyp- encoding alleles. Instead, they encode a truncated TRIM5 (TRIM5 ) or a TRIM5 protein that lacks exon 7 (TRIM5 ), and neither of these TRIM5s restricts HIV-1 (5). Interestingly, the exon 8 from these alleles does restrict HIV-1 when fused to human TRIM5 exons 2 to 7, indicating that it has the ability to interact with HIV-1 capsids if in the appropriate context (19). The fact that the mutation that is required to appropriately express TRIMCyp obviates the expression of full-length TRIM5 ␣ suggests that a single TRIM5 allele cannot encode antiviral TRIM5 ␣ and TRIMCyp, although this probably war- rants further investigation. The apparently inactive Mamu-6 TRIM5 ␣ is dominantnegative against Mamu-1 (Fig. 3). This suggests that Mamu-6, when expressed appropriately, is re- cruited into a TRIM5 trimer and that it cannot restrict due to a change in specificity resulting from polymorphisms in exon 6 and/or exon 8 (17).
Certain mutations within the protective antigen (PA) moiety of anthrax toxin endow the protein with a dominant-negative (DN) phenotype, converting it into a potent antitoxin. Proteolytically activated PA oligomerizes to form ring-shaped hep- tameric complexes that insert into the membrane of an acidic intracellular compartment and promote translocation of bound edema factor and/or lethal factor to the cytosol. DN forms of PA co-oligomerize with the wild-type protein and block the translocation process. We prepared and characterized 4 DN forms: a single, a double, a triple, and a quadruple mutant. The mutants were made by site-directed mutation of the cloned form of PA in Escherichia coli and tested by various assays conducted on CHO cells or in solution. All 4 mutant PAs were competent for heptamerization and ligand binding but were defective in the pH-dependent functions: pore formation, ability to convert to the SDS-resistant heptamer, and ability to translocate bound ligand. The single mutant (F427K) showed less attenuation than the others in the pH-dependent functions and lower DN activity in a CHO cell assay. The quadruple (K397D + D425K + F427A + 2 β 2-2 β 3) deletion showed the most potent DN activity at low concentrations but also gave indications of low stability in a urea-mediated unfolding assay. The double mutant (K397D + D425K) and the triple (K397D + D425K + F427A) showed strong DN activity and slight reduction in stability relative to the wild-type protein. The properties of the double and the triple mutants make these forms worthy of test- ing in vivo as a new type of antitoxic agent for treatment of anthrax.