Linker Insertion Mutations Research Articles

Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: effects on virion particle assembly, release, and infectivity

The phenotypes of a series of mutant human immunodeficiency virus type 1 proviruses with linker insertion and deletion mutations within the gag coding region were characterized. These mutants were tested for their ability to make and release viral particles in COS7 cells and for their viability in vivo. Of the 12 mutant proviruses, 4 did not make extracellular virion particles when transfected into COS7 cells. All four of these mutants had mutations in the C-terminal domain of CA. These mutants appeared to have defects both in the ability to accumulate high-molecular-weight intracellular structures containing Gag and Pol products and in the ability to release virion particles. Seven of the mutant proviruses retained the ability to make, release, and process virion particles from COS7 cells. These particles contained the Env glycoprotein, viral genomic RNA, and the mature products of the Gag and Gag-Pol polyproteins, yet they were noninfectious or poorly infectious. The defect in these mutants appears to be in one of the early steps of the viral life cycle. Thus, multiple regions throughout Gag appear to be important in mediating the early steps of the viral life cycle.

Human foamy virus Bel1 transactivator contains a bipartite nuclear localization determinant which is sensitive to protein context and triple multimerization domains.

The Bel1 protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bel1 from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; F. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bel1 (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bel1-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bel1 containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bel1 consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bel1 NLS. On the other hand, Bel1 residues 199 and 200 were not required to direct the Bel1-beta-galactosidase chimeric protein to the nucleus when the Bel1 NLS was appended to the amino terminus of beta-galactosidase. These results indicate that the function of the Bel1 NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bel1 protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bel1, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bel1 from residues 202 to 226 prevents Bel1 from forming a multimeric complex.

Mutational analysis of cauliflower mosaic virus gene VI: changes in host range, symptoms, and discovery of transactivation-positive, noninfectious mutants.

Gene VI of cauliflower mosaic virus (CaMV) has been shown to be a determinant of host specificity of the virus as well as a factor influencing symptom development in infected plants. In addition, it plays a crucial role in viral gene expression through a process of posttranscriptional transactivation. In the present study, linker-insertion mutations within gene VI of a cloned, recombinant cauliflower mosaic virus genome were constructed and tested for infectivity, symptom development on solanaceous plants, and the ability of transactivate viral gene expression. Certain mutations in the first third of the gene resulted in changes in symptoms shown by test plants. Another mutation, also in the first third of the gene, blocked infectivity in the Nicotiana species tested and systemic movement in Datura stramonium. The mutants were also tested in protoplasts for the ability to transactivate virus gene expression. Infectious mutants were invariably positive for transactivation and mutants negative for transactivation were noninfectious. Interestingly, two mutants positive for transactivation were noninfectious, suggesting a second function for gene VI in the infection process. These results further suggest a role for gene VI, as yet not fully understood, in systemic movement of the virus in infected plants.

Characterization of regions of herpes simplex virus type 1 glycoprotein E involved in binding the Fc domain of monomeric IgG and in forming a complex with glycoprotein I.

Glycoprotein E (gE) and glycoprotein I (gI) of herpes simplex virus type 1 form a molecular complex that binds the Fc domain of monomeric IgG. Two approaches were used to define regions of gE-1 involved in monomeric IgG binding and complex formation with gI-1. First, we constructed 22 in-frame gE-1 linker-insertion mutants and, in cotransfection experiments with gI-1, assayed each mutant for IgG monomer binding and the ability to complex with gI-1. Nine mutants with insertions between gE-1 amino acids 235 and 380 failed to bind IgG monomers, whereas mutants outside this region retained binding activity. Each mutant reacted with several gE-1 mAbs, was detected at the cell surface, and was fully processed. Only two gE-1 mutants with insertions at residues 235 and 264 lost the ability to co-immunoprecipitate with gI-1, which defines a region of gE-1 that complexes with gI-1. As an additional approach, we assayed 8 gE-1/gD-1 fusion proteins containing large overlapping gE-1 peptides inserted within the ectodomain of gD-1 for binding of IgG monomers and complex formation with gI-1. Three fusion proteins containing gE-1 peptides that overlap at residues 183-402 bound monomeric IgG. This region of gE-1 includes the Fc binding region defined by linker insertion mutagenesis. Five fusion proteins containing gE-1 peptides that overlap at residues 183-288 were co-immunoprecipitated with gI-1, confirming results of gE-1 linker insertion mutagenesis. These studies define two regions on gE-1 involved in Fc binding activity, one that interacts with gI-1, and another that binds IgG.

Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells.

We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination. These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site. We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination. Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference. Next, to inquire into the fate of double-strand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homology or by end-joining. Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining. These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S. cerevisiae, wherein only products of homologous events are detected.

Mutational Analysis of Murine Mx1 Protein: GTP Binding Core Domain Is Essential for Anti-Influenza A Activity

Interferon-induced resistance to influenza virus in murine cells is mediated by the Mx1 protein, which inhibits viral mRNA synthesis in the nucleus. Murine Mx1 protein is a GTPase specifically targeted into the cell nucleus and it has a C-terminal leucine zipper domain that mediates its oligomerization. In order to determine functionally important elements of the protein we created several substitution, linker insertion, and deletion mutants of murine Mx1 protein. The antiviral activity of mutant Mx1 proteins was analyzed by a transient transfection/influenza A infection assay in COS cells by indirect immunofluorescence. Mx1 proteins carrying mutations in the vicinity or within the consensus GTP binding elements exhibited markedly reduced, but not completely lost, antiviral activity. Baculovirus produced, GTP binding element substitution mutant Mx1 proteins showed very low (<10%) GTPase activity as compared to wild-type Mx1 protein. Mutations in other portions of the molecule had less effect on antiviral activity, except one mutant, which was situated six amino acids from the C-terminal end. This mutation evidently interrupted the nuclear localization signal rendering the protein cytoplasmic and clearly reduced the anti-influenza activity. Deletions of various sizes and locations further suggested that the N-terminal half of the molecule is more important in the antiviral activity than other regions of the molecule. These results indicate that the GTP binding domain of Mx1 protein is essential for its anti-influenza activity, correlating to the low GTPase activity of the GTP binding element substitution mutants, but other portions of the molecule such as the leucine zipper and the nuclear localization signal are of importance, too.

The Interaction of Glycoprotein C of Herpes Simplex Virus Types 1 and 2 with the Alternative Complement Pathway

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) binds the human complement fragment C3b, but the two proteins differ in their ability to bind C3b on infected cell surfaces. In addition, gC-1, but not gC-2, accelerates the decay of the alternative pathway C3 convertase, thereby affecting later steps of the complement cascade. Previously, we constructed linker insertion and deletion mutants of gC-1 and gC-2 and used transient transfection to express mutant proteins in uninfected cells. In spite of the differences between gC-1 and gC-2, C3b binding was localized to residues within the central portion of both proteins, encompassing the first four cysteines. For gC-1, deletion mutants lacking amino acids 33 to 123 or 367 to 469 or lacking both regions still bound C3b. We recombined these deleted forms of gC-1 into gC-39, an HSV-1 strain lacking the gC gene. The altered forms of gC-1 were incorporated into virions, expressed on the surface of infected cells, and bound C3b. We used these proteins to investigate the structural basis for the inhibitory action of gC-1 on the complement cascade. We found that gC-1 does not inhibit formation of the alternative pathway C3 convertase. This convertase is stabilized by the serum protein properdin. Purified gC-1, but not gC-2, inhibits the binding of properdin to C3b, suggesting that this destabilizes the convertase. The mutant lacking amino acids 367 to 449 was able to inhibit properdin binding to a limited extent when present at high concentrations, although it bound to C3b more weakly than wild-type gC. In contrast, the protein lacking amino acids 33 to 123 was unable to inhibit properdin binding to C3b. Thus, gC-1 contains two structural domains, one for C3b binding, residues 124 to 366, and another, residues 33 to 133, which interferes with properdin binding to C3b.

Specificity and sequence requirements for interactions between various retroviral Gag proteins

We previously established a genetic assay for retroviral Gag polyprotein multimerization (J. Luban, K. B. Alin, K. L. Bossolt, T. Humaran, and S. P. Goff, J. Virol. 66:5157-5160, 1992). Here we use this assay to demonstrate homomeric interactions between Gag polyproteins encoded by six different retroviruses. Of the Gag polyproteins tested, only those encoded by closely related retroviruses formed heteromultimers. To determine the primary sequence requirements for human immunodeficiency virus type 1 Gag polyprotein multimerization, we studied the effects on multimerization of deletion and linker insertion mutations. Sequences necessary for this process were located between the C-terminal one-third of the capsid domain and the C terminus of the nucleocapsid domain.

Insertional mutations in the yeast HOP1 gene: evidence for multimeric assembly in meiosis.

The HOP1 gene of Saccharomyces cerevisiae has been shown to play an important role in meiotic synapsis. In this study we analyzed the mechanism of this function by phenotypic characterization of novel in-frame linker-insertion mutations located at various sites throughout the HOP1 coding sequence. Among 12 mutations found to cause defects in meiotic recombination and spore viability, three were temperature-sensitive for the spore viability defect. Although substantial meiotic recombination was found for these conditional alleles at the restrictive temperature, the level of exchange measured in spo13 meiosis was reduced in some of the monitored intervals, indicating that nondisjunction resulting from a deficit in crossing over could account for SPO13 spore inviability. Intragenic complementation between linker-insertion alleles was assessed by testing the viability of spores generated from heteroallelic diploids after SPO13 meiosis. Complex patterns of complementation and enhancement of the spore-inviability phenotype indicate that HOP1 functions in a multimeric complex. In addition, the ability of alleles which map near the carboxyl terminus to complement several other alleles provides evidence for a functional domain in this region of the protein. Two previously identified multicopy suppressors of the conditional hop1-628ts allele were tested for their effects in cells bearing the linker-insertion hop1 alleles. Overexpression of REC104 from a 2 mu plasmid was shown to enhance the spore viability of every allele tested, including a hop1 disruption allele. On the other hand, suppression by overexpression of RED1 from a 2 mu plasmid was found only for allele hop1-628ts. Surprisingly, similar overexpression of RED1 in strains bearing several other conditional hop1 linker-insertion alleles caused enhanced spore lethality. This finding, in conjunction with the evidence for a carboxy-terminal domain, provides new insight into the nature of interactions between the HOP1 and RED1 products in meiosis.

Linker Insertion Mutagenesis Of Drosophila Topoisomerase II : Probing The Structure Of Eukaryotic Topoisomerase II

The sequences of all type II DNA topoisomerases, and possibly some of their key structural features, are conserved. The N-terminal and middle regions of the eukaryotic DNA topoisomerase II are homologous to the bacterial gyrase subunits B and A, respectively, and the hydrophilic C-terminal region is more divergent among these enzymes. To gain further insights into the structure of eukaryotic topoisomerase II, we constructed 23 linker insertion mutants of Drosophila DNA topoisomerase II. These mutant proteins were expressed in a heterologous yeast system, in which we have previously demonstrated that Drosophila DNA topoisomerase II could he functionally expressed and complement yeast top2 mutations. The linker insertion mutants were characterized genetically by testing for complementation of yeast, top2 13 mutation at the non-permissive temperature and complementation of yeast top2 null mutation using a color sector assay. We also partially purified the mutant proteins and examined their enzymatic activity by unknotting the P4 knotted DNA. There appears to be a good correlation between the in vivo and in vitro activities. There are nine fully active, six partially active, and eight negative linker insertion mutants. All five linker insertion mutants in the C-terminal region are active and two linker insertion mutants located in the junction of the two regions homologous to gyrB/gyrA subunits are also active. In addition, we also mapped the trypsin-sensitive sites in Drosophila DNA topoisomerase II. The C-terminal region is extremely sensitive to trypsin digestion. Another major trypsin-sensitive site is located between Lys406 and Thr407, which is near the protease sites also observed in the bacterial gyrB subunit and yeast topoisomerase II. We discuss the possible structural and functional implications of these results.

Analysis of deletions and thermosensitive mutations in Rous sarcoma virus gag protein p10.

Rous sarcoma virus protein p10 is a gag component of the virion present in stoichiometric amount but of unknown function. To characterize this protein, a series of mutants of p10 with linker insertions or deletions was generated by site-directed mutagenesis of a cloned proviral DNA. The deletions and two of the linkers insertions, which disrupted proline pairs, reduced the yield of virus particles upon transfection. These two linker insertion mutants were moreover thermosensitive for this phenotype, producing fewer virus particles at 41 degrees C than at 36 degrees C. Examination of the intracellular viral proteins demonstrated that for all mutants, the amount of gag precursor was similar to the wild-type level. Moreover, the amount of mature gag CA that could be detected by this analysis was similar between each of the mutants and the wild type. This finding suggests that the transport of gag to the membrane and the initial stages of maturation were not affected by the mutations. The virus particles contained normal amounts of active reverse transcriptase, showing that the gag-pol polyprotein was incorporated and cleaved properly. Viral RNA was quantitatively and qualitatively similar in mutant and wild-type virions. However, the infectivity of the mutants virions differed; one of the thermosensitive linker insertions that had no effect on particle production at 36 degrees C was nevertheless noninfectious at that temperature. Together, these data suggest that the p10 protein is involved in a late steps of virus maturation, possibly budding, and perhaps also in an early event of viral infection.

Analysis of the terminal repeat binding abilities of mutant adeno-associated virus replication proteins.

The adeno-associated virus (AAV) Rep78 and Rep68 proteins play essential roles in viral DNA replication, trans activation of viral gene expression, and suppression of oncogene-mediated cellular transformation. By using an extensive set of linker insertion and deletion mutations in the replication gene, we mapped the regions of the Rep78 protein that mediate binding to the AAV origin of replication in vitro. Deletions that removed amino acid codons 25 to 62, 88 to 113, 125 to 256, and 346 to 400 abolished binding. Alterations in several other regions of the protein affected the binding affinity of the mutant proteins. All of the mutant proteins that support AAV DNA replication or p40 trans activation bound to the terminal repeat sequence, thus verifying the importance of binding for these functions. Several mutant rep genes that failed to suppress oncogene-mediated cellular transformation produced proteins that were capable of binding to the AAV terminal repeat sequences.

Functional domains of the yeast STE12 protein, a pheromone-responsive transcriptional activator.

The pheromone response pathway of the yeast Saccharomyces cerevisiae is necessary for the basal level of transcription of cell-type-specific genes, as well as the induced level observed after pheromone treatment. The STE12 protein binds to the DNA sequence designated the pheromone response element and is a target of the pheromone-induced signal. We generated 6-nucleotide linker insertion mutants, internal-deletion mutants, and carboxy-terminal truncation mutants of STE12 and assayed them for their ability to restore mating and transcriptional activity to a ste12 delta strain. Two of these mutant proteins retain the capacity to mediate basal transcription but show little or no induced transcription upon pheromone treatment. Cells producing these proteins cannot mate, formally demonstrating that the ability to respond to pheromone by increasing gene expression is essential for the mating process. Since distinct domains of STE12 appear to be required for basal versus induced transcription, we suggest that the pheromone-induced signal is likely to target residues of the protein different from those targeted by the basal signal because of the constitutive activity of the response pathway. Our analysis of mutant STE12 proteins also indicates that only the DNA-binding domain is sensitive to the small changes caused by the linker insertions. In addition, we show that, while the carboxy-terminal sequences necessary for STE12 to form a complex with the transcription factor MCM1 are not essential for mating, these sequences are required for optimal transcriptional activity.

Functional domains of the yeast STE12 protein, a pheromone-responsive transcriptional activator.

The pheromone response pathway of the yeast Saccharomyces cerevisiae is necessary for the basal level of transcription of cell-type-specific genes, as well as the induced level observed after pheromone treatment. The STE12 protein binds to the DNA sequence designated the pheromone response element and is a target of the pheromone-induced signal. We generated 6-nucleotide linker insertion mutants, internal-deletion mutants, and carboxy-terminal truncation mutants of STE12 and assayed them for their ability to restore mating and transcriptional activity to a ste12 delta strain. Two of these mutant proteins retain the capacity to mediate basal transcription but show little or no induced transcription upon pheromone treatment. Cells producing these proteins cannot mate, formally demonstrating that the ability to respond to pheromone by increasing gene expression is essential for the mating process. Since distinct domains of STE12 appear to be required for basal versus induced transcription, we suggest that the pheromone-induced signal is likely to target residues of the protein different from those targeted by the basal signal because of the constitutive activity of the response pathway. Our analysis of mutant STE12 proteins also indicates that only the DNA-binding domain is sensitive to the small changes caused by the linker insertions. In addition, we show that, while the carboxy-terminal sequences necessary for STE12 to form a complex with the transcription factor MCM1 are not essential for mating, these sequences are required for optimal transcriptional activity.

Mutational analysis of the envelope gene of Moloney murine leukemia virus.

The env gene products of Moloney murine leukemia virus are required for binding and entry of the virus into the target cell. Thirty-three linker insertion mutations were constructed throughout the env gene of Moloney murine leukemia virus. Twenty of the mutations were located in the surface protein (SU), and the remaining thirteen were located in the transmembrane protein (TM). The viability of the viruses containing these env gene mutations was determined by performing transient transfections and screening for the release of reverse transcriptase. Eleven viable mutants were isolated, nine in SU and two in TM. Three of the viable mutants were temperature sensitive. Four of the viable mutants were clustered in the carboxy terminus of SU. The env gene products of transfected cell lines which produced viable virus were analyzed. Our results indicated two regions of SU important for the stability of the SU/TM heteropolymer and one region important for the interaction of the env gene products with the viral core.

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis

A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step.

Mutational analysis of the adeno-associated virus rep gene.

The replication (rep) gene of the human parvovirus adeno-associated virus (AAV) is a pleiotropic effector of numerous viral functions and experts profound effects on cellular transformation. Of the four Rep proteins, the primarily nuclear Rep78 and Rep68 direct AAV DNA replication, trans activation of the capsid (cap) gene promoter, and inhibition of cellular proliferation mediated by various oncogenes. In an initial attempt to define functional domains in Rep78, we have constructed a comprehensive set of XhoI linker insertion and deletion mutations in the rep gene. Each of the mutant genes has been expressed in cell culture and assayed for the following functions: (i) nuclear localization, (ii) AAV DNA replication, (iii) trans activation of the AAV capsid gene transcription promoter, and (iv) suppression of cellular transformation mediated by the adenovirus E1a and an activated ras oncogene pair. Modest disruptions in the normal conformation of Rep78 inactivated its AAV DNA replication function and trans activation of the cap gene promoter. Linker insertion mutations in the amino-terminal one-third of the protein inactivated Rep78's ability to suppress oncogene-mediated cellular transformation. The transformation suppression domains are not limited to the amino-terminal regions, however, since deletions throughout the protein altered its suppression capabilities. A putative nuclear localization signal that is essential for each of the above functions was found in the Rep proteins. These results provide a preliminary screening of the functional domains in the AAV Rep proteins and pave the way for more subtle mutational analysis.

Mutations in two cysteine-histidine-rich clusters in adenovirus type 2 DNA polymerase affect DNA binding.

Several point and linker insertion mutations in two Cys-His-rich regions of adenovirus (Ad) DNA polymerase (Pol) gene have been expressed in recombinant vaccinia virus. The resulting mutant enzymes were analyzed in vitro for their effects on DNA synthesis activity, on Ad-specific initiation assays, on gel shifts of Ad origin sequences, and on interactions with adenovirus preterminal protein (pTP) and nuclear factor I (NFI). In general, mutants in downstream Cys-His sequences had a pronounced effect in these assays. Mutants in the upstream Cys-His region had a moderate effect on DNA synthesis and elongation but failed to make dCMP-pTP initiation complexes and failed to make specific shifted complexes in a gel retardation assay. These mutants could still bind to pTP and NFI in a coimmunoprecipitation experiment, suggesting that this upstream Cys-His region of Ad Pol is involved either in specific Ad DNA origin binding or in nonspecific DNA binding. Changing residues within Cys doublets in the downstream Cys-His region had pronounced effects on many Ad Pol functions such as DNA synthesis, DNA binding, and in vitro initiation; however, these mutants showed little reduction in binding to pTP and NFI; mutants at other cysteines or histidines within this region of Ad Pol did not appear to have an effect on enzyme function. This observation suggests that the downstream Cys-His region of Ad Pol is important for DNA binding and might fold into a Zn finger motif.

Structural basis of C3b binding by glycoprotein C of herpes simplex virus.

Glycoproteins C (gC) from herpes simplex virus type 1 (HSV-1) and HSV-2, gC-1 and gC-2, bind the human complement fragment C3b, although the two glycoproteins differ in their abilities to act as C3b receptors on infected cells and in their effects on the alternative complement pathway. Previously, we identified three regions of gC-2 (I, II, and III) which are important for C3b binding. In this study, our goal was to identify C3b-binding sites on gC-1 and to continue our analysis of gC-2. We constructed a large panel of mutants by using the cloned gC-1 and gC-2 genes. Most of the mutant proteins were transported to the surface of transiently transfected L cells and reacted with one or more monoclonal antibodies to discontinuous epitopes. By using 31 linker insertion mutants spread across the coding region of gC-1, we identified four regions in the ectodomain of gC-1 which are important for C3b binding, three of which are similar in position to C3b-binding regions I, II, and III of gC-2. Region III shares some similarities with the short consensus repeat found in CR1, the human complement receptor. These were, in part, the targets for construction of 20 single amino acid changes in region III of gC-1 and gC-2. These mutants identified similarities and differences in the C3b-binding properties of gC-1 and gC-2 and suggest that the amino half of region III is more important for C3b binding. However, our results do not support the concept of a structural relationship between the short consensus repeat of CR1 and gC, since mutations of some of the conserved residues, including three of four cysteines in region III, had no effect on C3b binding. Finally, we constructed four deletion mutants of gC-1, including one which lacked residues 33 to 123, as well as residues 367 to 449. This severely truncated molecule, lacking four cysteines and five potential N-linked glycosylation sites, was transported to the cell surface and retained its ability to bind monoclonal antibodies as well as C3b. Thus, the four distinct C3b-binding regions of gC-1 and several epitopes within two different antigenic sites are localized within residues 124 to 366.

The GC box and TATA transcription control elements in the P38 promoter of the minute virus of mice are necessary and sufficient for transactivation by the nonstructural protein NS1.

To further define the transcriptional regulation of the P38 promoter in the minute virus of mice (MVM) genome, we constructed a series of internal deletion and linker scanning mutations. The mutant P38 constructs were assayed for transcriptional activity in vitro by primer extension analysis with nuclear extracts from murine A92L fibroblasts. Mutations which disrupted the GC box and TATA box severely reduced transcription in vitro. DNase I footprinting analysis confirmed that the murine transcription factor Sp1 bound to the GC box; however, no factors were observed interacting with a putative transcriptional activation regulatory element, termed the TAR element. The linker scanning mutations were analyzed in vivo by using a chloramphenicol acetyltransferase expression assay system, in both the presence and absence of constructs expressing the viral nonstructural protein, NS1. The ability of NS1 to transactivate the P38 promoter (up to 1,000-fold) depended entirely on the presence of intact GC and TATA box sequences. Disruption of the TAR element by either linker insertion mutations or an internal deletion did not inhibit transactivation of the P38 promoter. These results suggest that NS1 transactivates the P38 promoter indirectly by interacting with one or more components of the P38 core-transcription complex.