Analyses of single-amino-acid substitution mutants of adenovirus type 5 E1B-55K protein

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.     

Limited temperature-sensitive transactivation by mutant adenovirus type 2 E1a proteins.

A series of linker-scanning and deletion mutations was generated in the transactivating domain of the larger, 289-amino-acid-residue E1a protein of adenovirus type 2. Mutant genes were recombined into virus to assay the ability of the variant E1a proteins to activate expression of an E1a-dependent viral gene during infection. Results of assays performed at 32, 37, and 40 degrees C indicated that at least 2 of the 10 mutants tested showed limited temperature sensitivity for transactivation.     

A large compressibility change of protein induced by a single amino acid substitution.

The adiabatic compressibility (beta s) was determined, by means of the precise sound velocity and density measurements, for a series of single amino acid substituted mutant enzymes of Escherichia coli dihydrofolate reductase (DHFR) and aspartate aminotransferase (AspAT). Interestingly, the beta s values of both DHFR and AspAT were influenced markedly by the mutations at glycine-121 and valine-39, respectively, in which the magnitude of the change was proportional to the enzyme activity. This result demonstrates that the local change of the primary structure plays an important role in atomic packing and protein dynamics, which leads to the modified stability and enzymatic function. This is the first report on the compressibility of mutant proteins.     

Transformation-defective mutant of adenovirus type 5 containing a single altered E1a mRNA species.

A mutant of adenovirus type 5 containing an octanucleotide insert in region E1a of the viral genome was constructed. The insert was present in only one (13s) of the three overlapping mRNA's synthesized from this region. The insert was within the sequences removed by RNA splicing during the production of the other two nRNA's. The insertion resulted in a shift in the translational reading frame of the 13s mRNA and the probable premature termination of translation. The mutant was defective for viral DNA replication in HeLa cells and the transformation of rat embryo and baby rat kidney cells, indicating that a product encoded by the 13s nRNA is required for these two processes. Other early regions of the genome were expressed in HeLa cells infected by this mutant although in some cases the expression was decreased as compared with wild-type-infected cells.     

A single amino acid substitution reduces the superhelicity requirement of a replication initiator protein.

The origin of rolling circle replication in filamentous coliphage consists of a core origin that is absolutely required and an adjacent replication enhancer sequence that increases in vivo replication 30 to 100-fold. The core origin binds the initiator protein (gpII) which either nicks or relaxes negatively superhelical replicative form DNA (RFI). Nicking at the origin, but not relaxation, leads to initiation of DNA replication. Our results indicate that the ratio of nicking to relaxation (nicking-closing) in vitro depends on the superhelical density of the substrate. We have studied the effect of a single amino acid substitution in gpII, which allows wild-type levels of replication in the absence of the enhancer, on origin nicking and binding. The enhancer-independent mutation yields more nicking and less relaxation of RFI, compared to the wild-type protein. The mutant gpII also shows a reduced requirement for superhelicity of the substrate in the nicking reaction. At the same time, the mutant gpII increases the cooperativity of protein-protein interactions in origin binding. We propose that the relaxation activity of gpII negatively regulates replication initiation, and that both increase in the negative superhelicity of the substrate and action of the replication enhancer may antagonize the relaxation activity.     

A single amino acid substitution in yeast eIF-5A results in mRNA stabilization.

Most factors known to function in mRNA turnover are not essential for cell viability. To identify essential factors, approximately 4000 temperature-sensitive yeast strains were screened for an increase in the level of the unstable CYH2 pre-mRNA. At the non-permissive temperature, five mutants exhibited decreased decay rates of the CYH2 pre-mRNA and mRNA, and the STE2, URA5 and PAB1 mRNAs. Of these, the mutant ts1159 had the most extensive phenotype. Expression of the TIF51A gene (encoding eIF-5A) complemented the temperature-sensitive growth and mRNA decay phenotypes of ts1159. The tif51A allele was rescued from these cells and shown to encode a serine to proline change within a predicted alpha-helical segment of the protein. ts1159 also exhibited an approximately 30% decrease in protein synthesis at the restrictive temperature. Measurement of amino acid incorporation in wild-type cells incubated with increasing amounts of cycloheximide demonstrated that a decrease in protein synthesis of this magnitude could not account for the full extent of the mRNA decay defects observed in ts1159. Interestingly, the ts1159 cells accumulated uncapped mRNAs at the non-permissive temperature. These results suggest that eIF-5A plays a role in mRNA turnover, perhaps acting downstream of decapping.     

An amino acid substitution that blocks the deacylation step in the enzyme mechanism of penicillin-binding protein 5 of Escherichia coli

A mutant of Escherichia coli has been described that produces an altered form of penicillin-binding protein 5 which still binds penicillin but is unable to catalyse the release of the bound penicilloyl moiety. We show that the mutation is caused by a single nucleotide transition that results in a change from glycine at residue 105 of the wild-type sequence of penicillin-binding protein 5 to aspartate in the mutant.     

Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein.

The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells.     

A single amino acid substitution in SecY stabilizes the interaction with SecA.

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.     

A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus

Two different morphogenic processes of retroviral capsid assembly have been observed: the capsid is either assembled at the plasma membrane during the budding process (type C), or preassembled within the cytoplasm (types B and D). We describe here a gag mutant of Mason-Pfizer monkey virus, a type D retrovirus, in which a tryptophan substituted for an arginine in the matrix protein results in efficient assembly of capsids at the plasma membrane through a morphogenic process similar to that of type C retroviruses. We conclude that a type D retrovirus Gag polyprotein contains an additional, dominant signal that prevents immediate transport of precursors from the site of biosynthesis to the plasma membrane. Instead, they are directed to and retained at a cytoplasmic site where a concentration sufficient for self-assembly into capsids occurs. Thus, capsid assembly processes for different retroviruses appear to differ only in the intracellular site to which capsid precursors are directed.     

A single amino acid substitution in envelope protein E of tick-borne encephalitis virus leads to attenuation in the mouse model.

We have determined the virulence characteristics of seven monoclonal antibody escape mutants of tick-borne encephalitis virus in the mouse model. One of the mutants with an amino acid substitution from tyrosine to histidine at residue 384 revealed strongly reduced pathogenicity after peripheral inoculation of adult mice but retained its capacity to replicate in the mice and to induce a high-titered antibody response. Infection with the attenuated mutant resulted in resistance to challenge with virulent virus. Assessment of nonconservative amino acid substitutions in other attenuated flaviviruses suggests that a structural element including residue 384 may represent an important determinant of flavivirus virulence in general.     

An amino acid substitution matrix for protein conformation identification

Amino acid substitution matrices play an essential role in protein sequence alignment, a fundamental task in bioinformatics. Most widely used matrices, such as PAM matrices derived from homologous sequences and BLOSUM matrices derived from aligned segments of PROSITE, did not integrate conformation information in their construction. There are a few structure-based matrices, which are derived from limited data of structure alignment. Using databases PDB_SELECT and DSSP, we create a database of sequence-conformation blocks which explicitly represent sequence-structure relationship. Members in a block are identical in conformation and are highly similar in sequence. From this block database, we derive a conformation-specific amino acid substitution matrix CBSM60. The matrix shows an improved performance in conformational segment search and homolog detection.     

A single amino acid substitution in the movement protein enables the mechanical transmission of a geminivirus

Begomoviruses of the Geminiviridae are usually transmitted by whiteflies and rarely by mechanical inoculation. We used tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, to address this issue. Most ToLCNDV isolates are not mechanically transmissible to their natural hosts. The ToLCNDV-OM isolate, originally identified from a diseased oriental melon plant, is mechanically transmissible, while the ToLCNDV-CB isolate, from a diseased cucumber plant, is not. Genetic swapping and pathological tests were performed to identify the molecular determinants involved in mechanical transmission. Various viral infectious clones were constructed and successfully introduced into Nicotiana benthamiana, oriental melon, and cucumber plants by Agrobacterium-mediated inoculation. Mechanical transmissibility was assessed via direct rub inoculation with sap prepared from infected N. benthamiana. The presence or absence of viral DNA in plants was validated by PCR, Southern blotting, and in situ hybridization. The results reveal that mechanical transmissibility is associated with the movement protein (MP) of viral DNA-B in ToLCNDV-OM. However, the nuclear shuttle protein of DNA-B plays no role in mechanical transmission. Analyses of infectious clones carrying a single amino acid substitution reveal that the glutamate at amino acid position 19 of MP in ToLCNDV-OM is critical for mechanical transmissibility. The substitution of glutamate with glycine at this position in the MP of ToLCNDV-OM abolishes mechanical transmissibility. In contrast, the substitution of glycine with glutamate at the 19th amino acid position in the MP of ToLCNDV-CB enables mechanical transmission. This is the first time that a specific geminiviral movement protein has been identified as a determinant of mechanical transmissibility.     

A single amino acid substitution in sigma E affects its ability to bind core RNA polymerase.

We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis. sigma E is required for the production of endospores in B. subtilis but not for vegetative growth. Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores. Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase. The substitution at position 68 did not affect the stability of the protein in B. subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor. The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.