Structure, stability, and biological activity of bacteriophage T4 gene product 9 probed with mutagenesis and monoclonal antibodies

Gene product (gp) 9 connects the long tail fibers and triggers the structural transition of T4 phage baseplate at the beginning of infection process. Gp9 is a parallel homotrimer with 288 amino acid residues per chain that forms three domains. To investigate the role of the gp9 amino terminus, we have engineered a set of mutants with deletions and random substitutions in this part. The structure of the mutants was probed using monoclonal antibodies that bind to either N-terminal, middle, or C-terminal domains. Deletions of up to 12 N-terminal residues as well as random substitutions of the second, third and fourth residues yielded trimers that failed to incorporate in vitro into the T4 9(-)-particles and were not able to convert them into infectious virions. As detected using monoclonal antibodies, these mutants undergo structural changes in both N-terminal and middle domains. Furthermore, deletion of the first twenty residues caused profound structural changes in all three gp9 domains. In addition, N-terminally truncated proteins and randomized mutants formed SDS-resistant "conformers" due to unwinding of the N-terminal region. Co-expression of the full-length gp9 and the mutant lacking first 20 residues clearly shows the assembly of heterotrimers, suggesting that the gp9 trimerization in vivo occurs post-translationally. Collectively, our data indicate that the aminoterminal sequence of gp9 is important to maintain a competent structure capable of incorporating into the baseplate, and may be also required at intermediate stages of gp9 folding and assembly.     

A variant protein from phi29 replication gene, gene 1, did not form homo-polymer due to a single amino acid substitution near the carboxyl terminus

For study of the self-association of the product of psi29 gene 1, one variant which has a substitution at the 71(st) amino acid was used. By glycerol gradient sedimentation, the product of wild-type gene 1 existed both as large aggregate and oligomer, whereas the variant was detected as a single peak of monomer size. According to experiments using His-tagged proteins and Ni-NTA magnetic beads, the variant made only a little self-associated complex. From these results, a site essential for self-association was suggested to exist close to the carboxyl terminus of the product of psi29 gene 1.     

Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding

We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.     

In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity.

Site-specific mutagenesis was used to introduce amino acid substitutions at the asparagine codons of four conserved potential N-linked glycosylation sites within the gp120 envelope protein of human immunodeficiency virus (HIV). One of these alterations resulted in the production of noninfectious virus particles. The amino acid substitution did not interfere with the synthesis, processing, and stability of the env gene polypeptides gp120 and gp41 or the binding of gp120 to its cellular receptor, the CD4 (T4) molecule. Vaccinia virus recombinants containing wild-type or mutant HIV env genes readily induced syncytia in CD4+ HeLa cells. These results suggest that alterations involving the second conserved domain of the HIV gp120 may interfere with an essential early step in the virus replication cycle other than binding to the CD4 receptor. In long-term cocultures of a T4+ lymphocyte cell line and colon carcinoma cells producing the mutant virus, revertant infectious virions were detected. Molecular characterization of two revertant proviral clones revealed the presence of the original mutation as well as a compensatory amino acid change in another region of HIV gp120.     

Conserved residue Lys574 in the cavity of HIV-1 Gp41 coiled-coil domain is critical for six-helix bundle stability and virus entry

The fusion-active HIV-1 gp41 core structure is a stable six-helix bundle (6-HB) formed by its N- and C-terminal heptad-repeat sequences (NHR and CHR). A highly conserved, deep hydrophobic cavity on the surface of the N-helical trimer is important for stability of the 6-HB and serves as an ideal target for developing anti-human immunodeficiency virus (HIV) fusion inhibitors. We have recently identified several small molecule HIV-1 fusion inhibitors that bind to the gp41 cavity through hydrophobic and ionic interactions and block the gp41 6-HB formation. Molecular docking analysis reveals that these small molecules fit inside the hydrophobic cavity and interact with positively charged residue Lys574 to form a conserved salt bridge. In this study, the functionality of Lys574 has been finely characterized by mutational analysis and biophysical approaches. We found that substitutions of Lys574 with non-conserved residues (K574D, K574E, and K574V) could completely abolish virus infectivity. With a set of wild-type and mutant N36 peptides derived from the NHR sequence as a model, we demonstrated that non-conservative Lys574 substitutions severely impaired the stability and conformation of 6-HBs as detected by circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. The binding affinity of N36 mutants bearing non-conservative Lys574 substitutions to the peptide C34 derived from the CHR sequence dramatically decreased as measured by isothermal titration calorimetry. These substitutions also significantly reduced the potency of N-peptides to inhibit HIV-1 infection. Collectively, these data suggest that conserved Lys574 plays a critical role in 6-HB formation and HIV-1 infectivity, and may serve as an important target for designing anti-HIV drugs.     

In eukaryotic flap endonuclease 1, the C terminus is essential for substrate binding

Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease with important functions in DNA replication and DNA repair. It interacts like many other proteins involved in DNA metabolic events with proliferating cell nuclear antigen (PCNA), and its enzymatic activity is stimulated by PCNA in vitro. The PCNA interaction site is located close to the C terminus of Fen1 and is flanked by a conserved basic region of 35-38 amino acids in eukaryotic species but not in archaea. We have constructed two deletion mutants of human Fen1 that lack either the PCNA interaction motif or a part of its adjacent C-terminal region and analyzed them in a variety of assays. Remarkably, deletion of the basic C-terminal region did not affect PCNA interaction but resulted in a protein with significantly reduced enzymatic activity. Electrophoretic mobility shift analysis revealed that this mutant displayed a severe defect in substrate binding. Our results suggest that the C terminus of eukaryotic Fen1 consists of two functionally distinct regions that together might form an important regulatory domain.     

The highly conserved aspartic acid residue between hypervariable regions 1 and 2 of human immunodeficiency virus type 1 gp120 is important for early stages of virus replication.

Between hypervariable regions V1 and V2 of human immunodeficiency virus type 1 (HIV-1) gp120 lies a cluster of relatively conserved residues. The contribution of nine charged residues in this region to virus infectivity was evaluated by single-amino-acid substitutions in an infectious provirus clone. Three of the HIV-1 mutants studied had slower growth kinetics than the wild-type virus. The delay was most pronounced in a mutant with an alanine substituted for an aspartic acid residue at position 180. This aspartic acid is conserved by all HIV-1 isolates with known nucleotide sequences. Substitutions with three other residues at this position, including a negatively charged glutamic acid, all affected virus infectivity. The defect identified in these mutants suggests that this aspartic acid residue is involved in the early stages of HIV-1 replication.     

Interhelical interactions in the gp41 core: implications for activation of HIV-1 membrane fusion

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (gp120-gp41) promotes viral entry by mediating the fusion of viral and cellular membranes. Formation of a stable trimer-of-hairpins structure in the gp41 ectodomain brings the two membranes into proximity, leading to membrane fusion. The core of this hairpin structure is a six-helix bundle in which three carboxyl-terminal outer helices pack against an inner trimeric coiled coil. Here we investigate the role of these conserved interhelical interactions on the structure and function of both the envelope glycoprotein and the gp41 core. We have replaced each of the eight amino acids at the buried face of the carboxyl-terminal helix with a representative amino acid, alanine. Structural and physicochemical characterization of the alanine mutants shows that hydrophobic interactions are a dominant factor in the stabilization of the six-helix bundle. Alanine substitutions at the Trp628, Trp631, Ile635, and Ile642 residues also affected envelope processing and/or gp120-gp41 association and abrogated the ability of the envelope glycoprotein to mediate cell-cell fusion. These results suggest that the amino-terminal region of the gp41 outer-layer alpha-helix plays a key role in the sequence of events associated with HIV-1 entry and have implications for the development of antibodies and small-molecule inhibitors of this conserved element.     

The Bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage DNA replication

Mycobacteriophage Bxb1 encodes a serine-integrase that catalyzes both integrative and excisive site-specific recombination. However, excision requires a second phage-encoded protein, gp47, which serves as a recombination directionality factor (RDF). The viability of a Bxb1 mutant containing an S153A substitution in gp47 that eliminates the RDF activity of Bxb1 gp47 shows that excision is not required for Bxb1 lytic growth. However, the inability to construct a Δ47 deletion mutant of Bxb1 suggests that gp47 provides a second function that is required for lytic growth, although the possibility of an essential cis-acting site cannot be excluded. Characterization of a mutant prophage of mycobacteriophage L5 in which gene 54 - a homologue of Bxb1 gene 47 - is deleted shows that it also is defective in induced lytic growth, and exhibits a strong defect in DNA replication. Bxb1 gp47 and its relatives are also unusual in containing conserved motifs associated with a phosphoesterase function, although we have not been able to show robust phosphoesterase activity of the proteins, and amino acid substitutions with the conserved motifs do not interfere with RDF activity. We therefore propose that Bxb1 gp47 and its relatives provide an important function in phage DNA replication that has been co-opted by the integration machinery of the serine-integrases to control the directionality of recombination.     

Highly Conserved Residues in the Helical Domain of Dengue Virus Type 1 Precursor Membrane Protein Are Involved in Assembly,Precursor Membrane (prM) Protein Cleavage,and Entry

The envelope and precursor membrane (prM) proteins of dengue virus (DENV) are present on the surface of immature virions. During maturation, prM protein is cleaved by furin protease into pr peptide and membrane (M) protein. Although previous studies mainly focusing on the pr region have identified several residues important for DENV replication, the functional role of M protein, particularly the α-helical domain (MH), which is predicted to undergo a large conformational change during maturation, remains largely unknown. In this study, we investigated the role of nine highly conserved MH domain residues in the replication cycle of DENV by site-directed mutagenesis in a DENV1 prME expression construct and found that alanine substitutions introduced to four highly conserved residues at the C terminus and one at the N terminus of the MH domain greatly affect the production of both virus-like particles and replicon particles. Eight of the nine alanine mutants affected the entry of replicon particles, which correlated with the impairment in prM cleavage. Moreover, seven mutants were found to have reduced prM-E interaction at low pH, which may inhibit the formation of smooth immature particles and exposure of prM cleavage site during maturation, thus contributing to inefficient prM cleavage. Taken together, these results are the first report showing that highly conserved MH domain residues, located at 20–38 amino acids downstream from the prM cleavage site, can modulate the prM cleavage, maturation of particles, and virus entry. The highly conserved nature of these residues suggests potential targets of antiviral strategy.     

Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages

The filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram-negative bacterial proteins that are essential components of a widely distributed extracellular protein-export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non-functional, interfered with assembly of plV⁺ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non-functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.     

An efficient phage plaque screen for the random mutational analysis of the interaction of HIV-1 gp120 with human CD4.

A lambda phage expression methodology was adapted to dissect protein/ligand interactions efficiently through the creation and rapid screening of large numbers of mutants. Here we describe the method and its specific application to the interaction between the external envelope glycoprotein of the human immunodeficiency virus (HIV-1), gp120, and the human cell surface protein CD4. Random substitutions were introduced throughout the gp120 binding region (amino acids 38-62) in the amino-terminal domain of CD4 by oligonucleotide mutagenesis. These mutations were expressed within phage plaques and directly screened for their effect on binding of gp120 using a modified phage plaque lift procedure. Plaques showing increased, decreased, and no effect on binding were identified and mutations were verified by sequence analysis. In this manner, 25 unique mutations were identified that altered CD4 binding to gp120. A new site was identified at which mutations reduced binding to gp120 and several novel amino acid substitutions were defined at sites previously implicated in binding. Of particular interest, this in vitro genetic approach identified a mutation which significantly increased binding to gp120. The phenotypes of several of these mutants were further characterized by quantitative measurement of their binding affinity. The results confirmed the accuracy of the phenotypic selection and demonstrated that the sensitivity of the system allowed detection of a 3-4-fold increase or decrease in affinity. In the context of the recently determined atomic structure of CD4, these results further implicate residues in the CDR2-like region and in an adjacent loop in recognition of gp120. This methodology should be generally applicable to other high affinity protein/ligand interactions that are compatible with expression in Escherichia coli.     

An essential tryptophan of Escherichia coli DnaA protein functions in oligomerization at the E. coli replication origin

In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.     

Sequence and expression of the mouse mammary tumour virus env gene

We have determined the DNA sequence of the envelope gene region of the GR strain of mouse mammary tumour virus. The sequence extends for 3012 nucleotides from the single EcoRI site to beyond the PstI site in the 3' long terminal repeat (LTR) of the provirus. There is a major open reading frame from nucleotides 752 to 2818 which encompasses the entire env gene. This reading frame extends through a polypurine tract and into the LTR. There is another open reading frame from the first nucleotide to position 803, presumably corresponding to the end of the pol gene. The splice acceptor site which generates env mRNA has been mapped experimentally to nucleotide 750. The env gene products, gp52 and gp36, have been positioned on the sequence using the directly determined amino acid sequences of the amino terminus of gp52; and both the amino and carboxyl termini of gp36. The start of gp52 is preceded by a series of 19 uncharged amino acids which could function as a typical signal sequence, but this sequence is only part of a much longer leader peptide. The tetrad Arg-Ala-Lys-Arg is the presumed cleavage site in the gPr73env precursor, and occurs just before the gp36 amino terminus. There are five potential asparagine-linked glycosylation sites which agrees with previous experimental results. The gp36 has two long hydrophobic regions at its amino and carboxy termini, these are suggested to act as a fusion peptide and the trans-membrane anchor, respectively.     

Essential residues in the C terminus of the bacteriophage T7 gene 2.5 single-stranded DNA-binding protein

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA (ssDNA)-binding protein (gp2.5) that is an essential component of the phage replisome. Similar to other prokaryotic ssDNA-binding proteins, gp2.5 has an acidic C terminus that is involved in protein-protein interactions at the replication fork and in modulation of the ssDNA binding properties of the molecule. We have used genetic and biochemical approaches to identify residues critical for the function of the C terminus of gp2.5. The presence of an aromatic residue in the C-terminal position is essential for gp2.5 function. Deletion of the C-terminal residue, phenylalanine, is detrimental to its function, as is the substitution of this residue with non-aromatic amino acids. Placing the C-terminal phenylalanine in the penultimate position also results in loss of function. Moderate shortening of the length of the acidic portion of the C terminus is tolerated when the aromatic nature of the C-terminal residue is preserved. Gradual removal of the acidic C terminus of gp2.5 results in a higher affinity for ssDNA and a decreased ability to interact with T7 DNA polymerase/thioredoxin. The replacement of the charged residues in the C terminus with neutral amino acids abolishes gp2.5 function. Our data show that both the C-terminal aromatic residue and the overall acidic charge of the C terminus of gp2.5 are critical for its function.     

Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding.

Interaction with the CD4 receptor enhances the exposure on the human immunodeficiency type 1 gp120 exterior envelope glycoprotein of conserved, conformation-dependent epitopes recognized by the 17b and 48d neutralizing monoclonal antibodies. The 17b and 48d antibodies compete with anti-CD4 binding antibodies such as 15e or 21h, which recognize discontinuous gp120 sequences near the CD4 binding region. To characterize the 17b and 48d epitopes, a panel of human immunodeficiency virus type 1 gp120 mutants was tested for recognition by these antibodies in the absence or presence of soluble CD4. Single amino acid changes in five discontinuous, conserved, and generally hydrophobic regions of the gp120 glycoprotein resulted in decreased recognition and neutralization by the 17b and 48d antibodies. Some of these regions overlap those previously shown to be important for binding of the 15e and 21h antibodies or for CD4 binding. These results suggest that discontinuous, conserved epitopes proximal to the binding sites for both CD4 and anti-CD4 binding antibodies become better exposed upon CD4 binding and can serve as targets for neutralizing antibodies.     

Identification of individual human immunodeficiency virus type 1 gp120 amino acids important for CD4 receptor binding.

The binding of the CD4 receptor by the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein is important for virus entry and cytopathic effect. To investigate the CD4-binding region of the gp120 glycoprotein, we altered gp120 amino acids, excluding cysteines, that are conserved among the primate immunodeficiency viruses utilizing the CD4 receptor. Changes in two hydrophobic regions (Thr-257 in conserved region 2 and Trp-427 in conserved region 4) and two hydrophilic regions (Asp-368 and Glu-370 in conserved region 3 and Asp-457 in conserved region 4) resulted in significant reductions in CD4 binding. For most of the mutations affecting these residues, the observed effects on CD4 binding did not apparently result from global conformational disruption of the gp120 molecule, as assessed by measurements of precursor processing, subunit association, and monoclonal antibody recognition. The two hydrophilic regions exhibit a strong propensity for beta-turn formation, are predicted to act as efficient B-cell epitopes, and are located adjacent to hypervariable, glycosylated regions. This study defines a small number of gp120 residues important for CD4 binding, some of which might constitute attractive targets for immunologic intervention.