Forced evolution of a regulatory RNA helix in the HIV-1 genome.

The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.     

Functional interaction of constant and variable domains of human immunodeficiency virus type 1 gp120.

A previously reported amino acid substitution within the second conserved domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope results in the production of noninfectious particles. Molecular characterization of spontaneous revertant viruses, which arose during long-term cocultures of this env mutant, revealed that an amino acid change within another region of gp120 could functionally compensate for the mutation and restore infectivity. In the current study, we have introduced a conservative amino acid substitution at this second-site revertant codon and observed a marked reduction in HIV-1 infectivity. During the passage of this defective virus in cocultures, yet another revertant appeared which contained an amino acid change within a variable region of gp120 which restored infectivity to near wild-type levels. These results, in combination with other point mutations that have been introduced into the HIV-1 envelope, suggest that at least three discrete regions of gp120 may interact during the establishment of a productive viral infection. This critical step occurs subsequent to the adsorption of virions to the cell surface and either prior to or concomitant with the fusion of viral and cellular membranes.     

Interaction of Moloney murine leukemia virus matrix protein with IQGAP

The matrix protein (MA) of the Moloney murine leukemia virus (M-MuLV) was found to interact with IQGAP1, a prominent regulator of the cytoskeleton. Mutational studies defined residues of MA critical for the interaction, and tests of viruses carrying MA mutations revealed a near-perfect correlation between binding and virus replication. The replication-defective mutants showed defects in both early and late stages of the life cycle. Four viable second-site revertant viruses were isolated from three different replication-defective parental mutants, and in all cases the interaction with IQGAP1 was restored by the suppressor mutations. The interaction of MA and IQGAP1 was readily detected in vitro and in vivo. Virus replication was potently inhibited by a C-terminal fragment of IQGAP1, and impaired by RNAi knockdown of IQGAP1 and 2. We suggest that the IQGAPs link the virus to the cytoskeleton for trafficking both into and out of the cell.     

Isolation and characterization of temperature-sensitive and thermostable mutants of the human receptor-like protein tyrosine phosphatase LAR.

Human LAR is a transmembrane receptor-like protein whose cytoplasmic region contains two tandemly duplicated domains homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain I has enzymatic activity, the membrane-distal domain II has no apparent catalytic activity but seems to have a regulatory function. In order to study structure-function relationships of the LAR PTPase, LAR domain I was expressed in Escherichia coli, and mutants that have reduced catalytic activity or reduced thermostability were isolated and characterized. We isolated 18 unique hydroxylamine-induced missense mutations in the LAR domain I segment, of which three were temperature-sensitive. Five additional temperature-sensitive mutations were isolated using N-methyl-N'-nitro-N-nitrosoguanidine. All eight temperature-sensitive mutations are confined within a short segment of the LAR domain I sequence between amino acid positions 1329 and 1407. To examine whether this region is particularly prone to temperature-sensitive mutations, tyrosine at amino acid position 1379 was changed to a phenylalanine by oligonucleotide-directed mutagenesis. This mutant, Y1379-F, was indeed temperature-sensitive. We also isolated a revertant of a temperature-sensitive mutant. The revertant contained a second-site mutation (C1446-Y) that suppresses several temperature-sensitive mutations and also enhances the folding of LAR protein produced in E. coli.     

Repair of a Rev-minus human immunodeficiency virus type 1 mutant by activation of a cryptic splice site

We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the tat-rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5' splice site (ss) that, when used in conjunction with the regular HIV 3' ss #5, fuses the tat and rev reading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5' ss by mutational inactivation of an adjacent exon splicing silencer element.     

Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression.

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.     

Evaluation of the genetic stability of the temperature-sensitive PB2 gene mutation of the influenza A/Ann Arbor/6/60 cold-adapted vaccine virus.

A single-gene reassortant bearing the PB2 gene of the A/Ann Arbor/6/60 cold-adapted virus in the background of the A/Korea/82 (H3N2) wild-type virus is a temperature-sensitive (ts) virus with an in vitro shutoff temperature of 38 degrees C. A single mutation at amino acid (aa) at 265 (Asp-Ser) of the PB2 protein is responsible for the ts phenotype. This ts single-gene PB2 reassortant virus was serially passaged at elevated temperatures in Madin-Darby canine kidney cells to generate ts+ phenotypic revertant viruses. Four ts+ phenotypically revertant viruses were derived independently, and each possessed a shutoff temperature for replication in vitro of > 40 degrees C. Each of the four phenotypically revertant viruses replicated efficiently in the upper and lower respiratory tracts of mice and hamsters, unlike the PB2 single-gene reassortant virus, confirming that the ts phenotype was responsible for the attenuation of this virus in rodents. Mating the ts+ revertants with wild-type virus yielded ts progeny in high frequency, indicating that the loss of ts phenotype was due to a suppressor mutation which was mapped to the PA gene in each of the four independently derived ts phenotypic revertants. Nucleotide sequence analysis confirmed the absence of new mutations on the PB2 gene and the presence of predicted amino acid changes in the PA proteins of the revertant viruses. These studies suggest that single amino acid changes at aa 245 (Glu-Lys) or 347 (Asp-Asn) of the PA protein can completely suppress the ts and attenuation phenotypes specified by the Asp-Ser mutation at aa 265 of the PB2 protein of the A/Ann Arbor/6/60 cold-adapted virus.     

Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus.

The influenza A/Alaska/77 (H3N2) virus mutant 143-1 is temperature sensitive (ts) due to a spontaneous in-frame 36-nucleotide deletion in the nonstructural (NS) gene segment, which leads to a 12-amino-acid deletion in the NS1 protein. In addition, it has a small-plaque phenotype on MDCK cell monolayers. However, phenotypically revertant (i.e., ts+) viruses were isolated readily following replication of the 143-1 virus both in vitro and in vivo. In order to determine the genetic mechanism by which escape from the ts phenotype occurred, we performed segregational analysis and found that an intrasegmental suppressor mutation caused the loss of the ts phenotype. Nucleotide sequence analysis revealed the presence of an intragenic mutation in each of the ts+ phenotypic revertant viruses, involving a substitution of valine for alanine at amino acid 23 of the NS1 protein. This mutation resulted in acquisition of the ts+ phenotype and also in the large-plaque phenotype on MDCK cells, characteristic of the wild-type A/Alaska/77 parent virus. This amino acid substitution is predicted to generate an area of alpha helix in the secondary structure of the amino-terminal portion of the NS1 protein of the revertant viruses which may compensate for loss of an alpha-helical region due to the deletion of amino acids 66 to 77 in the NS1 protein of the 143-1 virus.     

Recombinant human immunodeficiency virus type 1 genomes with tat unconstrained by overlapping reading frames reveal residues in Tat important for replication in tissue culture.

Human immunodeficiency virus type 1 (HIV-1) Tat is essential for virus replication and is a potent trans activator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Extensive structure-function studies of Tat in subgenomic settings with point mutagenesis and transient transfection readouts have been performed. These reporter assays have defined certain amino acid residues as being important for trans activation of reporter plasmids. However, they have not directly addressed functions related to virus replication. Here, we have studied Tat structure-function in the setting of replicating viruses. We characterized mutations that emerged in Tat during HIV-1 infections of T lymphocytes. To ensure that the selection pressure for change was directed toward protein function, we constructed HIV-Is in which the Tat reading frame was freed from constraints exerted by overlapping with the reading frames of vpr, rev, and env. When these recombinant viruses were passaged in T cells, 26 novel nucleotide changes in tat were observed from sequencing of 220 independently isolated clones. Recloning of these changes into a pNL4-3 molecular background allowed for the characterization of residues in Tat important for virus replication. Interestingly, many of the changes that affected replication when they were assayed in transient trans activation of plasmid reporters were found to be relatively neutral. We conclude that the structure-function of Tat in virus replication is incompletely reflected by activity measurements based only on subgenomic transient transfections.     

A herpes simplex virus type 1 gamma34.5 second-site suppressor mutant that exhibits enhanced growth in cultured glioblastoma cells is severely attenuated in animals

We describe here the neurovirulence properties of a herpes simplex virus type 1 gamma34.5 second-site suppressor mutant. gamma34.5 mutants are nonneurovirulent in animals and fail to grow in a variety of cultured cells due to a block at the level of protein synthesis. Extragenic suppressors with restored capacity to replicate in cells that normally do not support the growth of the parental gamma34.5 deletion mutant have been isolated. Although the suppressor virus reacquires the ability to grow in nonpermissive cultured cells, it remains severely attenuated in mice and is indistinguishable from the mutant gamma34.5 parent virus at the doses investigated. Repairing the gamma34.5 mutation in the suppressor mutant restores neurovirulence to wild-type levels. These studies illustrate that (i) the protein synthesis and neurovirulence defects observed in gamma34.5 mutant viruses can be genetically separated by an extragenic mutation at another site in the viral chromosome; (ii) the extragenic suppressor mutation does not affect neurovirulence; and (iii) the attenuated gamma34.5 mutant, which replicates poorly in many cell types, can be modified by genetic selection to generate a nonpathogenic variant that regains the ability to grow robustly in a nonpermissive glioblastoma cell line. As this gamma34.5 second-site suppressor variant is attenuated and replicates vigorously in neoplastic cells, it may have potential as a replication-competent, viral antitumor agent.     

Characterization of human immunodeficiency virus type 1 matrix revertants: effects on virus assembly, Gag processing, and Env incorporation into virions.

The matrix protein of human immunodeficiency virus type 1 (HIV-1) has been postulated to serve a variety of functions in the virus life cycle. Previously, we introduced a large number of mutations into the HIV-1 matrix and determined the effects on virus replication. These studies identified domains involved in virus assembly and release and envelope glycoprotein incorporation into virions. Here we describe the identification and characterization of viral revertants containing second-site changes in the matrix which compensate for the effects of four of the original mutations on matrix function. Specifically, mutations at matrix residues 4 and 6 severely impaired virus assembly and release; substitutions at residues 4 and 6 reversed the phenotype of the amino acid 4 change while second-site mutations at matrix positions 10, 69, and 97 partially or fully reversed the phenotype of the amino acid 6 substitution. A mutation at matrix residue 62 reversed the effect of a position 34 change which blocks envelope glycoprotein incorporation into virions, and substitutions at residues 27 and 51 reversed the phenotype of a position 86 mutation which redirects virus assembly to the cytoplasm. In addition to determining the effects of the compensatory changes in the context of the original mutations, we also introduced and analyzed the second-site changes alone in the context of the wild-type molecular clone. The data presented here define potential intermolecular and intramolecular interactions which occur in the matrix during the virus life cycle and have implications for our understanding of the relationship between matrix structure and function.     

Spontaneous mutations in the human immunodeficiency virus type 1 gag gene that affect viral replication in the presence of cyclosporins.

Human immunodeficiency virus type 1 mutants that are resistant to inhibition by cyclosporins arise spontaneously in vitro during propagation in a HeLa-CD4+ cell line in the presence of a nonimmunosuppressive analog of cyclosporin A. Interestingly, the phenotype of all of the mutants examined is drug resistant and drug dependent, with both cyclosporin A and its analog. Four independently isolated mutants have been analyzed genetically by construction of recombinant proviruses in the NL4-3 parental strain background and subsequent testing of the chimeric viruses in HeLa cells. The cyclosporin-resistant, cyclosporin-dependent phenotype consistently transfers with a 1.3-kb fragment of gag, within which the four mutants share one of two possible single amino acid exchanges in a proline-rich stretch in the capsid domain of Pr55gag. These mutants provide the first evidence that mutations in human immunodeficiency virus type 1 gag confer resistance to cyclosporins; however, replication is conditional on the presence of the drug. In the T-cell line CEM, replication of the recombinant mutant viruses is also cyclosporin dependent. The drug-dependent replication in HeLa cells is stringent, and in the absence of cyclosporin only revertant viruses with the parental phenotype grow out of cultures infected with cyclosporin-dependent virus. In at least one isolate examined, the revertant phenotype appears to be due to suppressor mutations near the proline-rich region.     

Mutational analysis of HIV-1 Tat minimal domain peptides: identification of trans-dominant mutants that suppress HIV-LTR-driven gene expression

The HIV-1 Tat protein is a potent trans-activator essential for virus replication. We reported previously that HIV-1 Tat peptides containing residues 37-48 (mainly region II), a possible activating region, and residues 49-57 (region III), a nuclear targeting and putative nucleic acid binding region, possess minimal but distinct trans-activator activity. The presence of residues 58-72 (region IV) greatly enhances trans-activation. We postulate that Tat mutant peptides with an inactive region II and a functional region III can behave as dominant negative mutants. We synthesized minimal domain peptides containing single amino substitutions for amino acid residues within region II that are conserved among different HIV isolates. We identify four amino acid residues whose substitution within Tat minimal domain peptides leads to defects in transactivation. Some of these mutants are trans-dominant in several peptide backbones, since they strongly inhibit trans-activation by wild-type Tat protein added to cells or expressed from microinjected plasmid. Significantly, trans-activation of integrated HIV-LTRCAT is blocked by some trans-dominant mutant peptides. These results suggest an attractive approach for the development of an AIDS therapy.     

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.     

Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6Gag Protein Produces a Mutant That Fails To Package Its Envelope Protein

The p6^Gag protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6^Gag between Y³⁶ and P³⁷, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6^Gag proteins in HIV-1_MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6^Gag, site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6^Gag, Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41^TM cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6^Gag mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.