Natural variation of drug susceptibility in wild-type human immunodeficiency virus type 1.

Wild-type viruses from the ViroLogic phenotype-genotype database were evaluated to determine the upper confidence limit of the drug susceptibility distributions, or "biological cutoffs," for the PhenoSense HIV phenotypic drug susceptibility assay. Definition of the natural variation in drug susceptibility in wild-type human immunodeficiency virus (HIV) type 1 isolates is necessary to determine the prevalence of innate drug resistance and to assess the capability of the PhenoSense assay to reliably measure subtle reductions in drug susceptibility. The biological cutoffs for each drug, defined by the 99th percentile of the fold change in the 50% inhibitory concentration distributions or the mean fold change plus 2 standard deviations, were lower than those previously reported for other phenotypic assays and lower than the clinically relevant cutoffs previously defined for the PhenoSense assay. The 99th percentile fold change values ranged from 1.2 (tenofovir) to 1.8 (zidovudine) for nucleoside reverse transcriptase RT inhibitors (RTIs), from 3.0 (efavirenz) to 6.2 (delavirdine) for nonnucleoside RTIs, and from 1.6 (lopinavir) to 3.6 (nelfinavir) for protease inhibitors. To evaluate the potential role of intrinsic assay variability in the observed variations in the drug susceptibilities of wild-type isolates, 10 reference viruses with different drug susceptibility patterns were tested 8 to 30 times each. The median coefficients of variation in fold change for the reference viruses ranged from 12 to 18% for all drugs except zidovudine (32%), strongly suggesting that the observed differences in wild-type virus susceptibility to the different drugs is related to intrinsic virus variability rather than assay variability. The low biological cutoffs and assay variability suggest that the PhenoSense HIV assay may assist in defining clinically relevant susceptibility cutoffs for resistance to antiretroviral drugs.

Vertical transmission of multidrug-resistant human immunodeficiency virus type 1 (HIV-1) and continued evolution of drug resistance in an HIV-1-infected infant.

To confirm the vertical transmission of multidrug-resistant (MDR) human immunodeficiency virus type 1 (HIV-1) and to assess its impact on further evolution of drug-resistant virus in an infant, proviral DNA amplified from infected peripheral blood mononuclear cell cultures was sequenced to identify reverse transcriptase (RT) and protease (PR) mutations. The infant had proviral DNA with evidence of RT mutations (M41L, L74V, and T215Y) and 3 PR substitutions (K20R, M36I, and V82A). After delivery, the mother's proviral DNA had the same substitutions. Phylogenetic analyses of these HIV-1 RT and PR sequences indicated epidemiological linkage. Plasma drug susceptibility was determined by using a recombinant virus assay. Plasma HIV-1 obtained after the infant's birth demonstrated reduced susceptibility to zidovudine and ritonavir. Thus, vertical transmission of MDR HIV-1 was demonstrated in the setting of detectable maternal plasma viremia. Further accumulation of broad MDR in the infant's virus to 3 antiretroviral classes occurred, despite postnatal therapy.

A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1.

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Loss of antiretroviral drug susceptibility at low viral load during early virological failure in treatment-experienced patients.

BACKGROUND: Clinical studies have demonstrated a correlation between the response to second-line antiretroviral therapy and the number of drugs in the regimen to which the virus is susceptible. These studies have largely been performed in patients with viral loads over 1000 copies/ml. OBJECTIVES: To examine the evolution of resistance during early virological failure, and the potential role of susceptibility testing in patients with low viral loads (below 1000 copies/ml), in treatment-experienced patients. METHODS: Drug susceptibility and genotypes of HIV-1 from indinavir-experienced patients undergoing therapy with nelfinavir, saquinavir, abacavir and either a second nucleoside reverse transcriptase inhibitor (NRTI) or nevirapine were determined. RESULTS: Sixteen subjects were studied. Five of the ten subjects treated with nevirapine, and one of six treated with a second NRTI, achieved and maintained plasma HIV RNA < 500 copies/ml. Virus from the treatment failures lost susceptibility to one or more treatment drugs, including nelfinavir and/or saquinavir, after 4 to 36 weeks of treatment. In six of the ten failures, virus with new reductions in drug susceptibility was detected prior to failure. In five of the six failures who had at least one plasma sample with a viral load between 50 and 1000 copies/ml, reductions in susceptibility to one or more treatment drugs were detected (viral load range: 260 to 630 copies/ml). CONCLUSIONS: Drug resistance can be detected at viral loads below 1000 copies/ml which may be predictive of treatment failure. Failure of a second line regimen was typically associated with early evolution of resistance in HIV protease.

A mutation in human immunodeficiency virus type 1 protease, N88S, that causes in vitro hypersensitivity to amprenavir.

Amprenavir (Agenerase, 141-W94, VX-478) is a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PRI) recently approved for the treatment of HIV-1 infection in the United States. A major cause of treatment failure is the development of resistance to PRIs. One potential use for amprenavir is as salvage therapy for patients for whom treatment that includes one (or more) of the other four currently approved PRIs-saquinavir, indinavir, ritonavir, and nelfinavir-has failed. We evaluated the cross-resistance to amprenavir of viruses that evolved during treatment with the two most commonly prescribed PRIs, nelfinavir and indinavir. Unexpectedly, a dramatic increase in susceptibility (2.5- to 12. 5-fold) was observed with 20 of 312 (6.4%) patient viruses analyzed. The most pronounced increases in susceptibility were strongly associated with an N88S mutation in protease. All viruses that carried the N88S mutation were hypersensitive to amprenavir. Site-directed mutagenesis studies confirmed the causal role of N88S in determining amprenavir hypersensitivity. The presence of the N88S mutation and associated amprenavir hypersensitivity may be useful in predicting an improved clinical response to amprenavir salvage therapy.

Phenotypic changes in drug susceptibility associated with failure of human immunodeficiency virus type 1 (HIV-1) triple combination therapy.

The emergence of drug-resistant human immunodeficiency virus type 1 is a frequent cause of failure of combination therapies comprising reverse transcriptase and protease inhibitors. Rational design of salvage therapies requires new methods to assess drug susceptibility. A novel phenotypic drug susceptibility assay was developed and used to measure the drug susceptibilities of viruses obtained from 2 patients treated with zidovudine, lamivudine, and nelfinavir. Results showed that phenotypic drug resistance may be detectable before virus load rebound, treatment failure does not always imply viral resistance to all drugs in a treatment regimen, and persons with similar antiviral treatment histories and clinical courses may have different phenotypic drug resistance profiles at the time that treatment fails.

Novel four-drug salvage treatment regimens after failure of a human immunodeficiency virus type 1 protease inhibitor-containing regimen: antiviral activity and correlation of baseline phenotypic drug susceptibility with virologic outcome.

Twenty human immunodeficiency virus-infected patients experiencing virologic failure of an indinavir- or ritonavir-containing treatment regimen were evaluated in a prospective, open-label study. Subjects received nelfinavir, saquinavir, abacavir, and either another nucleoside analog (n=10) or nevirapine (n=10). Patients treated with the nevirapine-containing regimen experienced significantly greater virologic suppression at week 24 than those not treated with nevirapine (P=.04). Baseline phenotypic drug susceptibility was strongly correlated with outcome in both treatment arms. Subjects with baseline virus phenotypically sensitive to 2 or 3 drugs in the salvage regimen experienced significantly greater virus load suppression than those with baseline virus sensitive to 0 or 1 drug (median week-24 change=-2.24 log and -0.35 log, respectively; P=.01). In conclusion, non-nucleoside reverse transcriptase inhibitors may represent a potent drug in salvage therapy regimens after failure of an indinavir or ritonavir regimen. Phenotypic resistance testing may provide a useful tool for selecting more effective salvage regimens.

Genetically engineered live attenuated influenza A virus vaccine candidates.

We have generated new influenza A virus live attenuated vaccine candidates by site-directed mutagenesis and reverse genetics. By mutating specific amino acids in the PB2 polymerase subunit, two temperature-sensitive (ts) attenuated viruses were obtained. Both candidates have 38 degrees C shutoff temperatures in MDCK cells, are attenuated in the respiratory tracts of mice and ferrets, and have very low reactogenicity in ferrets. Infection of mice or ferrets with either mutant conferred significant protection from challenge with the homologous wild-type virus. Three tests for genetic stability were used to assess the propensity for reversion to virulence: 14 days of replication in nude mice, growth at 37 degrees C in tissue culture, and serial passage in ferrets. One candidate, which contains mutations intended to reduce the ability of PB2 to bind to cap structures, was stable in all three assays, whereas the second candidate, which contains mutations found only in other ts strains of influenza virus, lost its ts phenotype in the last two assays. This approach has therefore enabled the creation of live attenuated influenza A virus vaccine candidates suitable for human testing.

Temperature sensitive mutants of influenza A virus generated by reverse genetics and clustered charged to alanine mutagenesis.

Temperature sensitive (ts) mutants of influenza A virus have the potential to serve as live attenuated (att) virus vaccines. Previously, ts mutants were isolated by chemical mutagenesis or arose spontaneously, and most likely contained point mutations in one or more genes. While sufficiently attenuated, even the most genetically stable of these viruses was found to revert to a more virulent form in a seronegative vaccinee. Recently developed technology, however, allows the introduction of engineered mutations into the genome of influenza A and B viruses, permitting the rational design of attenuated mutants with the potential for increased genetic stability. To accomplish this goal, we have introduced ts mutations into the PB2 gene of A/Los Angeles/2/87 (H3N2) and rescued the mutated genes into infectious viruses. We have used clustered charged to alanine mutagenesis (substitution of alanine for charged amino acid residues which are present in clusters) of the PB2 gene to generate novel ts mutants. Viruses containing such ts PB2 genes were attenuated in mice and ferrets. This approach has thus yielded several vaccine candidates with ts and attenuated characteristics in animal models. Combination of these mutations with each other or with other ts mutations may lead to a high level of genetic stability.

Activity of Wnt-1 as a transmembrane protein.

The product of the Wnt-1 proto-oncogene is a cysteine-rich glycoprotein that plays a crucial role in the development of the vertebrate central nervous system. Wnt-1 protein is secreted but remains associated with the cell surface and extracellular matrix. The function of Wnt-1 in several different biological settings can be carried out by cells that receive the Wnt signal from adjacent cells. Ectopic expression of Wnt-1 in certain mammary gland cell lines, such as C57MG, causes morphological transformation; C57MG cells can also be transformed by a paracrine mechanism when mixed with other cell types secreting Wnt-1 protein. To ask whether Wnt-1 protein can function while bound to the cell of origin, a variety of cell types were programmed to produce chimeric proteins containing the complete sequence of mature Wnt-1 protein fused to part or all of the transmembrane protein CD4 or CD8. The chimeras were found at the cell surface of transfected cells and did not appear to be proteolytically processed. In autocrine and paracrine transformation assays with C57MG cells and in an axis induction assay in Xenopus laevis embryos, the Wnt-1/CD4 or CD8 fusions retained significant activity, as did a secreted chimera containing the CD8 extracellular domain but lacking the transmembrane domain. However, a chimera lacking a spacer between the Wnt-1 and the transmembrane domains was weakly active and only in autocrine transformation. These results show that tethering Wnt-1 to the cell surface still allows Wnt-1-mediated cell-to-cell signaling.

Human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo.

The human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion polyprotein is produced via ribosomal frameshifting. Previous studies in vitro and in Saccharomyces cerevisiae have argued against a significant role for RNA secondary structure 3' of the shift site, in contrast with other systems, in which such structure has been shown to be required. Here we show, by expressing the HIV-1 gag-pol domain in cultured vertebrate cells, that a stem-loop structure 3' of the HIV-1 shift site is indeed important for wild-type levels of frameshifting in vivo.

Control of the interferon-induced 68-kilodalton protein kinase by the HIV-1 tat gene product.

The tat-responsive region (TAR) of the human immunodeficiency virus-1 (HIV-1) exhibits a trans-inhibitory effect on translation in vitro by activating the interferon-induced 68-kilodalton protein kinase (p68 kinase). Productive infection by HIV-1 was shown to result in a significant decrease in the amount of cellular p68 kinase. The steady-state amount of p68 kinase was also reduced in interferon-treated HeLa cell lines stably expressing tat, as compared to the amount of the kinase in interferon-treated control HeLa cells. Thus, the potential translational inhibitory effects of the TAR RNA region mediated by activation of p68 kinase may be downregulated by tat during productive HIV-1 infection.

Structural requirements for trans activation of human immunodeficiency virus type 1 long terminal repeat-directed gene expression by tat: importance of base pairing, loop sequence, and bulges in the tat-responsive sequence.

In order to elucidate the molecular mechanisms of action of the tat-responsive sequence, mutational analysis of the tat-responsive sequence was carried out. The most critical region comprised nucleotides +18 to +44 and included the 3-nucleotide bulge at positions +23 to +25, the loop sequence, and an intact stem. In addition, base pairing up to nucleotide +52 was required for the full magnitude of the trans-activation response. Single-nucleotide bulges at positions +5 to +17 were dispensable. Analysis of truncated and full-length transcripts demonstrated that a transcriptional antitermination model does not fully account for trans activation.

Identification of a protein that binds specifically to RNA from the first exon of c-myc.

A 55 kilodalton protein present in HeLa and MEL cells, which binds specifically to RNA derived from the first exon of the human c-myc gene, has been identified using a UV-induced crosslinking assay. This protein, called p55, is found in both cytoplasmic and nuclear fractions. The binding site on the RNA, defined using deletion analysis and synthetic oligoribonucleotides, is a purine rich region located between the two major transcriptional initiation sites P1 and P2. Possible involvement of p55 in a number of regulatory events is discussed.

Mutational analysis of the 5' non-coding region of human immunodeficiency virus type 1: effects of secondary structure on translation.

The first 111 nt from the 5' end of human immunodeficiency virus type 1 (HIV-1) mRNAs are shown to have a strong inhibitory effect on the translation of mRNA in in vitro translation extracts as well as in Xenopus oocytes. Mutations in the sequence of the 5' untranslated region (UTR) designed to disrupt predicted secondary structure of this region relieve the inhibition. Inhibition is restored by mutations that reconstruct the predicted secondary structure. The accessibility of the 5'-terminal cap structure was also found to be increased by some of these mutations. We conclude that secondary structure in the 5' UTR of HIV-1 mRNAs and resultant inaccessibility of the cap structure is responsible for the inhibition of translation. The implications of these findings for the understanding of the life cycle of HIV-1 are discussed.