Resequencing microarray method for molecular diagnosis of human arboviral diseases.

BACKGROUND: Resequencing DNA microarray (RMA) technology uses probes designed to identify a panel of viral sequences. It can be used for detecting emerging viruses by revealing the nucleotide polymorphisms within the target of interest. OBJECTIVES/STUDY DESIGN: As a new tool for molecular diagnosis of arbovirus infection, high density PathogenID v2.0 RMA (PID2-RMA) was assessed for the detection and genetic analysis of dengue, West Nile, and Chikungunya viruses in spiked blood samples or sera from individuals infected with dengue virus. Viral RNAs extracted from biological samples were retrotranscribed into cDNA and amplified using the Phi 29 polymerase-based method. This amplified cDNA was used for hybridization on PID2-RMA. RESULTS: A good specificity of RMA-based detection was demonstrated using a panel of arboviruses including Dengue, West Nile and Chikungunya viruses. This technology was also efficient for the detection and genetic analysis of the different serotypes of dengue virus in sera of infected patients. Furthermore, the mixing of dengue, West Nile and Chikungunya prototype viruses within a single sample of human blood did not interfere with the sensitivity of PID2-RMA. CONCLUSIONS: Our data show that high density PID2-RMA was suitable for the identification of medically important arboviruses. It appears to be particularly adapted to the genetic analysis of dengue, West Nile, and Chikungunya viruses in urgent clinical situations where the rapid identification and characterization of the pathogen is essential.

Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation.

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5' m(7)GpppN cap and the 3' poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3' end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3'-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.

Analysis of HIV cross-resistance to protease inhibitors using a rapid single-cycle recombinant virus assay for patients failing on combination therapies.

OBJECTIVE: To assess the patterns of HIV phenotypic cross-resistance to protease inhibitors (PI) in patients experiencing viral load rebound on combination therapy including a PI. METHODS: Phenotypic analysis of sensitivity to indinavir, nelfinavir, saquinavir, ritonavir and amprenavir was carried out using a single-cycle recombinant virus assay. Viral protease was sequenced by automated dideoxynucleotide chain termination. RESULTS: Of the 108 patients studied, 68 had received indinavir, 50 ritonavir, 25 saquinavir and eight nelfinavir. The majority (71%) had received only one PI. The incidence of cross-resistance between indinavir, nelfinavir, ritonavir and saquinavir was high (60-90%). Cross-resistance to amprenavir was less frequent (37-40%). However there was some correlation between levels of sensitivity to amprenavir and indinavir (r2 = 0.34; P < 0.01). Conversely, the correlation between levels of sensitivity to indinavir and saquinavir was poor (r2 = 0.25), particularly for patients who had not received saquinavir. The degree of cross-resistance correlated with the level of resistance and with the total number of mutations in the protease gene (P < 0.05, chi square test) but could not be significantly correlated to any one particular mutation or combination of mutations. Mutation 184V was significantly associated with cross-resistance to amprenavir, with no mutations at codon 50 observed, while mutations associated with cross-resistance to saquinavir differed according to the treatment received. CONCLUSIONS: These results suggest that, although the total number of protease mutations correlates with the degree of cross-resistance, the specific mechanisms accounting for primary resistance and for cross-resistance may be different.

Effects of human immunodeficiency virus type 1 resistance to protease inhibitors on reverse transcriptase processing, activity, and drug sensitivity.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors often display a reduced replicative capacity as a result of an impairment of protease function. Such fitness-impaired viruses display Gag precursor maturation defects. Here, we report that some protease inhibitor-resistant viruses also display abnormalities in the processing of reverse transcriptase (RT) by the protease. In three recombinant viruses carrying resistant protease sequences from patient plasma, we observed a marked decrease in the amount of mature RT subunits and of particle-associated RT activity compared to their parental pretherapy counterparts. We investigated the possibility that a decrease in the amount of particle-associated mature RT could affect the sensitivity of the corresponding virus to RT inhibitors. We observed a twofold increase of sensitivity to zidovudine (AZT) when a virus which carried AZT mutations was processed by a resistant protease. Interestingly, the presence of AZT-resistance mutations partially rescued the replication defect associated with the mutated protease. The interplay between resistance to protease inhibitors and to RT inhibitors described here may be relevant to the therapeutic control of HIV-1 infection.

Constrained evolution of human immunodeficiency virus type 1 protease during sequential therapy with two distinct protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) variants that have developed protease (PR) inhibitor resistance most often display cross-resistance to several molecules within this class of antiretroviral agents. The clinical benefit of the switch to a second PR inhibitor in the presence of such resistant viruses may be questionable. We have examined the evolution of HIV-1 PR genotypes and phenotypes in individuals having failed sequential treatment with two distinct PR inhibitors: saquinavir (SQV) followed by indinavir (IDV). In viruses where typical SQV resistance mutations were detected before the change to IDV, the corresponding mutations were maintained under IDV, while few additional mutations emerged. In viruses where no SQV resistance mutations were detected before the switch to IDV, typical SQV resistance profiles emerged following the introduction of IDV. We conclude that following suboptimal exposure to a first PR inhibitor, the introduction of a second molecule of this class can lead to rapid selection of cross-resistant virus variants that may not be detectable by current genotyping methods at the time of the inhibitor switch. Viruses committed to resistance to the first inhibitor appear to bear the "imprint" of this initial selection and can further adapt to the selective pressure exerted by the second inhibitor following a pathway that preserves most of the initially selected mutations.

Loss of viral fitness associated with multiple Gag and Gag-Pol processing defects in human immunodeficiency virus type 1 variants selected for resistance to protease inhibitors in vivo.

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.

Extensive regions of pol are required for efficient human immunodeficiency virus polyprotein processing and particle maturation.

Human immunodeficiency type 1 particle maturation is dependent upon proteolytic cleavage of the gag and gag-pol precursors by the pol-encoded viral protease. We have investigated the importance of domains of pol other than the protease for particle maturation and gag proteolytic processing. Truncations of the gag-pol polyprotein precursor of HIV-1 were created by deleting segments of the reverse transcriptase coding region or by introducing stop codons in the integrase region of an HIV-1 infectious molecular clone. In these mutants, the protease coding sequence was left intact. Particles produced by all of the mutants displayed abnormal morphologies and impaired proteolytic processing of gag. The severity of particle morphology abnormalities and of gag polyprotein processing impairment appeared to be affected both by the size and by the position of the deletions in pol, suggesting that the integrity of several pol domains within the gag-pol precursor is required for optimal protease activation and particle maturation. Additionally, cotransfection of a deletion mutant with wild-type provirus led to a marked reduction in the titer of infectious virus, suggesting that truncated gag-pol precursors can interfere with wild-type virus assembly and maturation.

Resistance of human immunodeficiency virus type 1 to protease inhibitors: selection of resistance mutations in the presence and absence of the drug.

Inhibitors of the human immunodeficiency virus (HIV) protease are a promising class of antiviral agents that dramatically reduce HIV replication both in culture and in infected patients. However, as for many other antiviral compounds, long-term efficacy of these agents is impeded by the emergence of virus variants with increased resistance to their inhibitory action, following selection of specific mutations in the protease coding sequence. We have studied HIV-1 variants that emerged at different stages of selection in the presence of the C2-symmetrical protease inhibitor ABT-77003. The selection of variants was a gradual process during which mutations accumulated at different sites in the protease, generating virus populations with increasing levels of resistance to the drug. The initially selected viruses had a low level of resistance as well as a markedly reduced replicative capacity. Further accumulation of mutations at secondary sites led to an improvement in both drug resistance and replication. In spite of their reduced infectivity, partially selected virus populations did not readily revert to wild-type when serially passaged in drug-free conditions. Instead, even in the absence of drug, secondary mutations identical to those selected in the presence of the inhibitor continued to emerge. These mutations improved both the intrinsic replicative capacity of the virus and its level of resistance to the inhibitor, suggesting that once committed to drug resistance, readaptation of the enzyme to its natural substrate leads to a reduction of its sensitivity to the inhibitor.

HIV-1 reverse transcription. A termination step at the center of the genome.

During HIV-1 reverse transcription, the plus-strand of viral DNA is synthesized as two discrete segments. We show here that synthesis of the upstream segment terminates at the center of the genome after an 88 or 98 nucleotide strand displacement of the downstream segment, initiated at the central polypurine tract. Thus, the final structure of unintegrated linear HIV-1 DNA includes a central plus-strand overlap. In vitro reconstitution using only purified reverse transcriptase with appropriate DNA hybrids gave rise to efficient and accurate termination, which was dramatically amplified in the context of strand displacement. Mutation of the sequence immediately upstream of the termination sites almost completely abolished termination both in infected cells and in vitro. This mutation profoundly impaired replication of HIV-1. We conclude that proper central plus-strand termination, mediated by a novel cis-active termination sequence, is a key step in HIV-1 replication.

Amino acid changes in the fourth conserved region of human immunodeficiency virus type 2 strain HIV-2ROD envelope glycoprotein modulate fusion.

The fourth conserved region (C4) of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein has been shown to participate in CD4 binding and to influence viral tropism (A. Cordonnier, L. Montagnier, and M. Emerman, Nature [London] 340:571-574, 1989). To define the role of the corresponding region of HIV-2, we introduce single amino acid changes into the C4 sequence of HIV-2ROD. The effects of these mutations on glycoprotein function and on virus infectivity have been examined. We have shown that the tryptophan residue at position 428 is necessary primarily for CD4 binding. The isoleucine residue at position 421 is necessary for the establishment of productive infection in the promonocytic cell line U937, while it is dispensable to some extent for infection of primary T lymphocytes or the lymphocytic cell line SUP-T1. This replication defect correlated with the failure of the Ile-421-to-Thr (Ile-421-->Thr) mutant glycoprotein to form syncytia in U937 cells. DNA analysis of revertant viruses revealed that a strong selective pressure was exerted on residue 421 of the surface glycoprotein to allow HIV-2 infection of U937 cells. These results demonstrate that this region of HIV-2 plays an important role in determining fusion efficiency in a cell-dependent manner and consequently can influence viral tropism.

Functional mapping of the rev-responsive element of human immunodeficiency virus type 2 (HIV-2): influence of HIV-2 envelope-encoding sequences on HIV-1 gp120 expression in the presence or absence of Rev.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev stimulates expression of structural viral proteins via a target response element (RRE) located within gag-pol and env mRNAs. To analyse the HIV-2 Rev trans-activation effect on the expression of the envelope protein, we cloned a functionally active HIV-2 rev cDNA and showed that it contained four exons. Using transient expression assays, we mapped a 353 bp RRE fragment within the env gene of HIV-2 on which both HIV-1 and HIV-2 Rev could act. Interestingly, smaller fragments suppressed the use of additional splice sites within the env gene and caused envelope protein expression independent of Rev.