Atze T. Das

Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-Mediated Inhibition

ABSTRACT Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.

HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.

CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications.

Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter

Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline. Lentiviral vectors efficiently transfer genes to dividing and non-dividing cells with long-term gene expression both in vitro and in vivo. We compared regulatory function in a single lentiviral vector where rtTA was either expressed from a constitutive promoter or placed in an autoregulatory loop. Autoregulatory expression of rtTA was superior to constitutive promoter expression, resulting in higher viral titers, undetectable levels of both rtTA and transgene expression in the absence of doxycycline, improved induction kinetics and increased induction levels in all cells tested. We further expanded the utility of the autoregulatory vector by using an improved rtTA variant with an increased sensitivity to doxycycline. This lentiviral vector with doxycycline-regulated transgene expression may be useful for gene therapy applications and in experimental settings where strict temporal expression of a transgene is required.

Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping.

Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.

The TAR Hairpin of Human Immunodeficiency Virus Type 1 Can Be Deleted When Not Required for Tat-Mediated Activation of Transcription

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) RNA genome contains a terminal repeat (R) region that encodes the transacting responsive (TAR) hairpin, which is essential for Tat-mediated activation of gene expression. TAR has also been implicated in several other processes during viral replication, including translation, dimerization, packaging, and reverse transcription. However, most studies in which replication of TAR-mutated viruses was analyzed were complicated by the dominant negative effect of the mutations on transcription. We therefore used an HIV-1 variant that does not require TAR for transcription to reinvestigate the role of TAR in HIV-1 replication. We demonstrate that this virus can replicate efficiently upon complete deletion of TAR. Furthermore, evolution of a TAR-deleted variant in long-term cultures indicates that HIV-1 requires a stable stem-loop structure at the start of the viral transcripts in which the 5′-terminal nucleotides are base paired. This prerequisite for efficient replication can be fulfilled by the TAR hairpin but also by unrelated stem-loop structures. We therefore conclude that TAR has no essential function in HIV-1 replication other than to accommodate Tat-mediated activation of transcription.

The HIV-1 Tat Protein Has a Versatile Role in Activating Viral Transcription

ABSTRACT It is generally acknowledged that the Tat protein has a pivotal role in HIV-1 replication because it stimulates transcription from the viral long terminal repeat (LTR) promoter by binding to the TAR hairpin in the nascent RNA transcript. However, a multitude of additional Tat functions have been suggested. The importance of these functions is difficult to assess in replication studies with Tat-mutated HIV-1 variants because of the dominant negative effect on viral gene expression. We therefore used an HIV-1 construct that does not depend on the Tat-TAR interaction for transcription to reevaluate whether or not Tat has a second essential function in HIV-1 replication. This HIV-rtTA variant uses the incorporated Tet-On gene expression system for activation of transcription and replicates efficiently upon complete TAR deletion. Here we demonstrated that Tat inactivation does nevertheless severely inhibit replication. Upon long-term culturing, the Tat-minus HIV-rtTA variant acquired mutations in the U3 region that improved promoter activity and reestablished replication. We showed that in the absence of a functional TAR, Tat remains important for viral transcription via Sp1 sequence elements in the U3 promoter region. Substitution of these U3 sequences with nonrelated promoter elements created a virus that replicates efficiently without Tat in SupT1 T cells. These results indicate that Tat has a versatile role in transcription via TAR and U3 elements. The results also imply that Tat has no other essential function in viral replication in cultured T cells.

A conditionally replicating virus as a novel approach toward an HIV vaccine

Publisher Summary This chapter examines a conditionally replicating virus as a novel approach toward an HIV vaccine. Live-attenuated virus vaccines have proven to be highly successful at inducing protective immunity against pathogenic viruses, such as smallpox, polio, and measles. The development of an HIV-1 variant by replacing the natural gene expression mechanism of the virus with an inducible regulatory system is presented. The Tet–rtTA system seems to be the ideal regulatory system to control replication of a conditional live HIV-1 virus vaccine. It is found that controlling virus replication through rtTA instead of tTA will avoid the long-lasting administration of Tc or dox that would be required with tTA. It is observed that to transform the constitutively replicating HIV-1 virus into a conditional live variant, the viral genome was mutated to inactivate the Tat–TAR transcription regulation mechanism and to integrate the Tet system. The 5´TAR RNA hairpin is formed by base pairing of nucleotides from positions +1to +57 relative to the start site of transcription. The substitution of the nef gene with the rtTA gene is also elabroated.

Structural Requirements for the Binding of tRNA3Lys to Reverse Transcriptase of the Human Immunodeficiency Virus Type 1

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA3Lys molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA molecule that are required for interaction with the RT protein remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified RT to mutant forms of tRNA3Lys. The anticodon loop could be mutated without eliminating RT recognition. However, mutations in the TΨC stem were found to partially interfere with RT binding, and D arm mutants were completely inactive in RT binding. Interestingly, binding of the RT protein to tRNA3Lys facilitates the subsequent annealing of template strand to the 3′-terminus of the tRNA molecule. Consistent with this finding, we demonstrate that mutant HIV-1 virions lacking the RT protein do contain a viral RNA genome without an associated tRNA3Lys primer. We also found that a preformed primer tRNA-template complex is efficiently recognized by RT protein in vitro. Extension of the template molecule over the TΨC loop did result in complete inhibition of RT binding, suggesting the presence of additional recognition elements in the TΨC loop. These results, combined with a comparative sequence analysis of tRNA species present in HIV-1 virions and RNA motifs selected in vitro for high affinity RT binding, suggest that RT recognizes the central domain of the tRNA tertiary structure, which is formed by interaction of the D and TΨC loops.

Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control

BackgroundWe have previously constructed a doxycycline (dox)-dependent HIV-1 variant by incorporating the Tet-On gene regulatory system into the viral genome. Replication of this HIV-rtTA virus is driven by the dox-inducible transactivator protein rtTA, and can be switched on and off at will. We proposed this conditional-live virus as a novel vaccine approach against HIV-1. Upon vaccination, replication of HIV-rtTA can be temporarily activated by transient dox administration and controlled to the extent needed for optimal induction of the immune system. However, subsequent dox-withdrawal may impose a selection for virus variants with reduced dox-dependence.ResultsWe simulated this on/off switching of virus replication in multiple, independent cultures and could indeed select for HIV-rtTA variants that replicated without dox. Nearly all evolved variants had acquired a typical amino acid substitution at position 56 in the rtTA protein. We developed a novel rtTA variant that blocks this undesired evolutionary route and thus prevents HIV-rtTA from losing dox-control.ConclusionThe loss of dox-control observed upon evolution of the dox-dependent HIV-1 variant was effectively blocked by modification of the Tet-On regulatory system.