Search

Despite intensive research since the viral pathogen was discovered some 25 years ago, not much progress has been reported on the development of a safe vaccine that protects against human immunodeficiency virus type 1. A vaccine approach that has been abandoned because its safety cannot be guaranteed is the single vaccine candidate that provides good protection in the macaque model, a live-attenuated variant of the simian immunodeficiency virus. The attenuated virus will cause a low-grade, but persistent infection that allows optimization of viral replication kinetics over time by spontaneous virus evolution, which may increase viral pathogenicity. In this article, we discuss innovative strategies to overcome this hurdle, including the generation of "single-cycle" viruses and a conditionally replicating HIV-1 variant

RNA interference or RNAi-based gene therapy for the treatment of HIV-1 infection has recently emerged as a highly effective antiviral approach. The lentiviral vector system is a good candidate for the expression of antiviral short hairpin RNAs (shRNA) in HIV-susceptible cells. However, this strategy can give rise to vector problems because the anti-HIV shRNAs can also target the HIV-based lentiviral vector system. In addition, there may be self-targeting of the shRNA-encoding sequences within the vector RNA genome in the producer cell. The insertion of microRNA (miRNA) cassettes in the vector may introduce Drosha cleavage sites that will also result in the destruction of the vector genome during the production and/or the transduction process. Here, we describe possible solutions to these lentiviral-RNAi problems. We also describe a strategy for multiple shRNA expression to establish a combinatorial RNAi therapy

HIV-1 is an enveloped virus that derives its lipid envelope from the virus producing cell. The envelope glycoprotein complex (Env) mediates viral attachment to and entry in susceptible target cells and is therefore an attractive target for therapeutic intervention. The T20 peptide (Fuzeon, enfuvirtide), targeting the Env fusion machinery, was approved for clinical use in 2003 and served as salvage therapy for many HIV-1 infected individuals for whom no other effective inhibitors were available because of the development of viral resistance. The approval in 2007 of the small molecule inhibitor maraviroc (Selzentry, Celsentri), targeting the coreceptor CCR5, marked a new era for entry inhibitors, which now occupy a mature position in the repertoire of antiviral drugs available to clinicians

For almost three decades, researchers have studied the possibility to use nucleic acids as antiviral therapeutics. In theory, compounds such as antisense oligonucleotides, ribozymes, DNAzymes, and aptamers can be designed to trigger the sequence-specific inhibition of particular mRNA transcripts, including viral genomes. However, difficulties with their efficiency, off-target effects, toxicity, delivery, and stability halted the development of nucleic acid-based therapeutics that can be used in the clinic. So far, only a single antisense drug, Vitravene for the treatment of CMV-induced retinitis in AIDS patients, has made it to the clinic. Since the discovery of RNA interference (RNAi), there is a renewed interest in the development of nucleic acid-based therapeutics. Antiviral RNAi approaches are highly effective in vitro and in animal models and are currently being tested in clinical trials. Here we give an overview of antiviral nucleic acid-based therapeutics. We focus on antisense and RNAi-based compounds that have been shown to be effective in animal model systems

Mutational analysis of the viral genome is frequently used to study the role of sequence or structural elements in HIV-1 replication. Many laboratories that use this approach have occasionally come across revertant viruses that overcome an introduced defect either by restoration of the original sequence or by the introduction of additional mutations in the viral genome. Similarly, replication of a wild type virus under selective pressure, due to the presence of inhibitors or due to specific culture settings, may result in the appearance of evolved variants that replicate more efficiently under the applied conditions. We have developed in vitro HIV-1 evolution from an anecdotal event to a systematic research tool to study different aspects of the viral replication cycle. In this manuscript, we will briefly review the method of forced virus evolution to study HIV-1 biology and provide several examples that illustrate the power of this method, as it frequently yielded interesting and unexpected information about the mechanism of virus replication

Peptides based on the second heptad repeat (HR2) of viral class I fusion proteins are effective inhibitors of virus entry. One such fusion inhibitor has been approved for treatment of human immunodeficiency virus-1 (T20, enfuvirtide). Resistance to T20 usually maps to the peptide binding site in HR1. To better understand fusion inhibitor potency and resistance, we combined virological, computational, and biophysical experiments with comprehensive mutational analyses and tested resistance to T20 and second and third generation inhibitors (T1249 and T2635). We found that most amino acid substitutions caused resistance to the first generation peptide T20. Only charged amino acids caused resistance to T1249, and none caused resistance to T2635. Depending on the drug, we can distinguish four mechanisms of drug resistance: reduced contact, steric obstruction, electrostatic repulsion, and electrostatic attraction. Implications for the design of novel antiviral peptide inhibitors are discussed. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

RNA interference (RNAi) is an evolutionarily conserved mechanism in which double-stranded RNA (dsRNA) induces sequence-specific gene silencing. RNAi as a gene suppression tool holds great promise for basic research and multiple applications. RNAi strategies with a single inhibitor have received much attention and some are currently being tested in clinical trials as potential drug for infectious diseases, cancer and genetic disorders. Although very successful, such an RNAi mono-therapy is not always sufficient. For example, several proteins should be silenced simultaneously for the elucidation of cross-talk between signalling pathways. Furthermore, development of a durable RNAi therapy for viral infections and cancer may also require the use of multiple inhibitors, as this may prevent the selection of virus variants or cancer cells that escape from the RNAi therapy by mutation of the target gene. Therefore, several combinatorial RNAi strategies have been developed and recent reports demonstrated the potency of this approach by targeting multiple viral genes or oncogenes. At the same time, combinatorial RNAi may also increase the risk of saturation of the endogenous RNAi pathway and off-target effects. This review summarizes the latest experimental advances in combinatorial RNAi research and gene therapy approaches, in particular against escape-prone viruses, like the human immunodeficiency virus type 1 (HIV-1).

Publisher Summary This chapter focuses on the initiation step of reverse transcription as performed by human immunodeficiency virus type 1 (HIV‐1). During this step, a cellular tRNA primer is placed onto a complementary sequence in the viral genome, called the “primer‐binding site” (PBS). In this highly organized process, the cellular tRNALys3 molecule acts as primer. Retroviruses differ from other positive‐stranded RNA viruses in the process of reverse transcription. This process is an essential step in the viral life cycle as it converts the retroviral genomic RNA into a proviral DNA in a complex, multistep process. The viral enzyme reverse transcriptase (RT) recognizes the RNA–RNA complex and catalyzes the extension of the 3’ end of the tRNA primer, with the viral RNA (vRNA) acting as template. This initiation step is regulated and most retroviruses are restricted to the use of the cognate, self‐tRNA primer. Recent insight into additional contacts between the tRNA and the vRNA genome lead to a renewed attempt to change HIV‐1 primer specificity. The chapter discusses a study in which a replicating HIV‐1 variant is obtained that uses a nonself tRNA primer. The analysis of this variant confirms the role of multiple determinants of tRNA primer usage.