ABSTRACT Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5α did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5′ of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3′-to-5′ siRNA amplification and spreading. In contrast, HIV-1 RNA 3′ of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively “naked” cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5α, or other host restriction factors.

This chapter presents recent findings related to human immunodeficiency virus (HIV)‐1 drug resistance and fitness. Antiretroviral therapy is a form of extrinsic pressure applied against the HIV‐1 that reduces replication but also selects for variants with reduced sensitivity to this pressure. These HIV‐1 variants are typically present in the infecting virus population prior to treatment but are maintained at low frequency because of their low fitness. Fitness is defined by the replicative capacity of the virus in a given environment but it is important to note that this "environment" is in constant flux. The chapter examines the resistance to each class of antiviral drug and impact of these resistance mutations on replicative fitness in the absence of drug. The mutations conferring resistance to entry inhibitors (EIs) appear to have the most impact on the function of the Env glycoproteins in virus replication whereas resistance to protease inhibitors (PIs) and reverse transcriptase inhibitors (RTIs) is conferred by mutations that decrease the fitness of these enzymatic functions.