Reverse Transcriptase and Retroviral Replication

Within each viral particle, retroviruses package two copies of a single-stranded RNA genome of about 10 kb. All of the viral genomes contain three major genes, arranged in the order: 5 ́-gag–pol–env-3 ́, and some retroviruses may also have accessory genes (e.g. vif, vpr, tax, etc...). Structural proteins such as MA (matrix protein), CA (capsid protein) and NC (nucleocapsid protein) are encoded within gag. Envelope proteins that mediate viral entry (surface and transmembrane glycoproteins) derive from expression of the env gene. Virusencoded enzymes such as the protease, the reverse transcriptase (RT) and the integrase, required to complete the viral life cycle, usually derive from the expression of pol. The reverse transcription of the viral single-stranded (+) RNA genome into double-stranded DNA is an essential step in retroviral replication and an important target for therapeutic intervention (for reviews, see Telesnitsky & Goff, 1997; Abbink & Berkhout, 2008; Sarafianos et al., 2009). Reverse transcription is a relatively complex process that requires the intervention of at least three elements: (i) the viral genomic RNA (that serves as template); (ii) a specific primer (i.e. a transfer RNA); and (iii) the viral RT. Retroviral RTs are enzymes that possess two activities: (i) a DNA polymerase activity that uses either RNA or DNA as template, and (ii) an RNase H activity, which degrades RNA from RNA/DNA hybrids. Unlike eukaryotic DNA polymerases, retroviral RTs are devoid of 3 ́ 5 ́ exonucleolytic proofreading and show intrinsic error frequencies of around 10-4 to 10-5, well above the values reported for cellular DNA polymerases. Their lower accuracy together with their ability to switch templates during reverse transcription are major contributors to the extensive genetic variability observed in many retroviruses including human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). The diversification of retroviral genomes, based on the sequence of the pol gene (encoding for viral enzymes including RT) is illustrated in Fig. 1. Environmental factors as well as the molecular structure of retroviral RTs modulate their fidelity. In addition, retrovirus genetic variability can be affected by viral and cellular proteins. In this review, studies dealing with the molecular basis of fidelity of HIV-1 RT are summarized and discussed in the light of crystal structures of the enzyme. Structural information has been most useful in the design of antiretroviral drugs targeting the DNA polymerase activity of the RT. The last sections of this chapter summarize current