Understanding Retroviral Reverse Transcription, Recombination, and Replication

Division Of Basic Sciences - Nci

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Abstract

During replication, HIV-1 converts its packaged dimeric RNA genomes into DNA and generates one provirus in an infection event. In this process, HIV-1 needs to preserve its genetic information while the host innate immune response attempts to abolish the generation of proviruses capable of producing infectious progeny. HIV-1 uses several transcription start sites to generate several unspliced RNAs including those containing three or one guanosine at the 5 ft end, referred to as 3G and 1G RNA, respectively. The 1G RNA is preferentially selected for packaging, indicating that these 99.9% identical RNAs exhibit functional differences. To determine whether heterogenous transcription start site usage is unique to laboratory adapted HIV-1 strains, we examined 15 primate lentiviruses including transmitted founder viruses of HIV-1, HIV-2, and several simian immunodeficiency viruses (SIVs). We found that all 15 viruses used multiple transcription start sites to some extent. Most viruses also exhibited selective packaging of specific full-length viral RNA species into particles. These findings demonstrate that using multiple transcription start sites and selective packaging of certain full-length viral RNA species are conserved features of primate lentiviruses. In addition, an SIV strain closely related to the progenitor virus that gave rise to HIV-1 group M, the pandemic pathogen, exhibited transcription start site usage similar to some HIV-1 strains and preferentially packaged 1G RNA. These findings indicate that multiple transcription start site usage and selective packaging of a particular unspliced RNA species predate the emergence of HIV-1. To better understand the importance of using multiple transcription start sites, we have generated two HIV-1 mutants with distinct 2-nucleotide modifications that predominantly express 3G RNA or 1G RNA. Both mutants can generate infectious viruses and undergo multiple rounds of replication in T cells. However, both mutants exhibit replication defects compared to the wild-type virus. Our findings demonstrate that HIV-1 maximizes its replication fitness by using multiple transcription start sites to generate unspliced RNAs with different specialized roles in viral replication. The three consecutive guanosines at the junction of U3 and R may also maintain HIV-1 genome integrity during reverse transcription. These studies reveal the intricate regulation of HIV-1 RNA and complex replication strategy. Additionally, we have examined the adaptation of a HIV-1 that contained a NC domain that does not interact properly with packaging signals. These studies provide understanding to the HIV-1 replication.