Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted antiviral functions of APOBEC3G

Abstract

Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular anti-viral effectors, called restriction or resistance factors. The host anti-viral protein APOBEC3G (A3G) antagonizes the early steps of HIV-1 infection through the combined effects of inhibiting viral cDNA production, and cytidine-to-uridine driven hypermutation of this cDNA. In seeking to address the underlying molecular mechanism for inhibited cDNA synthesis, we developed a deep sequencing strategy to characterize nascent reverse transcription products and their precise 3'-termini in HIV-1 infected T cells. Our results demonstrate site- and sequence-independent interference with reverse transcription, which requires A3G’s specific interaction with reverse transcriptase (RT) itself. This approach also established, contrary to current ideas, that cellular uracil base excision repair (UBER) enzymes target and cleave A3G-edited uridine-containing viral cDNA. Together, these findings yield further insights into the regulatory interplay between RT, A3G and the cellular DNA repair machinery, and identify the suppression of HIV-1 RT by a directly interacting host protein as a new cell-mediated anti-viral mechanism.

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…A3 enzymes are able to restrict viral replication through deamination-independent mechanisms in addition to the well-characterized deamination-dependent mechanism [73,155,156,169,214,286,294,295]. A3G and A3F delay the initiation of primer extension, which leads to production of less full-length extension products [71,74].…”

“…In addition, A3G can interact with reverse transcriptase directly and inhibit polymerase activity [71,75]. The double-stranded proviral genome is imported into the nucleus and either the uracils cause DNA repair mediated cleavage of the provirus or the provirus is integrated through the action of HIV-1 integrase and host protein interactions [75] (Figure 1.2B, Figure 1.4B).…”

“…Throughout this process, the A3 enzymes also directly disrupt proviral DNA synthesis by oligomerizing on the template DNA resulting in a "roadblock" for the reverse transcriptase [71][72][73][74]. In addition, A3G can interact with reverse transcriptase directly and inhibit polymerase activity [71,75]. The double-stranded proviral genome is imported into the nucleus and either the uracils cause DNA repair mediated cleavage of the provirus or the provirus is integrated through the action of HIV-1 integrase and host protein interactions [75] (Figure 1.2B, Figure 1.4B).…”

“…However, certain A3s are able to additionally inhibit HIV through deamination-independent mechanisms that act in concert with the deamination activity [71-74, 155, 156, 159, 292]. A3 enzymes, such as A3G, A3F, A3H and A3C are able restrict both HIV-1 and the endogenous retroelement LINE-1 through deamination-independent modes [72,73,155,156,159,169,214,286,[293][294][295]. A3G and A3F delay the initiation of primer extension by HIV-1 reverse transcriptase (RT) to varying degrees by binding to the RNA template and acting as a physical block to the scanning mechanism of RT.…”

Antiretroviral APOBEC3 Cytidine Deaminases Alter HIV-1 Provirus Integration Site Profiles

“…A3 proteins can also restrict HIV-1 replication via mechanisms other than deamination (e.g. binding the viral RNA or to the viral reverse transcriptase (RT), which reduces vDNA synthesis) [14][15][16][17][18][19][20] . It was previously shown that A3G and A3F can interact with the viral integrase (IN) and RT, but the role of this binding on viral integration is not yet clear [21][22][23][24] .…”