Abstract
Retroviruses have evolved multiple means to counteract host restriction factors such as single-stranded DNA-specific deoxycytidine deaminases (APOBEC3s, A3s). These include exclusion of A3s from virions by an A3-unreactive nucleocapsid or expression of an A3-neutralizing protein (Vif, Bet). However, a number of retroviruses package A3s and do not encode apparent vif- or bet-like genes, yet they replicate in the presence of A3s. The mode by which they overcome deleterious restriction remains largely unknown. Here we show that the prototypic betaretrovirus, mouse mammary tumor virus (MMTV), packages similar amounts of A3s as HIV-1ΔVif, yet its proviruses carry a significantly lower level of A3-mediated deamination events than the lentivirus. The G-to-A mutation rate increases when the kinetics of reverse transcription is reduced by introducing a mutation (F120L) to the DNA polymerase domain of the MMTV reverse transcriptase (RT). A similar A3-sensitizing effect was observed when the exposure time of single-stranded DNA intermediates to A3s during reverse transcription was lengthened by reducing the dNTP concentration or by adding suboptimal concentrations of an RT inhibitor to infected cells. Thus, the MMTV RT has evolved to impede access of A3s to transiently exposed minus DNA strands during reverse transcription, thereby alleviating inhibition by A3 family members. A similar mechanism may be used by other retroviruses and retrotransposons to reduce deleterious effects of A3 proteins.
Highlights
Reverse transcription (RTN) is an essential step in the life cycle of all retroviruses and retrotransposons
We show that a point mutation in the DNA polymerase domain of mammary tumor virus (MMTV) reverse transcriptase (F120L), which reduced the rate of DNA synthesis, increased
A similar APOBEC3 sensitizing effect was detected for cell culture conditions that slow down the rate of reverse transcription reaction such as decreased dNTP levels within target cells or the presence of sub-optimal, non-lethal concentrations of reverse transcriptase inhibitors
Summary
Reverse transcription (RTN) is an essential step in the life cycle of all retroviruses and retrotransposons. During RTN, A3G extensively deaminates deoxycytidine to deoxyuridine residues (C-to-U) in the negative strand of retroviral DNA, resulting in G-to-A hypermutation in the newly synthesized plus DNA strand [3,4,5] As both human A3G and murine mA3 deaminate exclusively ssDNA intermediate products of RTN [6,7,8], their antiviral function can be exerted only during a finite period of time when the viral minus DNA strand remains single-stranded. This is determined by the time between synthesis of minus DNA strand, followed by degradation of the RNA template by the RT-associated RNase H activity, and synthesis of the plus DNA strand. As various regions of the minus DNA strand remain single-stranded for a different amount of time, the retroviral genomic DNA contains A3-induced mutational gradient peaking just 5’ (when considering plus strand sequence) to the polypurine tract (PPT) sequence [6, 8, 9]
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