Abstract
Characterizing how RNA elements and nucleotide biases in the HIV-1 genome regulate replication is necessary for a complete understanding of the viral life cycle. To characterise how the RNA sequence in gag controls HIV-1 replication, we codon modified an ~350nt region in this gene. These synonymous mutations inhibited viral replication by decreasing genomic RNA (gRNA) abundance, gRNA stability, Gag expression, virion production and infectivity. Using RNA-seq, we identified that the synonymous mutations activated a cryptic splice site that is the cause of the reduction in viral replication.
There is a much lower than expected frequency of CpG dinucleotides in HIV- 1 and codon modification introduced a substantial increase in CpG abundance. To determine if CpG dinucleotides are necessary for inhibition of HIV-1 replication, codons introducing CpGs were mutated back to the wild type codon, which restored efficient Gag expression and infectious virion production. To determine if they are sufficient to inhibit viral replication, CpG dinucleotides were inserted into gag in the absence of other point mutations. The results showed that while viral replication is greatly decreased, Gag expression, and virion production levels are similar to wild type HIV-1. Interestingly, gRNA abundance was decreased in the cell lysate and the media.
When we introduced CpG dinucleotides into nucleotides 22-1188 in gag we showed a reduction in viral replication that was the result of a decrease in Gag expression, virion production and infectivity. In addition, we showed that the inhibitory mechanism required the RNA-binding protein ZAP.
In summary, we have used HIV-1 as a model system to understand how CpG dinucleotides can inhibit viral replication. Understanding the mechanisms underlying the reduction in viral fitness may be extrapolated to different RNA viruses and could result in many applications.
| Date of Award | 1 Jun 2019 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Chad Swanson (Supervisor) & Michael Malim (Supervisor) |