Abstract
BackgroundThe human immunodeficiency virus type 1 (HIV-1) structural protein Gag is necessary and sufficient to form viral particles. In addition to encoding the amino acid sequence for Gag, the underlying RNA sequence could encode cis-acting elements or nucleotide biases that are necessary for viral replication. Furthermore, RNA sequences that inhibit viral replication could be suppressed in gag. However, the functional relevance of RNA elements and nucleotide biases that promote or repress HIV-1 replication remain poorly understood.ResultsTo characterize if the RNA sequence in gag controls HIV-1 replication, the matrix (MA) region was codon modified, allowing the RNA sequence to be altered without affecting the protein sequence. Codon modification of nucleotides (nt) 22-261 or 22-378 in gag inhibited viral replication by decreasing genomic RNA (gRNA) abundance, gRNA stability, Gag expression, virion production and infectivity. Comparing the effect of these point mutations to deletions of the same region revealed that the mutations inhibited infectious virus production while the deletions did not. This demonstrated that codon modification introduced inhibitory sequences. 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 they are necessary for inhibition of HIV-1 replication, codons introducing CpG dinucleotides 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 changes. The increased CpG dinucleotide content decreased HIV-1 infectivity and viral replication.ConclusionsThe HIV-1 RNA sequence contains low abundance of CpG dinucleotides. Increasing the abundance of CpG dinucleotides inhibits multiple steps of the viral life cycle, providing a functional explanation for why CpG dinucleotides are suppressed in HIV-1.
Highlights
The human immunodeficiency virus type 1 (HIV-1) structural protein Gag is necessary and sufficient to form viral particles
Synonymous mutations in gag inhibit HIV‐1 replication To analyze the functional relevance of RNA elements and nucleotide bias underlying the MA domain in Gag, we introduced 80 synonymous mutations into nt 22-261 of HIV-1NL4-3 gag (Fig. 1a)
The mutations were derived from pHDMHgpm2, a codon optimized Gag-Pol construct in which many of the HIV-1 codons are replaced with codons used in highly expressed human mRNAs [59, 68]
Summary
The human immunodeficiency virus type 1 (HIV-1) structural protein Gag is necessary and sufficient to form viral particles. In addition to encoding the amino acid sequence for Gag, the underlying RNA sequence could encode cis-acting elements or nucleotide biases that are necessary for viral replication. The HIV-1 genomic RNA (gRNA) has three major functions in the viral life cycle [1]. It serves as the premRNA that is spliced into over 70 different transcripts [2,3,4] It acts as the mRNA for the Gag and GagPol polyproteins that comprise the structural and enzymatic proteins, respectively [5, 6]. The 5′ UTR contains several cis-acting elements in complex stem-loop structures that regulate multiple stages of the viral life cycle including transcription, splicing, gRNA dimerization, encapsidation and reverse transcription [8, 10]. The central 8618 nt region encodes nine open reading frames: gag, pol, vif, vpr, tat, rev, vpu, env and nef
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