The HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication including, but not limited to transcription, splicing, nuclear export, translation, packaging and reverse transcription. Despite this extensive existing catalogue of RNA sequences that are critical to its replication, chemical probing and targeting mutagenesis studies suggest that the HIV-1 genome may contain many more RNA elements of unknown important function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we conducted a global synonymous mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice sites. This data from the Group 2 mutants indicates complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication. In silico analysis of the Group 3 mutants revealed that our mutagenesis had significantly increased the frequency of CG dinucleotides in sections of the viral genome to that of random sequence. This is important due to the remarkable CG suppression in both the HIV-1 and human genomes, and we had therefore disrupted the dinucleotide congruence that exists between HIV-1 and the genome of its host. We recoded these mutants to selectively remove either only the CG dinucleotides or only remove the mutations that did not encode a CG dinucleotide. Analysis of these mutants clearly demonstrated that the addition of CG dinucleotides were the causative mutations entirely responsible for the observed replication defects. qPCR analysis and smFISH microscopy revealed that the addition of CG dinucleotides to HIV-1 resulted in a depletion of the cytoplasmic mRNA molecules where the CG-dinucleotides were encoded as exons. A targeted siRNA screen for proteins that destabilize cytoplasmic RNA identified the Zinc-finger Antiviral Protein (ZAP) as responsible for the restriction of the CG-high HIV-1, specifically by targeting CGhigh viral RNA. CLIP-Seq experiments demonstrate that ZAP binds directly to CG dinucleotides in both cellular and viral RNA. Collectively these studies implicate ZAP as a cellular protein that can recognize CG-high viral RNA and is possibly a cellular mechanism for determining self from non-self RNA based on the CG composition. TRIM25 has previously been identified as a cofactor for two cytosolic RNA binding proteins that have antiviral functions, RIG-I where it is an essential cofactor, and ZAP where it functions as an enhancing cofactor. The mechanism by which TRIM25 enhances the antiviral activity of ZAP currently remains unclear. Through CLIP-Seq experiments in cells knocked out for TRIM25, we determined that ZAP does not require TRIM25 to recognize CG-high RNA. Using full length mutants of TRIM25 that are deficient for either RNA binding, E3 ligase activity, or formation of higher order multimers, our data suggest that the key biological activity required for TRIM25 to enhance ZAP is the formation of higher order multimers. Analyzing the replication of CG-high HIV-1 in different cell lines indicates that ZAP is not equally potent across all cell lines. The degree of potency ZAP possess against CG-high HIV-1 does not correlate with TRIM25 expression, suggesting the possibility of an additional ZAP cofactor that is heterogeneously expressed in varying cell lines. siRNA screens have been used in an attempt to identify a yet undiscovered cofactor, but so far these experiments have not yielded any such factor