Longitudinal HIV-1 single genome sequencing (SGS), which permits unambiguous genetic characterization of circulating viral strains without introduction of PCR error, can be used to identify sites in the viral genome that are under selective pressure. Following transmission, the earliest sites under positive selection often fall in cytotoxic T lymphocyte (CTL) epitopes. During escape from CTL immune pressure, viral sequences typically exhibit nonsynonymous mutations within the span of the cognate T cell epitope. I applied SGS to study sequence evolution in the HIV-1 5’ leader sequence, which is thought to be translationally silent. I observed mutational patterns consistent with CTL escape and demonstrated that the HIV-1 5’ leader expresses T cell antigens from non-canonical one-off AUG codons (e.g. CUG). While these non-canonical start codons can be mutated during CTL escape, a reverse transcriptase overextension error periodically restores a one-off AUG within the 5’ leader. As infection ensues, sites under selection within the gene encoding the viral envelope glycoprotein (Env) often fall within autologous neutralizing antibody epitopes. In a subset of individuals, the strain-specific neutralizing antibody response develops into a broadly cross-reactive neutralizing antibody (bnAb) response. To understand what factors influence bnAb ontogeny, I used SGS to study Env evolution both during natural infection and immunotherapy. I found viral diversification in bnAb contact residues and divergence of the virus population into multiple persistent lineages to precede bnAb development. Taken together, these data demonstrate that longitudinal HIV-1 SGS can be used to discover novel aspects of virus biology and host-pathogen interactions.