Project Summary/Abstract We have recently discovered dehydroamino acids (DHAAs) in the capsid and matrix proteins that make up HIV-1 virions. These dehydroalanine (DHA) and dehydrobutyrine (DHB) residues result from posttranslational modification of serine, threonine, or cysteine residues. We propose here to investigate the importance and origin of these fascinating protein modifications. Their high prevalence in these viral proteins, in marked contrast to their low levels in the human proteome generally, raises two key questions: a) why are they there? and b) how did they get there? We hypothesize that DHAAs are present in virions because they play important roles in the HIV virion assembly or capsid maturation. We hypothesize further that DHAAs are formed by a presently unknown enzymatic activity in viral or host proteins. To test these hypotheses, we will conduct mutation and inactivation studies of the amino acids that convert to dehydroamino acids and study the effects on the viral replication cycle and infectivity. We will use quantitative proteomics to determine how much of this modification is generated and whether it is before or after viral maturation. We will seek to discover intra- or intermolecular protein crosslinks, which dehydroamino acids are known to be able to form, using innovative proteomics approaches. Further, we will seek to discover the enzyme responsible for the formation of these modifications. The discovery of the enzyme responsible for DHAA formation would be interesting as fundamental biology, offer new insights into HIV replication, and potentially reveal a new therapeutic pathway for the treatment of HIV infection.