Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by viral nucleoprotein (NP) and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins (vRNPs) responsible for RNA synthesis. NP is a structural component of the vRNP but also interacts with both viral and host factors to regulate viral RNA expression. NP is conserved among influenza A isolates, making NP interactions compelling antiviral targets. Here I characterize mutations within 5 amino acids of NP that comprise an accessible region of the NP body domain, as determined by NP crystal structure. This region was selected for mutagenesis to target interaction between NP and RdRP. NPbd3 encodes glycine at 5 amino acids within the accessible NP body domain. Cellular fractionation and Western Blot, in addition to NP-GFP fusions and fluorescence, confirm NPbd3 was expressed and localized as WT-NP. Gel shift with purified NP protein confirm NPbd3 bound nucleic acids as WT-NP. Although NPbd3 was expressed, localized, and bound nucleic acid as WT-NP, I found NPbd3 was defective for RNA expression in reconstituted vRNPs, as evaluated by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). To investigate this NP body domain further, single and double amino acid mutations were cloned. Analysis of NP single mutants revealed that all were nearly as functional as WT-NP for RNA expression in reconstituted vRNPs, suggesting these accessible amino acids in the NP body domain play a redundant role. However, four different combinations of two amino acid mutations resulted in NP double mutants that displayed a significant defect in RNA expression in reconstituted vRNPs, confirming these accessible amino acids in the NP body domain play a significant role for viral RNA synthesis. A disruption in an essential NP interaction with the RdRP is likely the explanation for the RNA defect observed. In support of this, avian influenza virus passaged in human cells resulted in virus with one NP amino acid change in this domain consistently paired with specific changes in the PB2 subunit of the RdRP. I reason this accessible body domain of NP is a viable antiviral target. Indeed, two amino acids in this NP body domain comprise a novel groove implicated in binding the small molecule inhibitor nucleozin. My thesis highlights this conserved NP body domain as an important interaction surface essential for viral RNA synthesis and support further investigation of antiviral drugs that target this region of NP.