Abstract Quantitative sequencing of SARS-CoV-2 viral RNA modifications and identification of host modification enzymes critical to viral RNA replication A new coronavirus disease (known as COVID-19) has swept 200 countries and was declared a pandemic. The causative agent is named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There is no effective vaccine currently available for SARS-CoV-2. FDA has approved two anti-malaria drugs, chloroquine and hydroxychloroquine, for emergency use for treatment of COVID-19. In addition, remdesivir, a nucleotide analog used for treatment of Ebola virus disease, is now in clinical trials and clinical use for COVID-19 treatment. Understanding properties of SARS-CoV-2 and revealing cellular components essential to its infection are critical to development of effective therapies and vaccines in the near future. SARS-CoV-2 is an RNA virus. Its viral RNAs have been shown to be chemically modified. Previous studies from us and others have revealed crucial roles of viral RNA modifications in viral replication and immune evasion. Our most recent data indicate that an RNA m5C methyltransferase NSUN2 plays a vital role in human coronavirus replication inside host cells. In this administrative supplement application we propose to apply quantitative sequencing methods developed by our CEGS to map N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (Ψ), 2’-O-methylation (Nm), N7-methylguanosine (m7G) and N1-methyladenosine (m1A) in SARS-CoV-2 RNA. We will also assign modification enzymes and test their effects on viral infection using established infection models of SARS-CoV-2. We will specifically examined NSUN2 and its effect on viral RNA m5C methylation, and test known inhibitors for inhibition of viral infection. We will also examine potential roles of m6A and related modifications in protecting viral RNA from host innate immune responses.