Summary: In order to prevent epidemic/pandemic influenza, high growth reassortants (genetically mixed influenza viruses) are necessary for efficient preparation of inactivated influenza virus vaccines. Many viruses selected for use in vaccines do not grow well in eggs or in cell cultures in their native states. Generating high-growth influenza viruses is thus a priority for increasing vaccine production. In addition to the inactivated influenza virus vaccine, live attenuated influenza viruses are being evaluated for vaccines to stimulate not only the local and systemic antibody responses but also to have effects on cellular immune reactions, which could result in additional protection against influenza infection. Since replication is one of the potential factors in influenza virus virulence, understanding viral replication is a priority for preparing safe live attenuated vaccines. Thus, a better understanding of the mechanisms of replication of influenza viruses is central to improving both inactivated and live attenuated influenza vaccines. Although multiple genes may contribute to the attenuation phenotype of influenza viruses, the matrix gene by itself may confer attenuating properties. The matrix protein (M1) of influenza virus also plays a central role in viral replication. We have studied the RNP-binding activity of M1's from a high-growth strain and a relatively low-growth wild-type strain. Our results indicated that RNP-binding of M1 of the high-growth strain was more difficult to break than the RNP-binding of M1 of the low-growth strain. These studies also suggested that the binding between M1 and RNP may be one of the factors determining viral growth. Another of our studies also demonstrated that the interaction of NP with viral RNA and M1 in a system devoid of other viral proteins is sufficent for translocation of RNP from the nucleus to the cytoplasm, which indicates that viral RNA and M1 together are neccesary for maturation of the viral RNP and for final assembly of viral particle. To further understand the role of M1 in viral assembly and replication, mutations in M1 nuclear localization signal/RNP-binding sequences (amino acids 101-105 RKLKR) or M1 zinc-finger motif (which has RNA-binding properties) were constructed using PCR techniques and introduced into wild type influenza virus by reverse genetics. Altering the zinc-finger motif of M1 only slightly affected viral growth. Although substitution of Arg with Ser at position 101 or 105 of RKLKR did not have a major impact on nuclear export of RNP or viral replication, growth rates of these mutants were reduced. In contrast, deletion of RKLKR or substitution of Lys with Asn at position 102 or 104 of RKLKR were lethal. These results further refine the understanding of the importance of the RKLKR domain of M1 protein in viral replication, and also have implications for understanding viral attenuation.