Mumps virus (MuV) is highly neurotropic and was the leading cause of aseptic meningitis in the WesternHemisphere prior to widespread use of live attenuated MuV vaccines. Due to the absence of markers of virus neuroattenuation and neurovirulence, ensuring mumps vaccine safety has proven problematic, as demonstrated by the occurrence of aseptic meningitis in recipients of certain vaccine strains. Here we examined the genetic basis of MuV neuroattenuation and neurovirulence by generating a series of recombinant viruses consisting of combinations of genes derived from a cDNA clone of the neurovirulent wild-type 88-1961 strain (r88) and from a cDNA clone of the highly attenuated Jeryl Lynn vaccine strain (rJL). Testing of these viruses in rats demonstrated the ability of several individual rJL genes and gene combinations to significantly neuroattenuate r88, with the greatest effect imparted by the rJL nucleoprotein/matrix protein combination. Interestingly, no tested combination of r88 genes, including the nucleoprotein/matrix protein combination, was able to convert rJL into a highly neurovirulent virus, highlighting mechanistic differences between processes involved in neuroattenuation and neurovirulence.Mumps virus (MuV) is a paramyxovirus belonging to the Rubulavirus genus. The nonsegmented negative-sense genome of 15,384 nucleotides contains the following seven transcription units: the nucleoprotein (N), the V/phosphoprotein (V/P/ I), the matrix protein (M), the fusion protein (F), the small hydrophobic protein (SH), the hemagglutinin-neuraminidase protein (HN), and the large protein (L) genes. Each gene encodes a single protein, with the exception of the V/P/I gene (conventionally referred to as the P gene), which gives rise to additional mRNA species as a result of the cotranscriptional insertion of nontemplated G nucleotides between positions 461 and 466, the so-called editing site. Faithful transcription of the gene produces the V protein, whereas insertion of two G residues within the editing site produces an mRNA encoding the P protein, and insertion of four G residues produces an mRNA encoding the I protein, analogous to the W protein identified in other paramyxoviruses (9,20,28). The roles of the viral proteins in the life cycle of paramyxoviruses have been well described in the literature (9,12,20). Briefly, the N protein encapsidates the nascent viral RNA as it is being synthesized, forming a ribonucleoprotein (RNP) complex, which serves as the substrate for the viral RNA-dependent RNA polymerase (RdRp), formed by the P and L proteins. The HN glycoprotein mediates attachment of the virus to its cellular receptor (sialic acid) and, together with the F glycoprotein, mediates fusion of the virion membrane with the target cell membrane. Based on studies of related paramyxoviruses, the M protein is involved in assembling the RNP at the plasma membrane in regions containing the HN and F glycoproteins, presumably via interacting with the carboxy-terminal tail portion of the RNP-associated N protein and the cy...