Measles virus (MV) is a negative-strand RNA virus within the Morbillivirus genus of the Paramyxoviridae family. The MV transcriptional complex consists of the virus-encoded RNAdependent RNA polymerase (L), the polymerase cofactor (P), and a ribonucleoprotein template consisting of the singlestranded RNA genome and the nucleocapsid protein (N protein). N protein monomers assemble on the RNA genome during replication to form a single-start left-handed helix, protecting the genome from nuclease degradation. Encapsidation is initiated on specific sequences found within the leader RNA, drawing upon pools of soluble N-P heterodimers, whereas elongation of encapsidation occurs in an RNA sequence-independent manner (7, 24). Since genomic replication presumably is dependent upon concurrent encapsidation, functional motifs in the N protein required for genomic replication must include an RNA binding domain for the initiation of encapsidation, a binding site for P to form an N-P encapsidation complex, and N-N interaction sites required to drive nucleocapsid elongation and maintain nucleocapsid structural integrity. Additionally, N protein must contain a P binding site that is exposed on the nucleocapsid, thereby permitting the viral polymerase complex to interact with formed ribonucleoprotein templates during both transcription and genomic replication.The expression of MV N protein deletion mutants demonstrates that only the amino-terminal three-fourths of the molecule (i.e., amino acids 1 to 398) is required for the formation of organized nucleocapsid-like particles, localizing the N-N interaction domain to this highly conserved portion of the protein (1, 16). This conclusion is supported by the observation that selective proteolysis of the MV nucleocapsid can remove the C-terminal 15 kDa of the N protein while leaving nucleocapsid structural integrity and the amino-terminal 45-kDa N protein fragment intact (12). Also identified within the latter region are two sites necessary for the formation of soluble N-P complexes, localized to amino acids 4 to 188 and 304 to 373 (1). Enhanced sensitivity of the C terminus of the N protein to proteolysis is consistent with its exposure on the surface of the formed nucleocapsid. Within this exposed hypervariable domain is a third binding site for P, tentatively localized between amino acids 457 and 525 (1). For Sendai virus, the C-terminal domain of the N protein is required for template function in RNA replication assays (5) and contains a P binding site (4). Given the essential role of P in supporting viral polymerase function, a model for paramyxoviruses emerges in which P provides the link between L and N protein domains that are exposed on the nucleocapsid and that are necessary for both transcription and replication.The putative template function of the MV N protein C

HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2+ND1, Ad2+ND2, Ad2+ND4. and Ad2+ND5) svnthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methioninecontaining tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific pi'otein (100,000 daltons) from Ad2+ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2+ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virusinfected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and-transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, the 42,000-and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28

The two-dimensional peptide maps of the methionine-containing tryptic peptides of the 100,000-molecular-weight (100K) and 17K T antigens of simian virus 40 (SV40) have been compared. The two proteins share nine methionine-containing tryptic peptides in common. The 17K T antigen has two peptides not found in the 100K T antigen, and the 100K T antigen has 14 unique peptides. The peptide maps of the 100K and 17K T antigens were also compared with those of the SV40-specific proteins found in cells infected by the nondefective adenovirus type 2-SV40 hybrid viruses, which we have previously shown are encoded by defined sequences within the early region of SV40 (K. Mann, T. Hunter, G. Walter, and H. K. Linke, J. Virol. 24:151-169, 1977). This comparison shows that the lOOK and 17K T antigens share common N-terminal sequences coded for between 0.65 and 0.59 map units on the SV40 genome. Furthermore, none of the sequences in the 17K T antigen arises from the region between 0.54 and 0.18 map units. We deduce that the sequences unique to the 17K T antigen originate between 0.59 and 0.54 map units. This type of structural relationship between the 100K and 17K T antigens fits well with the proposed model (L. V. Crawford, C.

After infection of permissive human fetal brain cells by BK human papovavirus (BKV), the vast majority of the cells were killed by the virus, but rare survivors were recovered after frequent medium changes. These surviving cells grew and formed visible colonies after 5 to 6 weeks and were thereafter established as permanent cell lines. These cells, designated as BK-HFB cells, were persistently infected and shed BKV. Morphologically, they were small, polygonal cells and had transformed growth properties. Their plating efficiency on solid substrates or in semisolid medium was high, and they were tumorigenic in athymic nude mice. Cloning experiments in medium containing BKV antiserum revealed that BKV did not persist in the cultures in a simple carrier state. All cloned cell lines were initially T-antigen negative and virus-free. However, every clone began to release BKV and again became persistently infected within 3 weeks after removal of BKV antiserum. After rigorous antibody treatment, four of seven clones still released virus spontaneously upon removal of antiserum; three clones have remained virus-free and are apparently cured. Although these cloned cell lines are T-and V-antigen negative when grown in antiserum-containing medium, they retain "free" or episomal BKV genomes; integrated viral DNA was not detected in any of the clones. These free genomes are indistinguishable from prototype BKV DNA and are found in much larger amounts in virus-shedding cell lines.