Sandra M. Horikami

Host cell proteins required for measles virus reproduction

We have developed a cell-free system derived from measles virus-infected cells that supported the transcription and replication of measles virus RNA in vitro. The data suggest that tubulin may be required for these reactions, since an anti-beta-tubulin monoclonal antibody inhibited viral RNA synthesis and the addition of purified tubulin stimulated measles virus RNA synthesis in vitro. Tubulin may be a subunit of the viral RNA polymerase, since two different anti-tubulin antibodies, one specific for the beta- and another specific for the alpha-subunit of tubulin, coimmunoprecipitated the measles virus L protein as well as tubulin from extracts of measles virus-infected cells. Other experiments further implicated actin in the budding process during virus maturation, as there appeared to be a specific association of actin in vitro only with nucleocapsids that have terminated RNA synthesis, which is presumably a prerequisite to budding.

The Role of Viral and Host Cell Proteins in Paramyxovirus Transcription and Replication

The viruses of the Paramyxoviridae family contain a nonsegmented RNA genome of the negative (−)-strand sense [for earlier reviews see Kingsbury (1977), Choppin and Compans (1975), Kolakofsky and Roux (1987)]. In the case of Sendai virus, the prototype of the family, the genome RNA and its full-length complement, the antigenome RNA, are found both in the virion and in the infected cell as an RNase-resistant nucleocapsid due to the tight association of the major nucleocapsid protein, NP (M r 57, 000) with the RNA. Two other viral proteins, the P (M r 79, 000) and L (M r 240, 000) proteins, are associated less tightly with the nucleocapsid and function as subunits of the RNA-dependent RNA polymerase. Three additional viral proteins, the hemagglutinin-neuraminidase (HN, M r 72, 000, fusion (F o, M r 65, 000), and matrix (M, M r 34, 000) proteins, are associated with the lipid envelope of the virion.

DOUBLE-STRANDED RNA ADENOSINE DEAMINASE ACTIVITY DURING MEASLES VIRUS INFECTION

It has been postulated that the cellular double-stranded (ds) RNA adenosine deaminase enzyme is responsible for biased hypermutation during persistent SSPE measles infections in humans. As a test of this hypothesis we studied the effect of negative-strand RNA virus infection on enzyme activity. The adenosine deaminase activity was found in nuclear extracts of both uninfected CV-1 and A549 cells and in cytoplasmic extracts of A549, but not CV-1, cells. During measles or Sendai virus infection of either CV-1 or A549 cells the adenosine deaminase activity in the nucleus remained fairly constant up to 24 h post infection, and there was no apparent re-partitioning of the enzyme between the nucleus and the cytoplasm. Transcription complexes of Sendai virus in vitro or measles virus in vivo did not serve as substrates for the enzyme. These data suggest that even though some portion of the adenosine deaminase enzyme may be present in the cytoplasm of at least some cells during virus infection, modification of the viral RNAs by this enzyme, if it occurs at all, must be at a very low level not directly detectable by biochemical analysis.