Characterization of Two Recombination-Complementation Groups of Uukuniemi Virus Temperature-sensitive Mutants

Abstract

SUMMARYWith the aim of isolating temperature-sensitive (ts) mutants defective in virus maturation or glycoprotein transport, Uukuniemi virus, a bunyavirus, was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. Out of 13 initial clones unable to grow at 39 °C (non-permissive temperature), five mutants which grew to titres above 107 p.f.u./ml at 32 °C (permissive temperature) were selected for further studies. The mutants fell into two coinciding recombination-complementation groups. Three group I mutants (ts7, 8 and 12) and two group II mutants (ts6 and 11) synthesized all three RNA segments and were able to form the corresponding nucleoproteins at 39 °C. Thus, members of these two recombination groups had a RNA-positive phenotype. All five mutants showed immunofluorescence when cells were stained at 39 °C using a doublestaining technique employing monoclonal antibodies against the glycoproteins G 1 or G2, and polyclonal antibodies against the nucleoprotein, N. We have previously shown that in cells infected with wild-type virus both the G1/G2 and the N proteins accumulate in the Golgi complex, the site of virus maturation. In cells infected with tsl2, accumulation of G1 and G2, but not N protein, was observed in the Golgi complex at 39 °C. The N protein was found evenly scattered in the cytoplasm, suggesting lack of interaction between the G1/G2 and N proteins. With ts6 and 11, G1 and G2 appeared to accumulate and aggregate in the endoplasmic reticulum (ER) at 39 °C. The location of the N protein coincided with that of the aggregated glycoproteins, suggesting that the N protein interacted with G 1/G2 already in the ER.

Bunyaviruses: From Transmission by Arthropods to Virus Entry into the Mammalian Host First-Target Cells

“…Thereby one can imagine that virus variants have proteins correctly folded and functional in the typical range of temperature associated with these hosts (28-32°C), but not at a higher temperature, such as that in mammalian bodies (37°C). Temperature-sensitive virus mutants obtained in laboratory have been reported for UUKV and RVFV [124,125]. While remaining important for the infection of the first target-cells, these mutants might be unable to propagate throughout mammalian hosts.…”

Membrane Insertion and Transport of Viral Glycoproteins: A Mutational Analysis

“…Several viruses assemble at points within the secretory pathway, and since in many cases the location of the viral glycoproteins defines the virus maturation point (Roth et al, 1983b;Jones et al, 1985;Gottlieb et al, 1986;Gahmberg, 1984;Kabcenell and Atkinson, 1985) these systems are proving useful in investigating the signals that target proteins to specific subcellular locations. Figure 3 is a schematic summary of the assembly points for the major groups of enveloped viruses.…”

“…During the genetic analysis of enveloped virus replication through the isolation and biochemical characterization of spontaneous and mutageninduced variants, complementation groups were established for several viruses that contained mutants defective in normal transport of the viral glycoproteins (Knipe et al, 1977a,b;Zilberstein et al, 1980;Pesonen et al, 1981;Gahmberg, 1984;Ueda and Kilbourne, 1976). The existence of conditional lethal mutants that were blocked at different stages of virus glycoprotein maturation suggested that the viral polypeptides themselves might contain the signals necessary for normal sorting by the cells' transport machinery and raised the possibility that such mutants could be used tq dissect the maturation pathway of a glycoprotein.…”

“…The mutants of VSV, together with equivalent mutants in other viral systems that were blocked at different stages of the secretory pathway (Pesonen et al, 1981;Gahmberg, 1984;Kuismanen et al, 1984), raised the possibility of identifying and understanding the nature of sorting signals in secreted polypeptides. With the advent of recombinant DNA and rapid nucleotide sequence techniques, it has been possible to determine at the amino acid level the basis for these defects (Gallione and Rose, 1985;Arias et al, 1983), but the interpretation of this information with regard to protein transport has been less than straightforward.…”

The Proteins and RNAs of St. Abb's Head Virus, a Scottish Uukuvirus

“…The Uukuvirus genus comprises a single serogroup containing seven viruses, and in nature uukuviruses have been isolated from ticks, birds and certain rodents (Bishop & Shope, 1979;Bishop et al, 1980). The prototype of the genus is Uukuniemi virus (Oker-Blom et al, 1964) which has been the subject of many biochemical analyses (Gahmberg, 1984;Kuismanen, 1984;Kuismanen et al, 1982Kuismanen et al, , 1984Parker & Hewlett, 1981 ;Pesonen et aL, 1982;Pettersson & K/ifiri~inen, 1973 ;Pettersson et al, 1971Pettersson et al, , 1977von Bonsdorff & Pettersson, 1975;Ulmanen et al, 1981). The other six recognized members of the genus, Grand Arbaud, Manawa, Oceanside, Ponteves, Zaliv-Terpeniya and EgAn1825-61, are serologically related to Uukuniemi virus but no structural or biochemical analyses of these viruses have been reported.…”