Isolation of influenza virus ribonucleoprotein from infected cells. Demonstration of the presence of negative-stranded RNA in viral RNP

1982

SUMMARYThe synthesis of influenza virus ribonucleoprotein structures (RNPs) in infected chick embryo cells was analysed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium deoxycholate which resolves the RNPs into five size classes. A relatively small proportion of total RNPs accumulated in the nucleus but free NP protein was found there in large amounts over the period 1.5 to 4 h post-infection. In contrast, by 4 h post-infection, all cytoplasmic NP was complexed into RNP structures. At early times, during a 15 min pulse of [aSSlmethionine, nearly all the newly synthesized NP was incorporated into RNPs but by 4 h the majority of pulse-labelled NP was present as free protein. However, the proportion of free NP:NP in RNPs remained constant over the 1.5 to 4 h post-infection period, indicating that there was a delay before the NP synthesized later in infection was assembled into RNP structures. Individual RNP size classes were predominantly cytoplasmic and accumulated at similar rates but were not produced in equimolar amounts. The rates of synthesis of individual RNPs were in general agreement with their rates of accumulation with the remarkable exception of RNP d (containing RNA 7, the matrix protein gene). This was synthesized nearly 10-fold faster but accumulated at the same rate as the other RNPs. Possibly RNP d is more rapidly degraded than the other RNPs.The influenza virus genome consists of eight separate segments of single-stranded negative-sense RNA (RNA-, McGeoch et al., 1976;Palese & Schulman, 1976). In the infected cell these RNA segments are transcribed into two forms of complementary plus-sense RNA (RNA+): a truncated copy which serves as messenger RNA (mRNA) to direct the synthesis of virus proteins and a complete transcript (cRNA) which serves as template for the synthesis of negative-sense virus RNA (Hay et al., 1977a, b). Where the various forms of virus-coded RNA are synthesized is unclear, but there is evidence that the cell nucleus is the probable site of transcription of virus mRNA (Armstrong & Barry, 1974;Taylor et al., 1977;Mark et al., 1978Mark et al., , 1979 Barrett et al., 1979).Within the virion the RNA segments are complexed with nucleoprotein (NP; Pons et al., 1969) to form separate ribonucleoprotein (RNP) complexes (Duesberg, 1969; Compans et al., 1972). In extracts from infected cells, RNPs containing both RNA + and RNA-have been found (Pons, 1971(Pons, , 1975 and it seems reasonable to suppose that all intracellular virus-induced RNA may be present in RNP complexes. Apart from trace amounts of other virus proteins the principal component of intracellular RNPs is the NP protein (Caliguiri & Gerstein, 1978; Rees & Dimmock, 1981 b).A number of studies has been made of the kinetics of synthesis and accumulation of virus-specific RNA (Scholtissek & Rott, 1970; Hay et al., 1977a, Barrett et al., 1978Mark et al., 1978) and proteins (Skehel, 1972(Skehel, , 1973 Meier-Ewert & Compans, 1974;Lamb & Choppin, 1976; Inglis et al., 1976 Inglis et al., , 1978 Inglis & Mahy,...

Section: Discussionmentioning

confidence: 99%

Kinetics of Synthesis of Influenza Virus Ribonucleoprotein Structures

Rees

1982

“…During infection at 4 °C, which allows events to proceed up to viral transcription, influenza virus RNP accumulates in the nucleus, but when cells are warmed to 37 °C the majority of the RNP migrates from the nucleus (Hudson et al, 1978). Newly synthesized RNPs with properties similar to those obtained from disrupted virus particles have been detected in both the nucleus and cytoplasm of infected cells (Krug, 1971), while Pons (1971) has shown the presence of what is now known as RNA + in RNPs isolated from infected cells, whereas RNPs from virions contain little or no RNA +. In addition, other data suggest that influenza virus mRNA exists on polysomes, not as free RNA but in the form of an RNP (Pons, 1975).…”

Section: Introductionmentioning

confidence: 96%

“…RNPs were extracted from purified virus contained in low salt buffer (0-2 M-NaC1, 0.02 M-tris pH 7.4, 2 mM-EDTA; Duesberg, 1969), by disruption with 0.2 vol. 5 % Nonidet P40 (NP40; BDH), 2.5% sodium deoxycholate (DOC; BDH) followed by incubation at 37 °C for 4 min (Pons, 1971) before loading on to the gel. Gradient slab gels of 3 to 4% or 3 to 5 % polyacrylamide were formed from the following solutions: 3 % (w/v) acrylamide, 0.14% (w/v) bisacrylamide, 10% (v/v) glycerol, 0.048% (v/v) N,N,N',N'-tetramethylenediamine (TEMED), 0.48% (w/v) ammonium persulphate and 4% (or 5%) acrylamide, 0.19% (0.23% for 5% gel) bisacrylamide, 30% glycerol, 0.02% TEMED, 0.2% ammonium persulphate.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Electrophoretic Separation of Influenza Virus Ribonucleoproteins

Rees

1981

SUMMARYA new procedure for the separation of influenza virus particle ribonucleoproteins (RNPs) by electrophoresis on a slab of polyacrylamide gel resolves five discrete bands. The largest is a triplet containing the three largest RNAs (1 to 3), the intermediate sized RNP is a doublet containing RNAs 5 and 6 and the others each contain a single RNA. In addition, each RNP is composed of NP protein and an amount of M which is independent of the size of the RNP. This suggests that some virus particle M is specifically associated with influenza ribonucleoprotein.

“…Analysis of radiolabelled proteins following the fractionation of infected cells showed that after synthesis in the cytoplasm, the majority of NP migrates to the nucleus where it associates with the nucleoplasm (Taylor et al, 1969(Taylor et al, , 1970Lazarowitz et al, 1971;Krug & Etkind, 1973;Hay & Skehel, 1975;Flawith & Dimmock, 1979;Briedis et al, 1981). The situation is complicated because NP is found in both nucleus and cytoplasm as 'free' protein and associated with viral RNA, and the latter ribonucleoproteins (RNPs) contain both negative and positive strand viral RNA (Pons, 1971(Pons, , 1975Caliguiri & Gerstein, 1978;. Also NP appears to be able to cross the nuclear envelope in either direction (Hudson et al, 1978;Flawith & Dimmock, 1979).…”

Section: Introductionmentioning

confidence: 99%

Location of Influenza Virus M, NP and NS1 Proteins in Microinjected Cells

Davey

Colman

1985

SUMMARYWhen microinjected as cloned DNA, the nucleoprotein (NP) of influenza virus A/NT/60/68 (H3N2) accumulated in the nuclei of Xenopus laevis oocytes, and cultured cells of rodent and primate origin. This accumulation appeared to be specific and a property of the NP itself (or conceivably NP in association with unknown cellular constituents) since no other influenza virus components were present in DNA-injected cells. In the oocyte nucleus, clonally derived NP achieved an eightfold concentration over that in the cytoplasm. Such NP was full-length as judged by its mobility during PAGE and had the native conformation of H3N2 virus NP according to its reaction with a panel of monoclonal antibodies. NP appeared to be in the soluble fraction of the nucleus as it did not sediment under conditions which removed particulate matter from nuclear extracts. Microinjection of extracts of chick embryo fibroblast cells infected with A/FPV/Rostock/34 (H7N1) showed that exogenous NP had an affinity for the nucleus similar to that synthesized intracellularly from cloned NP DNA. This conclusion was supported by an experiment in which cloned NP from the oocyte nucleus re-entered the nucleus after injection into the cytoplasm of fresh oocytes. Injection of mRNA, extracted from chick embryo fibroblast cells infected with A/FPV/Rostock/34, into oocytes directed the synthesis of the viral proteins M (Mr 28000), and NS1 (MT 27000) as well as NP (Mr 56000). While NP from this source concentrated in the nucleus as before, M merely associated with the nucleus without exceeding the cytoplasmic level. Even more remarkable was NS1 ; although in injected cells this protein is concentrated in nucleoli, in microinjected oocytes its nuclear concentration was threefold less than that in the cytoplasm, despite the very large number (> 1500) of nucleoli present in Xenopus oocytes. It seems likely that the karyophilic nature of M and NS1, unlike that of NP, is a property not of the proteins themselves, but of a complex which they form with some other product of the infected cell. These findings were repeated when extracts from infected chick embryo cells containing NP, M and NS1 proteins radiolabelled in vivo, were injected into the cytoplasm of oocytes.