Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus

Abstract

Specific single stranded DNA probes have been obtained for both influenza virion RNA (vRNA) and complementary RNA (cRNA) by cloning a hemagglutinin gene fragment in the single stranded DNA phage M13. These probes were used for hybridization with the total labeled RNA from cytoplasmic extracts of infected cells. MDCK cells were infected with temperature-sensitive mutants of Influenza HK/68 and the production of the virus specific RNA species was analysed at both permissive and restrictive temperatures. Results show that two NP mutants which undergo intracistronic complementation exhibit two different phenotypes at the non permissive temperature: ts2C is poly A cRNA and vRNA negative whereas ts463 is RNA positive. Two mutants of P genes were also analysed and we discuss the relationship existing between the synthesis of the three RNA species especially between poly A and non poly A cRNA.

Studies of an Influenza A Virus Temperature-Sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions

“…Most NP ts mutants that have been previously characterized show defects in vRNA synthesis at the NPT (35,40,43,48,61,63). However, although there are abundant data showing that NP plays a key role as a structural and functional component of viral RNPs, it is also likely to be important at later time points in the replication cycle.…”

Functional Analysis of the Influenza Virus H5N1 Nucleoprotein Tail Loop Reveals Amino Acids That Are Crucial for Oligomerization and Ribonucleoprotein Activities

“…Briefly, an excess of DNA primer (about 10 5 cpm), labeled at its 5Ј end with 32 P, was mixed with 5 g of total RNA in 5 l of water and denatured at 95°C for 3 min. The mixture was cooled on ice and subsequently incubated at 45°C for 1 h with the addition of 50 U SuperScript II RNase H Ϫ reverse transcriptase (Invitrogen) in first-strand buffer (Invitrogen).…”

“…NP has been implicated in the transcription-replication processes of influenza virus, as temperature-sensitive NP mutants resulted in defective transcription at nonpermissive temperatures (13,16,29,32). NP may act as a cofactor that cotranscriptionally coats the newly synthesized cRNA (30).…”

The influenza virus RNA synthesis machine

“…96,106 This is in line with the proposal that initiation on cRNA-or vRNA-containing polymerase complexes is carried out by distinct mechanisms: 107 Whether synthesis of cRNA is initiated using as template the 3'-terminus of the vRNA template, initiation of vRNA synthesis occurs three nucleotides downstream and the pppApG initial dinucleotide should re-align to the template terminus to lead to complete replication. These observations, as well as the phenotype of temperature-sensitive and engineered NP mutants that are defective in either cRNA or vRNA synthesis 88,102 suggest that the vRNP and cRNP replication complexes are structurally and functional distinct. These differences may be at the basis of the diverse kinetics and extent of cRNA and vRNA synthesis during infection.…”

“…3,9 This observation, together with the phenotype of virus mutants that are temperature-sensitive for vRNA synthesis but show normal accumulation of cRNA at restrictive temperature, [100][101][102] suggest that most of the cRNA synthesis that takes place in the infection uses the parental RNPs as templates. The large differences in cRNA and 115,116 and in vivo, 88,102 although replication of short RNA templates can proceed in the absence of NP. 61,117 The incorporation of NP into new vRNPs is facilitated by the activity of cellular factors UAP56 and Tat-SF1, that may act as chaperones.…”