Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer

Abstract

The two steps in influenza virus RNA replication are (i) the synthesis of template RNAs, i.e., full-length copies of the virion RNAs, and (ii) the copying of these template RNAs into new virion RNAs. We prepared nuclear extracts from infected HeLa cells that catalyzed both template RNA and virion RNA synthesis in vitro in the absence of an added primer. Antibody depletion experiments implicated nucleocapsid protein molecules not associated with nucleocapsids in template RNA synthesis for antitermination at the polyadenylation site used during viral mRNA synthesis. Experiments with the WSN influenza virus temperature-sensitive mutant ts56 containing a defect in the nucleocapsid protein proved that the nucleocapsid protein was indeed required for template RNA synthesis both in vivo and in vitro. Nuclear extracts prepared from mutant virus-infected cells synthesized template RNA at the permissive temperature but not at the nonpermissive temperature, whereas the synthesis of mRNA-size transcripts was not decreased at the nonpermissive temperature. Antibody depletion experiments showed that nucleocapsid protein molecules not associated with nucleocapsids were also required for the copying of template RNA into virion RNA. In contrast to the situation with the synthesis of transcripts complementary to virion RNA, no discrete termination product(s) were made during virion RNA synthesis in vitro in the absence of nucleocapsid protein molecules.

Ubiquitination and deubiquitination of NP protein regulates influenza A virus RNA replication

“…Furthermore, NP can interact with several cellular proteins, including filamentous (F) actin (Digard et al, 1999), nuclear import receptors of the importin-a class (O'Neill and Palese, 1995), the nuclear export receptor CRM1 (Elton et al, 2001) The RNP complex is the basic functional unit for replication and transcription of influenza viral genome. Previous data have shown that the template-size RNA can not be synthesized if there is no NP in the RNP complex (Honda et al, 1988), and NP is essential for the synthesis of vRNA and cRNA (Shapiro and Krug, 1988). Several hypotheses have been proposed for the role of NP in the switch between mRNA and cRNA synthesis (Portela and Digard, 2002).…”

Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication

“…However, the mechanisms of this broad inhibition of some siRNAs are still not very clear yet. From the standpoint of viral target choice in RNAi based antiviral therapy, NP protein is required for elongation and antitermination of nascent cRNA and vRNA transcripts [33,34]. Without newly synthesized NP, further viral transcription and replication are blocked.…”

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM

“…Previous reports showed that NP is important for virus genome replication (Shapiro and Krug, 1988;Medcalf et al, 1999). NP is required for elongation of RNA synthesis (Honda et al, 1988).…”

“…NP is required for elongation of RNA synthesis (Honda et al, 1988). Moreover, the in vitro cRNA synthesis using infected cell extracts as an enzyme source depends on a supply of non-RNP-associated NP (Shapiro and Krug, 1988). The latter finding may be interpreted as that NP functions in co-transcriptional encapsidation of nascent RNA for prevention of discrete RNA synthesis.…”

“…Recently, we found that PA is involved in the assembly of a functional polymerase (Kawaguchi et al, 2005). It was reported that NP is also important for the replication process (Shapiro and Krug, 1988;Medcalf et al, 1999). However, the precise function of NP in the replication remains uncertain.…”