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RNA virus infection results in expression of type 1 IFNs, especially IFN-α/β, which play a crucial role in host antivirus responses. Type 1 IFNs are induced in a cell type-specific manner through TLR and RIG-I–like receptor pathways, both of which activate IFN regulatory factors (IRFs) and NF-κB transcription factors. Although NF-κB activation and association with the IFN-β promoter after RNA virus infection is well documented, our previous work showed that, surprisingly, NF-κB is not essential for IFN-β gene expression. Thus, the actual function of NF-κB in IFN-β expression and virus replication is not clear. In this study, we found Newcastle disease virus and vesicular stomatitis virus replication is enhanced in mouse embryonic fibroblasts (MEFs) lacking the NF-κB RelA subunit. Increased virus replication was traced to a specific requirement for RelA in early virus-induced IFN-β expression. At these time points, when IFN-β expression is ~100-fold less than peak levels, impaired IFN-β production delayed IFN-induced gene expression, resulting in increased virus replication in RelA−/− MEFs. Importantly, our results show that RelA requirement is crucial only when IRF3 activation is low. Thus, high levels of activated IRF3 expression are sufficient for induction of IFN-β in RelA−/− MEFs, transcriptional synergism with the coactivator CREB-binding protein, and rescue of susceptibility to virus. Together, these findings indicate that NF-κB RelA is not crucial for regulating overall IFN-β production, as previously believed; instead, RelA is specifically required only during a key early phase after virus infection, which substantially impacts the host response to virus infection.

Reverse transcription, an essential step in the life cycle of all retroelements, is a complex, multistep process whose regulation is not yet clearly understood. We have recently shown that reverse transcription in the pararetrovirus duck hepatitis B virus is associated with complete dephosphorylation of the viral core protein, which forms the nucleocapsid wherein reverse transcription takes place. Here we present a genetic study of the role of this dynamic nucleocapsid phosphorylation in regulating viral reverse transcription. Detailed analyses of the reverse transcription products synthesized within nucleocapsids composed of core phosphorylation site mutants revealed that alanine substitutions, mimicking the nonphosphorylated state, completely blocked reverse transcription at a very early stage. In contrast, aspartate substitutions, mimicking the phosphorylated state, allowed complete first-strand DNA synthesis but were severely defective in accumulating mature doublestranded DNA. The latter defect was due to a combination of mutant nucleocapsid instability during maturation and a block in mature second-strand DNA synthesis. Thus, the reversible phosphorylation of the nucleocapsids regulates the ordered progression of reverse transcription.Reverse transcription, the synthesis of a double-stranded (DS) DNA copy of an RNA, is an essential step in the life cycle of all reverse-transcribing elements, including classical retroviruses, retrotransposons, and pararetroviruses, such as the hepadnaviruses. The family Hepadnaviridae, which includes the global human pathogen Hepatitis B virus (HBV) and closely related animal viruses, such as Duck hepatitis B virus (DHBV), is a group of small hepatotropic DNA viruses (7,8). All hepadnaviruses replicate their DNA genomes via an RNA intermediate termed the pregenomic RNA (pgRNA) (31). Viral assembly begins with the packaging of the pgRNA, along with the virally encoded reverse transcriptase (RT), into an immature nucleocapsid (NC), followed by reverse transcription of the pgRNA within the NC into the characteristic, partially DS relaxed circular (RC) DNA genome. The DNA-containing mature NC is then enveloped and secreted extracellularly as a complete virion (10,26,31,36).The icosahedral NC consists of 180 or 240 copies of a single viral protein, the capsid or core protein (2,6,38). The Nterminal two-thirds of the core protein forms the capsid shell (1), while its basic, arginine-rich C-terminal domain is dispensable for capsid assembly but required for pgRNA packaging and reverse transcription (30,39). Both the HBV and DHBV core proteins are known to be phosphorylated at the C-terminal domain in the host cells, which has been shown to modulate core protein functions, including pgRNA packaging and DNA synthesis (9,18,20,22,24,28,30,40,41).Recently, we reported that the phosphorylation state of the DHBV NC undergoes a dynamic change during reverse transcription such that the phosphorylated, immature NCs become dephosphorylated as they mature (27). The immature NCs consist of co...

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