Dynamics of Viral RNA Synthesis during Measles Virus Infection

Abstract

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until ϳ24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is ϳ3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5-end abortive replication fragment RNAs.Viruses are obligate intracellular parasites. To understand the complexity of virus replication and to develop potent antiviral drugs, there is a growing need for a systematic analysis of the underlying molecular mechanisms. Although the kinetics of protein expression has been established for many viruses and a model of the dynamics of virus transcription and replication for bacteriophage Q has been proposed (see reference 10 for a review), knowledge in this area is very limited for all eucaryotic viruses. We report the first kinetics model of the transcription and replication of a negative-sense, singlestranded RNA virus.Measles virus (MV) belongs to the order Mononegavirales (33). The replication strategy involves a viral RNA-dependent RNA polymerase (vRdRp), which uses as a template a nucleocapsid (NC) made of a single strand of RNA in tight complex with the nucleoprotein (N). The negative-strand genome contains six transcription units encoding the N, phospho (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) or polymerase protein, in that order (Fig. 1). The transcription units are flanked by short leader (Le) and trailer (Tr) sequences containing the genomic promoter (on the minus strand) and the antigenomic promoter (on the plus strand), respectively. The P gene also encodes the nonstructural V and C proteins by RNA editing and alternative overlapping open reading frame, respectively.Every N protein binds to 6 nucleotides so that the N polymer entirely covers the 15,894-nucleotide genome (5) and makes it resistant to nucleases (33). NC comprises 2,649 N, ϳ300 P, and ϳ20 to 50 L proteins (33) and is enclosed within a membrane envelope containing H and F glycoproteins. After fusion with the plasma membrane at neutral pH, intact NCs are released into the cytoplasm (24), where they serve as a template for both primary transcription from and replication of the genomic RNA (Fig. 1). Transcription is initiated from a single promoter at the 3Ј end of the genome (for a review, see reference 23) by vRdRp made of L and P proteins. Immediately prior to an intergenic junction...

Suppression of the Sendai Virus M Protein through a Novel Short Interfering RNA Approach Inhibits Viral Particle Production but Does Not Affect Viral RNA Synthesis

“…SeV multiplies efficiently in cells in culture, making it one of the most productive viruses of the family. The number of SeV-M messengers per infected cell has been estimated to amount to ϳ30,000 (23), a number that exceeds by almost 10-fold that measured in measles virus-infected cells (10,39). It was therefore comforting to realize that efficient suppression of SeV-M was feasible.…”

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus

“…This positive-stranded RNA (antigenome) does not serve as a template for transcription; its unique role is to provide an intermediate in genome replication. The intracellular concentration of the N protein is the main element controlling the relative level of transcription versus replication (9). When N is limiting, the polymerase functions preferentially as a transcriptase, thus leading to an increase in the intracellular concentration of viral proteins including N. When N levels are high enough to allow encapsidation of the nascent RNA chain, the polymerase functions preferentially as a replicase (see Refs.…”

“…by sedimentation on a discontinuous sucrose gradient. Sucrose gradientpurified viruses were characterized by SYBR Green real time RT-PCR analysis of the L-trailer/F genome ratio as a measure of defective interfering particle content (9,53).…”

“…Viral mRNA and genome production was defined by Northern blot analysis of total cell RNA to assess the integrity of viral transcript and genome production and by SYBR Green realtime RT-PCR analysis to assess the kinetics of viral RNA production (9,53). Vero cells were infected at an m.o.i.…”

“…These RT reactions included 25 nmol of dNTP and 1 l of reverse transcriptase (RT from Stratagene) in a final volume of 20 l. Reactions were incubated for 90 min at 48°C. SYBR Green quantitative PCR was performed in a Roche 480 LightCycler using previously reported N, L-trailer, and negative strand F genespecific primers and PCR cycle conditions (9,53). Serial dilutions of pϩMV were used as the RT-PCR standard for genome levels, and in vitro transcribed N mRNA was the standard for transcript analysis.…”