Sendai virus C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis

Kurotani, Atsushi; Kiyotani, Katsuhiro; Kato, Atsushi; Shioda, Tatsuo; Sakai, Yuko; Mizumoto, Kiyohisa; Yoshida, Takemi; Nagai, Yoshinori

doi:10.1046/j.1365-2443.1998.00170.x

Cited by 113 publications

(155 citation statements)

References 44 publications

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“…SeV-C has been shown to inhibit viral RNA synthesis (7,30,46), it is known to interact with the RNA polymerase L protein (21,24), and it harbors a membrane-targeting signal (D. Garcin and D. Kolakofsky, unpublished data) that could serve to bring the nucleocapsids to the site of assembly, where M would come into play. This scenario is consistent with a clear overtranscription phenotype associated with SeV-C gene mutants (12,28,30) and with the observation that an rSeV deficient in the four C proteins was claimed to exhibit a defect in infectious virus production (28).…”

Section: Discussionsupporting

confidence: 83%

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

Mottet-Osman

Iseni

Pelet

et al. 2007

J Virol

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Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3 untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50-to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.Enveloped viruses derive their envelope from cellular membranes after the viral components have assembled at the lipid bilayer. The assembly process brings together the glycoproteins spanning the lipid bilayer with the inner core of the virus particle. The inner layer of the membrane generally contains a viral protein that bridges the glycoproteins and the inner core, dubbed the matrix or M protein. M is generally considered an essential protein, without which the production of virus particle production is highly impaired if not impossible.The M protein of Sendai virus (SeV-M), a member of the Paramyxovirinae subfamily, Paramyxoviridae family, is no exception to the rule. It is synthesized in the cytoplasm and self-associates to form a leaflet at the inner face of the plasma membrane (for a recent review, see reference 45). In the virus particle, it similarly carpets the inner part of the viral envelope, interacting with the two surface glycoproteins, HN and F, on the one hand and with the viral ribonucleoprotein complex (N protein plus viral RNA) associated with the L and P proteins on the other hand (for a review, see reference 29). In addition to its role in virus particle formation, paramyxovirus M has been reported to participate in the regulation of RNA synthesis (19,27,38,40,44). Such a role for M in viral transcription control has been described for other negative-stranded RNA viruses, such as vesicular stomatitis virus (VSV) and rabies virus (both members of the Rhabdoviridae family) (9,11,26,31,49) as well as for the influenza viruses (Orthomyxovirus family) (32, 48). In addition, VSV-M has been implicated in the shutoff of cellular transcription (3, 4), and rabies virus-M has been implicated in the stimulation of viral replication in vivo (14, 15).Our laboratory has long been interested in the SeV-M protein and, in particular, in its function in virus particle formation (13,35,36,43). To perform a structure-function analysis, one would ideally like to silence expression of the resident SeV-M gene and replace it with M mutants in a search for residues or domains that can modulate its functions. One approach would con...

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Section: Discussionsupporting

confidence: 83%

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

Mottet-Osman

Iseni

Pelet

et al. 2007

J Virol

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“…LLC-MK 2 and CV1 cells were grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum. Wild-type SeV derived from a cDNA of the Z strain (16) and its 4C(Ϫ) mutant virus, in which all four C proteins were knocked out (20), were propagated in embryonated chicken eggs, and infectivity was measured using an immunofluorescent infectious focus assay (19) and expressed as cell infectious units (CIU)/milliliter.…”

Section: Methodsmentioning

confidence: 99%

AIP1/Alix Is a Binding Partner of Sendai Virus C Protein and Facilitates Virus Budding

Sakaguchi

Kato

Sugahara

et al. 2005

J Virol

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“…In contrast to the well-characterized P protein, little is known about the functions of the C and V proteins. The C proteins have been shown to be indispensable for efficient virus multiplication and pathogenicity (18), and they have also been shown to block interferon-mediated antivirus responses (11,12). The SeV V protein is reportedly able to suppress virus genome RNA replication in vitro with a defective interfering minigenome model (5).…”

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confidence: 99%

Involvement of the Zinc-Binding Capacity of Sendai Virus V Protein in Viral Pathogenesis

Huang

Kiyotani

Fujii

et al. 2000

J Virol

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The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C 341 S and V-C 365 R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV V ⌬C , which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.Sendai virus (SeV), which belongs to the genus Respirovirus in the family Paramyxoviridae, is a respiratory tract pathogen of rodents. Like other members within the order Mononegavirales, it is an enveloped virus with a single-stranded, negativesense RNA genome of approximately 15.4 kb. The arrangement of the SeV genome starts with a short 3Ј leader sequence, followed by six genes encoding the structural proteins, including N (nucleocapsid), P (phosphoprotein), M (matrix), F (fusion), HN (hemagglutinin-neuraminidase), and L (large) proteins and terminates with a 5Ј trailer sequence (19).Among the six genes, the P gene was found to be unique in that it encodes not a single but multiple proteins. The colinear transcript encodes the P protein as well as the C, CЈ, Y 1 , and Y 2 proteins; these four proteins are translated in a shifted frame by alternative translational starts. The P gene also directs synthesis of an accessory mRNA for the V protein by inserting a pseudotemplated G residue at the specific editing site (30, 31). Consequently, the V and P proteins have the same 317 residues at the amino terminus (the P/V common region), while the V protein contains a 67-residue unique carboxyl terminus (the Vu region). This Vu region features a motif formed by seven cysteine residues (CX 3 CX 11 CXCX 2 CX 3 CX 2 C, where X refers to any amino acid residue), which are highly conserved in almost all the members of the subfamily Paramyxovirinae. The number and spacing of these cysteine residues resemble those of zinc finger structures and metalloproteins, and the V proteins of other members of the subfamily Paramyxovirinae, including simian virus 5 (SV5), measles virus, ...

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Sendai virus C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis

Cited by 113 publications

References 44 publications

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

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

AIP1/Alix Is a Binding Partner of Sendai Virus C Protein and Facilitates Virus Budding

Involvement of the Zinc-Binding Capacity of Sendai Virus V Protein in Viral Pathogenesis

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