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

Abstract

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...

Nipah Virus Matrix Protein Influences Fusogenicity and Is Essential for Particle Infectivity and Stability

“…While the fusogenicity of NiVeG⌬M was slightly enhanced, replication kinetics was significantly impaired. Together with the release of large amounts of viral and cellular proteins and the severely reduced infectivity and stability, our data indicate that the M protein is essential for proper NiV particle assembly, consistent with its role in other paramyxoviruses (21,(31)(32)(33).…”

“…While some attempts to generate members of the Paramyxoviridae family without complementation of functional M proteins were not successful (31)(32)(33), M protein-deleted measles (MV⌬M) and respiratory syncytial (M-null HRSV) viruses have been recovered (20,21). While M-null HRSV was completely defective in virus budding (21), limited amounts of infectious MV⌬M were found in the supernatant (20), indicating a varying importance of the M protein contribution among different genera.…”

The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments

“…These differences may be the result of the unique properties of closely related viruses. In the case of Sendai virus, another paramyxovirus, it was shown that VLPs were not or were poorly generated when M expression was suppressed (40). While our data show that the production of infectious HRSV progeny was nearly abrogated in the absence of M, we cannot exclude the possibility that noninfectious VLPs were released.…”

“…An important role for M in virion production was anticipated based on a previously reported HRSV minireplicon system (58) and analogy to the roles of M proteins from other paramyxoviruses (12,29,40,46). The growth analysis shown in Fig.…”

“…Studies of other paramyxoviruses have shown that their M proteins play a role in the assembly of virus particles (12,29,40,46). A requirement for HRSV M for cell culture propagation was previously reported, based on an HRSV minireplicon system (58).…”