BackgroundParamyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood.Methodology/Principal FindingsTo analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV), rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP). The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594.Conclusions/SignificanceCollectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural components to the assembly sites of enveloped viruses.

No abstract

Human parainfluenza virus type 1 (HPIV-1) and Sendai virus (SeV) are highly homologous in structure and sequence, whilst maintaining distinct host ranges. These viruses express accessory proteins from their P/C gene that are known to have activities against innate immunity. The accessory proteins expressed from the P/C gene of these viruses are different. In addition to the nested set of C proteins, SeV expresses V protein from edited P mRNA, which is not expressed by HPIV-1. This study evaluated the host specificity and role of the P/C gene products in antiinterferon (IFN) and anti-apoptosis activity by characterizing a recombinant SeV, rSeVhP, in which the SeV P/C gene was replaced with that of HPIV-1. Unlike SeV, rSeVhP infection strongly activated IFN regulatory transcription factor (IRF)-3 and nuclear factor-kB, resulting in an increased level of IFN-b induction compared with SeV in murine cells. In contrast, activation of IRF-3 was not observed in rSeVhP-infected human A549 cells. rSeVhPSV, which expressed SeV V protein from an inserted gene in rSeVhP, induced less IFN-b than rSeVhP, suggesting that V contributes to the suppression of IFN production in murine cells. Furthermore, rSeVhP induced apoptotic cell death in murine but not in A549 cells. These data indicate the functional difference in P/C gene products from SeV and HPIV-1 in antagonizing IFN induction and apoptosis, which is likely to be one of the major factors for pathogenicity in specific hosts.

Paramyxovirinae, a subfamily of Paramyxoviridae, are negative strand RNA viruses comprised of many important human and animal pathogens, which share a high degree of genetic and structural homology. The accessory proteins expressed from the P/V/C gene are major factors in the pathogenicity of the viruses, because of their ability to abrogate various facets of type I interferon (IFN) induction and signaling. Most of the paramyxoviruses exhibit a commonality in their ability to antagonize innate immunity by blocking IFN induction and the Jak/STAT pathway. However, the manner in which the accessory proteins inhibit the pathway differs among viruses. Similarly, there are variations in the capability of the viruses to counteract intracellular detectors (RNA helicases, mda-5 and RIG-I). Furthermore, a functional specificity in the antagonism of the IFN response has been reported, suggesting that specificity in the circumvention of innate immunity restricts viral host range. Available evidence indicates that paramyxoviruses employ specific strategies to antagonize the IFN response of their specific hosts, which is one of the major factors that determine viral pathogenicity and host range.

Parainfluenza viruses are enveloped viruses that contain nonsegmented negative‐strand genomic ribonucleic acid (RNA). Replication of these viruses begins with entry by attachment and fusion, followed by genomic transcription and replication. Next, the de novo synthesised viral components are trafficked to assembly sites at the plasma membrane where newly formed virions bud out from the cell. These viruses are respiratory pathogens which act as the causative agents for croup, bronchiolitis and pneumonia. As a group, the parainfluenza viruses are second only to human respiratory syncytial virus as the cause of acute paediatric respiratory tract disease and are responsible for hundreds of thousands of additional U.S. hospitalisations per year. Despite their clinical importance, licensed vaccines or antiviral compounds for prevention or treatment of parainfluenza viruses are not currently available, making understanding the pathogenesis of these viruses critical for antiviral drug and vaccine development. Key Concepts: Parainfluenza viruses are enveloped, nonsegmented negative‐strand RNA viruses, which cause respiratory infections. The structure of the virion consists of glycoprotein spikes protruding from a lipid envelope under which lies the matrix protein surrounding the viral nucleocapsid, consisting of viral RNA encapsidated by nucleoprotein and associated with the polymerase complex. Specific interactions between the two envelope glycoproteins HN and F are necessary for efficient virus infection. Because the viral genome is negative‐stranded RNA, an intermediate step is required for production of progeny viral genomes. Newly synthesised viral nucleocapsids are transported to the assembly site at the plasma membrane utilising the microtubule network. Pathogenicity of parainfluenza viruses is highly dependent on the anti‐interferon activity of the viral accessory proteins. Infectious virus can be created from a complementary deoxyribonucleic acid (cDNA) clone of the viral RNA genome, which is termed reverse genetics. The development of vaccines against parainfluenza viruses currently include intranasally administered vaccines attenuated by cold‐passage, host range attenuation, chimeric vaccine constructs, or the introduction of attenuating mutations through the use of reverse genetics.