Human parainfluenza virus type 2 polymerase complex recognizes leader promoters of other species belonging to the genus Rubulavirus.

Leader sequence, located at the 3' terminus of paramyxovirus genomes, determines the degree of viral transcription and replication. The essential nucleotides in the leader sequence that influence viral propagation, however, have not been investigated in detail. In the present study, we show that polymerase complex of human parainfluenza virus type 2 (hPIV2) uses a luciferase-encoding hPIV2 mini-genome possessing the leader sequence from other closely related viruses as a template. Furthermore, we demonstrate that although hPIV2 polymerase complex can recognize the leader sequence of hPIV4B, mumps virus (MuV) and PIV5 as well as Newcastle disease virus (NDV), it cannot recognize measles virus, hPIV1, Sendai virus (SeV) or hPIV3. We could obtain the chimeric hPIV2 possessing the leader sequence from hPIV4B, MuV and PIV5, but not from other species, including NDV and SeV. These results reveal that although hPIV2 polymerase complex can recognize the leader sequence from rubulaviruses to achieve efficient viral infection, this does not apply to viruses belonging to other genus. A comparison of leader sequence nucleotides among paramyxoviruses highlights the importance of the conservation in the first 13 nucleotides for infectious hPIV2 growth.

Identification of two essential aspartates for polymerase activity in parainfluenza virus L protein by a minireplicon system expressing secretory luciferase.

Gene expression of nonsegmented negative-strand RNA viruses (nsNSVs) such as parainfluenza viruses requires the RNA synthesis activity of their polymerase L protein; however, the detailed mechanism of this process is poorly understood. In this study, a parainfluenza minireplicon assay expressing secretory Gaussia luciferase (Gluc) was established to analyze large protein (L) activity. Measurement of Gluc expression in the culture medium of cells transfected with the minigenome and viral polymerase components enabled quick and concise calculation of L activity. By comparing the amino acid sequences in conserved region III (CRIII), a putative polymerase-active domain of the L protein, two strictly conserved aspartates were identified in all families of nsNSV. A series of L mutants from human parainfluenza virus type 2 and parainfluenza virus type 5 showed that these aspartates are necessary for reporter gene expression. It was also confirmed that these aspartates are important for the production of viral mRNA and antigenome cRNA, but not for a polymerase-complex formation. These findings suggest that these two aspartates are key players in the nucleotidyl transfer reaction using two metal ions.

Human parainfluenza virus type 2 hemagglutinin-neuramindase gene: sequence and phylogenetic analysis of the Saudi strain Riyadh 105/2009.

BACKGROUND: Although human parainfluenza type 2 (HPIV-2) virus is an important respiratory pathogen, a little is known about strains circulating in Saudi Arabia. FINDINGS: Among 180 nasopharyngeal aspirates collected from suspected cases in Riyadh, only one sample (0.56%) was confirmed HPIV-2 positive by nested RT-PCR. The sample that was designated Riyadh 105/2009 was used for sequencing and phylogenetic analysis of the most variable virus gene; the haemagglutinin-neuramindase (HN). Comparison of HN gene of Riyadh 105/2009 strain and the relevant sequences available in GenBank revealed a strong relationship with Oklahoma-94-2009 strain. Phylogenetic analysis indicated four different clusters of HPIV-2 strains (G1-4). Twenty-three amino acid substitutions were recorded for Riyadh 105/2009, from which four are unique. The majority of substitutions (n=18) had changed their amino acids characteristics. By analyzing the effect of the recorded substitutions on the protein function using SIFT program, only two located at positions 360 and 571 were predicted to be deleterious. CONCLUSIONS: The presented changes of Riyadh 105/2009 strain may possess potential effect on the protein structure and/or function level. This is the first report that describes partial characterization of Saudi HPIV-2 strain.

Virus-receptor interactions of human parainfluenza viruses types 1, 2 and 3.

Human parainfluenza viruses types 1, 2 and 3 (HPF 1, 2 and 3) are important pathogens in children. While these viruses share common structures and replication strategies, they target different parts of the respiratory tract; the most common outcomes of infection with HPF3 are bronchiolitis and pneumonia, while HPF 1 and 2 are associated with croup. While the HPF3 fusion protein (F) is critical for membrane fusion, our previous work revealed that the receptor binding hemagglutinin-neuraminidase (HN) is also essential to the fusion process; interaction between HN and its sialic acid-containing receptor on cell surfaces is required for HPF3 mediated cell fusion. Using our understanding of HPF3 HN's functions in the cell-binding and viral entry process, we are investigating the ways in which these processes differ in HPF 1 and 2, in part by manipulating receptor availability. Three experimental treatments were used to compare the HN-receptor interaction of HPF 1, 2 and 3: infection at high multiplicity of infection (m.o.i.); bacterial neuraminidase treatment of cells infected at low m.o.i.; and viral neuraminidase treatment of cells infected at low m.o.i. (using Newcastle disease virus [NDV] neuraminidase or UV irradiated HPF3 as sources of neuraminidase). In cells infected with HPF3, we have shown that infection with high m.o.i. blocks fusion, by removing sialic acid receptors for the viral HN. However, in cells infected with HPF 1 and 2, infection with high m.o.i. did not block fusion; the fusion increases with increasing m.o.i. In cells infected with HPF 1 and 2, neither bacterial nor NDV neuraminidase blocked cell fusion, using amounts of neuraminidase that completely block fusion of HPF3 infected cells. However, when inactivated HPF3 was used as a source of viral neuraminidase, the treatment inhibited fusion of cells infected with HPF 1 and 2 as well as 3. The differences found between these viruses in terms of their interaction with the cell, ability to modulate cell-cell fusion and response to exogenous neuraminidases of various specificities, may reflect salient differences in biological properties of the three viruses.

Distinct hemagglutinin and neuraminidase epitopes involved in antigenic variation of recent human parainfluenza virus type 2 isolates.

A panel of fourteen neutralizing anti-HN monoclonal antibodies (mAbs) to the prototype Greer strain of human parainfluenza virus type 2 (PI2) was used to determine the extent of antigenic variation in recent virus isolates. Competitive binding analysis with the mAbs indicated the presence of at least five distinct antigenic sites (I to V) on the HN glycoprotein molecule. MAbs recognizing different antigenic sites were found to be associated with the hemagglutinin (sites I, IV and V), hemagglutinin and neuraminidase (site II), or neuraminidase (site III) activities. The location of two distinct epitopes identifying the neuraminidase sites (II and III) was further verified from the generation of escape mutants. Antibodies directed to sites I and III failed to show any detectable binding or neutralizing activity against a number of natural PI2 virus isolates collected in Texas between 1986 and 1987. Interestingly, these natural variants, unlike the prototype virus, did not show any detectable neuraminidase activity with fetuin as a substrate and the enzyme activity was only detected with N-acetylneuramin-lactose as an alternative substrate. Despite the observed variation in the antigenic sites, primary infection with the prototype virus or the natural variants generated a protective immune response against challenge infection with the other virus strains.