Kenneth Dimock

The fusion and hemagglutinin-neuraminidase glycoproteins of human parainfluenza virus 3 are both required for fusion

Recombinant vaccinia viruses, VF and VHN, expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of human parainfluenza virus 3 (HPIV3) were constructed. Infection of HeLa T4 cells with VF and VHN led to the synthesis of glycoproteins, with the correct apparent molecular weights, that were recognized by monoclonal antibodies specific for HPIV3F and HN. The HN glycoprotein was present on the surface of cells infected with VHN and these cells demonstrated both hemadsorbing and neuraminidase activities. The F glycoprotein was present in cleaved and uncleaved forms and was also expressed on the surface of VF-infected cells. Fusion activity, however, as evidenced by syncytium formation and lysis of human erythrocytes, could only be demonstrated when HeLa T4 cells were coinfected with VF and VHN. Fusion events that are mediated by HPIV3, therefore, require both the F and HN glycoproteins.

Numerous transitions in human parainfluenza virus 3 RNA recovered from persistently infected cells

The nucleotide sequences at the 3-termini of human parainfluenza virus 3 (HPIV3) genomic RNAs recovered from two lines of persistently infected LLC-MK2 cells were determined following PCR amplification. After 29 months of persistence the 3-end of the HPIV3 genome was found to be highly mutated. Interestingly, the only types of nucleotide changes observed were U to C and A to G transitions. Both U to C and A to G transitions were present on individual RNA molecules. The data indicate that biased hypermutational activity leading to U To C and A to G mutations operates in cultured cells during persistent HPIV3 infections.

Nucleotide Sequence of the Coding and Flanking Regions of the Human Parainfluenza Virus 3 Hemagglutinin – Neuraminidase Gene: Comparison with Other Paramyxoviruses

The nucleotide sequence of the human parainfluenza virus 3 (HPIV3) hemagglutinin-neuraminidase (HN) gene has been determined using cDNA clones derived from both HPIV3 genomic RNA and mRNA. The HN mRNA contains 1,882 nucleotides, not including the poly(A) tail. Primer extension experiments were carried out to locate the 5 terminal nucleotide of the HN mRNA. The 3 end of the mRNA was located at a putative polyadenylation signal. The HPIV3 HN mRNA has one large open reading frame that codes for 572 amino acids with a deduced molecular weight of 64,178. Potential polymerase recognition signals for the HN and L genes were located in the flanking regions. The HN protein of HPIV3 shares some common features with the previously sequenced HN proteins of Sendai virus and Simian virus 5. The features include: an N-terminal membrane anchor, two regions of highly conserved amino acid sequence and strong conservation in the positions of the cysteine residues. The relationship is closest between Sendai virus and HPIV3.

Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus.

The Urabe AM9 mumps vaccine is composed of a mixture of variants distinguishable by a difference at nucleotide (nt) 1081 of the hemagglutinin-neuraminidase (HN) gene (Brown, E.G., Dimock, K., Wright, K.E., 1996. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid (aa) 335 of the hemagglutinin-neuraminidase gene with one form associated with disease. J. Infect. Dis. 174, 619-622.). Further genetic and biological variation was detected in plaque purified viruses from the Urabe AM9 vaccine by examining the HN gene sequence, plaque morphology, cytopathic effects and growth in Vero cells, and temperature sensitivity (ts). Infection of Vero cells with plaque purified viruses with a G at nt 1081 of the HN gene produced large, clear plaques, caused significant CPE early after infection but yielded lower titres of virus than other purified viruses. None of these viruses were ts. In contrast, half of the plaque purified viruses with an A at nt 1081 were sensitive to a temperature of 39.5 degrees C. These viruses produced small plaques, caused significant CPE and grew to low titres. Two ts viruses possessed a unique aa substitution at aa 468 of HN. The remaining A(1081) viruses were not ts, produced large plaques but little CPE, and grew to titres 10-fold higher than the G(1081) viruses. Isolates of Urabe AM9 associated with post-vaccination illness were similar to these non-ts A(1081) viruses, but could be further sub-divided into two groups on the basis of a difference at aa 464 of HN. The post-vaccination isolates may represent insufficiently attenuated components of the vaccine, while the G(1081) and ts subset of A(1081) viruses may be more fully attenuated.

Evolution of the fusion protein gene of human parainfluenza virus 3.

The nucleotide sequences of the fusion (F) gene of 15 clinical strains of human parainfluenza virus 3 (HPIV3) isolated between 1959 and 1987 were compared with the F gene sequence of the prototype strain, Wash/47885/57. Nucleotide sequence diversity was greatest in the noncoding regions of the F gene; however, regions believed to function as transcriptional signals were completely conserved. Amino acid sequences were highly conserved and all but a few amino acid substitutions were conservative in nature. Sequence comparisons indicate heterogeneity in HPIV3 F genes; however, a significant proportion of nucleotide changes are maintained after they first appear and seem to be accumulating with time. Phylogenetic analysis suggests that there are 2 lineages of HPIV3 in North America. The two lineages can be distinguished by specific amino acid differences in the F protein, which correlate with differences in antigenic properties and neutralization patterns of HPIV3. The pattern of HPIV3 evolution, based on the analysis of F gene sequences, most closely resembles that of influenza virus B, vesicular stomatitis virus and Newcastle disease virus.

Function and immunogenicity of human parainfluenza virus 3 glycoproteins expressed by recombinant adenoviruses.

Human parainfluenza virus type 3 fusion (F) and hemagglutinin-neuraminidase (HN) cDNA sequences were inserted into the E3 region of the adenovirus type 5 genome. Cells infected with recombinant adenoviruses containing HPIV3 F (AdF) and HN (AdHN) sequences were shown to express HPIV3 F and HN proteins that were functional and immunogenic. The HN protein produced following AdHN infection was glycosylated, expressed on the surface of infected cells and exhibited both hemagglutinin and neuraminidase activities. AdF infection led to the synthesis of both the HPIV3 F0 precursor and its proteolytic cleavage product, F1. F proteins produced by AdF were glycosylated and expressed on the infected cell surface. Syncytium formation was observed in HeLa T4 cell monolayers upon coinfection with AdF and AdHN. The F and HN proteins expressed by recombinant adenoviruses were recognized by HPIV3 F- and HN-specific monoclonal antibodies. Mice injected intraperitoneally with AdF or AdHN produced antibodies that immunoprecipitated the appropriate HPIV3 glycoproteins and sera from immunized mice effectively neutralized HPIV3 virions. These results support future work using recombinant adenoviruses to study the immune response to individual HPIV3 glycoproteins as well as in protection studies using animal models.

Viral RNA and protein synthesis in two LLC-MK2 cell lines persistently infected with human parainfluenza virus 3

Two lines of LLC-MK2 cells persistently infected with human parainfluenza virus 3 (HPIV-3) have been maintained in culture for approximately 3 years. Subgenomic RNAs (putative defective interfering particle genomes) were detected in virions released from both persistently infected cultures. In one of the persistently infected cell lines cyclic variation in the production of virions containing standard virus genomic-size (50S) RNA and subgenomic RNA was observed. The molar ratio of subgenomic RNA to 50S RNA ranged from less than 0.1/1 to 8.7/1. Northern blot analyses revealed that the patterns of viral mRNA synthesis in persistently infected cells from both cultures were similar to those of standard virus infected cells. Furthermore, the intracellular viral-specific proteins had electrophoretic mobilities similar to the corresponding proteins in standard virus-infected cells. Nucleotide sequence analysis of cloned M gene from virus after 29 months of persistence (147 passages) revealed only one variable conservative amino acid change in two clones analyzed from each cell line, indicating that the M protein is not likely to be involved in the maintenance of the persistent infections. The possible mechanisms by which the persistent state is maintained are discussed.

Defective interfering particles of human parainfluenza virus 3

A cyclic pattern of virus production was observed when human parainfluenza virus 3 (HPIV3) was serially passaged nine times in LLC-MK2 cells. Viruses produced from serial passages 8 and 9 interfered with the replication of standard HPIV3. Three subgenomic RNA species (DI-1, DI-2, and DI-3) and virus genomic RNA were detected in the progeny virions produced from cells mixedly infected with standard virus and virus from either serial passages 5 or 8. Northern blot analysis with probes representing all six HPIV3 structural protein genes revealed that DI-1 and DI-2 RNAs contain sequences from the 5 end of the standard virus genome. DI-1 RNA contains L, HN, and F specific sequences, while DI-2 RNA contains only L and HN sequences. DI-3 RNA did not hybridize with any of the probes used. The possibility that DI-3 RNA contains sequences from the 5 end of the standard virus genome is discussed. These results demonstrate that 5 defective interfering particles are generated during serial passage of HPIV3.

Characterization of the Cyno-EBV LMP1 homologue and comparison with LMP1s of EBV and other EBV-like viruses.

EBV latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation and has been associated with several cases of malignancies. EBV-like viruses in Cynomolgus monkeys (Macaca fascicularis) have been associated with high lymphoma rates in immunosuppressed monkeys. In the study, the entire coding region of the Cyno-EBV LMP1 gene was cloned, sequenced and expressed in human embryonic kidney (HEK) cells 293. The Cyno-EBV LMP1 homologue sequence predicted a 588 amino acid (a.a.) protein with a short 19 a.a. N-terminus, six transmembrane domains and a long carboxy tail of 404 a.a. The protein contained a series of seven 9 a.a.-tandem repeats and two 20 a.a.-repeats, which harbored two potential TRAF binding motifs, PxQxT/S. These repeats shared no homology with the repeats in any other LMP1. However, the proline-rich sequence GPxxPx(6) found within the 11 a.a.-repeats of EBV LMP1 was conserved in Cyno-EBV carboxy tail and contained two consensus JAK/STAT sequences PxxPxP. A cluster of eight histidine residues was found in proximity to the last transmembrane domain of Cyno-EBV LMP1 and was exploited as a natural protein tag in expression studies. Western blot analysis revealed a major polypeptide of 110 kDa. Comparative functional studies showed that Cyno-EBV LMP1 expressed in HEK 293 cells shares the same ability as EBV LMP1 to induce NFkappaB driven CAT activity.