Marcel W. Pons

Molecular cloning and sequence analysis of the human parainfluenza 3 virus gene encoding the matrix protein

The sequence of the matrix (M) protein gene and contiguous intergenic regions of the human parainfluenza 3 virus (PF3) was determined by molecular cloning. The encoded M protein contains 354 amino acids and has a predicted mol wt of 39,506. The M protein amino acid sequence was compared to the homologous proteins from other members of the Paramyxoviridae family. The PF3 protein shared 61% homology with the Sendai virus protein and approximately 35% homology with measles and canine distemper virus proteins. Little homology was observed with respiratory syncytial virus. The M protein appears to be the most highly conserved among the Paramyxoviridae proteins.

Molecular cloning and sequence analysis of the human parainfluenza 3 virus RNA encoding the nucleocapsid protein

The sequence of 1690 nucleotides from the 5 end of the viral complementary RNA for the human parainfluenza 3 virus was determined by molecular cloning. One large open reading frame consisting of 1548 nucleotides was demonstrated. The encoded protein, the nucleocapsid protein (NP), consists of 515 amino acids, and has a predicted molecular weight of 57,819. A noncoding 5 sequence of 51 nucleotides is present at the end of the NP-mRNA. Two consensus sequences were identified which are homologous with sequences found in Sendai virus. One of these sequences, AGGATTAAAG, was located at the 5 end of the nucleocapsid mRNA and may function in transcription initiation. The other consensus sequence, GTAAGGGAA, was found in the viral genomic leader sequence. The nucleocapsid protein amino acid sequence was compared to other members of the Paramyxoviridae family. The parainfluenza 3 virus protein nucleocapsid amino acid sequence demonstrated a high degree of homology with the Sendai virus nucleocapsid protein. Seventy percent of the first 387 amino acids from the amino termini were identical. Little homology was observed in the distal carboxy termini.

Human parainfluenza virus 3: Purification and characterization of subviral components, viral proteins and viral RNA

A simple method was established that allowed large quantities of human parainfluenza 3 (PF3) virions to be isolated from tissue culture cells. The purity of the virus was sufficient for biochemical analysis of virion proteins. The density of PF3 virions was 1.18-1.20. Purified virions contained seven viral proteins with estimated molecular weights of: L, 180 000; P, 83 000; HN, 69 000; NP, 66 000; F0, 60 000; F1, 51 000; and M, 38 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. There were three phosphoproteins, P, NP and M, and two glycoproteins, HN and F (includes F0 and F1). F1.2, the activated, cleaved, fusion glycoprotein (60 000 Da), consisting of two disulfide-linked subunits, F1 and F2, was seen only under nonreducing conditions. Because of its small size (approximately 9000 Da) F2 could be seen only on gels with high acrylamide concentrations. As in other enveloped viruses, cellular actin (43 000 Da) was present in purified virions. Several minor bands migrating between NP and M represented breakdown products of NP. Solubilization of the virion membrane in low salt buffer with non-ionic detergent resulted in the loss of HN and F. In high salt buffer, the M protein was also removed. Nucleocapsids isolated by CsCl centrifugation contained L, P, NP and small amounts of M. Nucleocapsids isolated in the presence of the ionic detergent, sarcosyl, contained only the NP protein. The density of nucleocapsids was 1.29-1.30. Genomic 50S RNA isolated from nucleocapsids had an estimated molecular weight of 5 X 10(6).

A simple method for increased recovery of purified paramyxovirus virions

A simple method involving vigorous agitation of infected cell monolayers prior to collection of culture medium is described to greatly increase the recovery of purified virions of measles virus, respiratory syncytial virus and human parainfluenza virus. Vigorous agitation of the flasks containing monolayers of infected cells increased the recovery of purified virions by at least 3- to 10-fold as judged by the intensity of [35S]methionine labeled viral proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). These protein profiles also indicated that these virions were as clean as those purified from gently collected medium. Analysis of titers of infectious virus recovered from medium of agitated and non-agitated flasks showed similar increases. These results suggest that the cell associated nature of these viruses may be at least partly due to either partially budded virions or mature virions sticking to the cell membrane, since these both might be expected to be freed from the cell by mechanical shearing.

The genome of incomplete influenza virus

Abstract After three high-multiplicity passages of the WSN strain of influenza virus in primary chick embryo fibroblast monolayers, the virus produced contained much less of the largest RNA segment and reduced amounts of the next two largest segments as compared with normal virus. The double-stranded RNA isolated from the cells which produced these particles also showed a similar decrease in these three segments as did the cRNA, both that associated with polysomes and non-polysome associated. Virions lacking these RNAs did not transcribe these segments in vitro but did transcribe the other five segments. An analysis of the molar ratios of segments indicated that the three largest segments are the most seriously affected. There was no evidence for random loss of segments either by these analyses or by our inability to enhance infectivity by clumping defective virions.

The in vivo Inhibition by β-Phenylserine of Rabies, Myxoma, and Vaccinia Viruses.

Abstract The daily intraperitoneal administration of 10–15 mg of β-phenylserine protected rats against death from lethal amounts of rabies virus. The protective effect was most marked when the animals were treated for 3 days prior to the injection of the virus, although initiation of treatment 24 hours after the virus was injected also gave protection. The administration of dl -phenylalanine or dl -tyrosine in combination with β-phenylserine reversed the inhibition of rabies virulence. Rabies-infected animals protected with β-phenylserine and challenged with a second injection of rabies virus 6–7 weeks later did not show any immunity. The xanthine oxidase activity of infected rat brain increased during the course of rabies. The increased activity paralleled the titer of virus in the brain. There was no alteration in the xanthine oxidase activity when animals injected with the virus were treated with β-phenylserine. β-Phenylserine inhibited the propagation of vaccinia virus in rabbit skin when the compound and virus were mixed prior to injection. The daily intraperitoneal injection of 75 mg of β-phenylserine delayed the development of myxomatosis in rabbits. The compound had no apparent effect on the multiplication of influenza A (PR8), eastern equine encephalitis, poliovirus (Lansing strain), or mouse encephalomyelitis (FA strain) viruses in mice. The compound was inactive also against influenza A virus in embryonated eggs.

Ultraviolet inactivation of influenza virus RNA in vitro and in vivo

Abstract The uv inactivation of influenza virus RNA within the infected cell indicated that each segment was being transcribed individually. That is, each segment carried its own promoter region. Similar results were obtained when RNA synthesis by uv-irradiated virus was tested in vitro.

Immediate persistent infection by human parainfluenza virus 3: unique fusion properties of the persistently infected cells

We describe here a persistently infected cell system with unique properties. Cells infected with human parainfluenza virus type 3 (PF3) at high multiplicities of infection showed little or no cytopathic effects (cell fusion). Unlike other paramyxovirus persistent infections that require a long development time, the majority of the cells survived the initial infection and formed persistently infected cell cultures that were immediately available for study. In addition, unlike other paramyxovirus persistent infections, the PF3 system described here produced high levels of infectious virus and did not undergo periodic crises. Although cells persistently infected with PF3 contained large amounts of the cleaved, active form of the viral fusion protein, F1, the persistently infected cells did not fuse with each other. However, they did fuse with uninfected cells within minutes of cell-to-cell contact. Other persistent paramyxovirus infections do not have this property. Fusion occurred with all cells tested, including red blood cells, and was not dependent on protein synthesis. The unique fusion properties of these PF3 persistently infected cells make this an interesting system for the study of mechanisms of viral fusion and mechanisms of inhibition of viral fusion.