- Volume 64, Issue 8, 1983

The 11.8 kilobase pair (7.8 × 106 mol. wt.) genome of mycoplasma virus L2 in lysogenic Acholeplasma laidlawii cells was examined. For this study, DNAs were analysed by agarose gel electrophoresis and viral DNA sequences identified by DNA—DNA hybridization. L2 DNA was found to be integrated into the lysogenic host cell chromosome at a unique site in both viral and cellular DNA. The viral DNA site was roughly mapped and the approximate time of integration during the L2 non-cytocidal infectious cycle was determined.

Monoclonal antibodies against the major glycoprotein (HN) and the nucleoprotein (NP) of paramyxovirus SV5 and human paramyxovirus (PF) type 3 have been obtained. Two different epitopes have been detected on the SV5 HN polypeptide, one being specific to SV5 and the other also cross-reacting with PF-2 HN polypeptide. Monoclonal antibody against SV5 NP polypeptide also reacted with PF-2 NP polypeptide. Similar epitopes showing both specific and group cross-reactions were detected with PF-3 HN and NP monoclonal antibodies against PF-3 and Sendai viruses. No cross-reactions were detected between these two groups of paramyxoviruses.

By the use of horseradish peroxidase-labelled Fab fragments of monoclonal antibodies, five major structural components of Sendai virus, namely the nucleocapsid (NP), polymerase (P), matrix (M), fusion (F), and haemagglutinin—neuraminidase (HN) proteins were localized in infected Vero cells. The P and NP proteins were found in association with large clusters of ribosome-like particles and nucleocapsids in the cell cytoplasm. They were not concentrated at the cytoplasmic membrane, except in nucleocapsids within budding viral particles. F and HN proteins, on the other hand, were found in connection with ribosomes and endoplasmic reticulum in the cell cytoplasm, but not in nucleocapsids. Both proteins were evenly distributed on the outer cytoplasmic membrane and appeared on the surface clearly before budding of viral particles. The M protein was seen in connection with both nucleocapsids and ribosomes. It was not found at the cell surface except in budding viral particles.

DNA copies of segments of Sendai virus genes P, NP and M, obtained by reverse transcription of virus mRNA species extracted from infected cells, were cloned in plasmid pBR322. Genes were identified by hybrid-arrested translation of viral mRNAs in vitro. Hybrid selection of NP mRNA confirmed the identity of an NP gene clone. Partial sequencing of this insert showed that it represents the 5′-terminal region of the gene, containing transcription termination signals. Hybridization of DNA inserts to blots of electrophoretically separated, denatured mRNA species indicated that the P, NP and M messages had sizes of 2400, 2100 and 1500 nucleotides, respectively. Specific T1 ribonuclease-resistant oligonucleotides, previously identified in the NP and M genes, were selected by hybridization to the respective inserts. Although most of the inserts are smaller than 500 base pairs, representing no more than 16% of the P gene and 24% of the NP gene, one of the M gene inserts, comprising 700 base pairs, represents almost half of that gene. These cloned virus gene segments will assist further investigations of the molecular biology of this model paramyxovirus.

Six commonly used strains of lymphocytic choriomeningitis virus (LCMV) [Armstrong (Arm) CA 1371, Arm E-350, WE, UBC, Traub and Pasteur C1PV 76001] were examined for distinctive genetic and biological properties. Agarose gel electrophoresis yielded no detectable differences among the L or S RNAs of these six strains. The RNase T1 fingerprint patterns of LCMV Arm CA 1371 and E-350 RNAs were similar, but in contrast, those of the WE, UBC, Traub and Pasteur strains differed from each other and from the pattern of LCMV Arm CA 1371 and E-350. There were also differences among LCMV strains in their biological properties. LCMV Arm CA 1371, E-350 and Pasteur caused severe vasculitis and focal necrotizing hepatitis in the livers of neonatally infected BALB/WEHI mice in contrast to LCMV WE which caused minimal lesions. LCMV Arm CA 1371 and E-350 were lethal for neonatal C3H/St mice. In contrast, LCMV WE, Traub and Pasteur induced persistent infections in C3H/St mice. Adult guinea-pigs resisted infection by Arm CA 1371, E-350, Traub and Pasteur but succumbed to WE and UBC LCMV strains. Our results show a wide variation in the RNA genomes of LCMV strains commonly used in research laboratories, and these genomic differences are accompanied by variations in the biological properties of LCMV strains.

Monoclonal antibodies have been used to show that an epitope is present on the G1 glycoprotein of prototype La Crosse virus that is absent or significantly altered on several isolates of La Crosse virus made in New York State, U.S.A. The portion of the G1 protein where this epitope is located plays a role in both virus neutralization and haemagglutination. Additional experiments revealed that under the appropriate assay conditions the monoclonal antibodies permitted discrimination between representatives of the North American members of the California serogroup.

It has been shown previously, by sequence analysis of the S RNA segment of snowshoe hare (SSH) bunyavirus, that two overlapping open reading frames in the viral complementary sequence code for proteins with molecular weights of 26.8 × 103 and 10.5 × 103 respectively. In addition to the viral nucleocapsid (N) protein, which is coded by the S RNA, analyses of parental and reassortant bunyavirus-infected cell extracts have shown that the viral S RNA and M RNA species each code for nonstructural proteins (NSS and NSM, respectively). In the present report, in vitro translation analyses of the S mRNA species recovered from virus-infected cells indicate that a single size class of mRNA directs the synthesis of N and NSS. Compositional analyses of selected tryptic peptides of N and NSS have provided proof that N is the product of the first open reading frame, and NSS the product of the second.

The properties of Kunjin virus produced during acute infections of Aedes albopictus (Aal) mosquito cells were compared with those of the virus progeny from the C6/36 clone of mosquito cells and from Vero cells. Titres of 108 p.f.u./ml or greater were obtained from all cells, but significant haemagglutinin activity was associated only with progeny from Vero and C6/36 cells. Kunjin virus from Aal cells adsorbed to goose erythrocytes and blocked haemagglutination by virus from Vero or C6/36 cells. High titres of virus from Aal cells were obtained only when the cells were grown in the presence of 10% or higher concentrations of foetal calf serum, and large losses were encountered when this virus was concentrated by pelleting or by precipitation with polyethylene glycol, and during rate-zonal sedimentation through sucrose gradients. Because of these losses, and because of poor incorporation of [35S]methionine in Aal cells, only analytical amounts of labelled structural proteins were recovered. Electropherograms showed that the presumptive envelope protein E of Kunjin virus from Aal cells migrated more rapidly than E of virions grown in C6/36 and Vero cells, and that the core (C) and membrane-like (M) proteins were not detectably labelled. All the observed changes in phenotype were reversed during one cycle of growth in Vero cells.

Coxsackieviruses A2, A5 and B3 did not replicate in L8CL3-U cells (a non-fusing variant of the rat L8 myogenic cell line) although these cells possessed a common receptor for coxsackieviruses A2 and A5, and a different receptor for coxsackievirus B3. The restriction in replication was identified as a block in viral eclipse, since 6 M-LiCl treatment permitted recovery of the coxsackievirus A2 inoculum from L8CL3-U cells after 2 h at 37°C, and the cells could be transfected by viral RNA. Cellular fusion which was induced in L8CL3-U cultures by herpes simplex virus type 1 (HF strain) facilitated coxsackievirus A2 and A5 replication. Differentiating myogenic L8 cells acquired full susceptibility to infection concurrently with the appearance of acetylcholine receptors, the muscle-specific isoenzyme of creatine phosphokinase, prominent myotube formation and the acquired capacity of the cells to eclipse virus.

L cells grown for 18 to 22 months in the presence of interferon (IFN) retained their sensitivity to the antiviral effect of IFN but were less sensitive to cell growth inhibition by IFN. Moreover, the parental and variant lines became hyporesponsive (i.e. less responsive to virus induction of IFN) with different kinetics after IFN treatment. Experiments using mitomycin C to inhibit cell division suggested that cell division is required to enter the hyporesponsive state.

The cell growth inhibitory efficacies of preparations of human α and γ interferons (IFNs) have been tested on a variety of human cell lines. Human IFN-γ was found to be more effective at inhibiting the growth of HeLa and U-amnion cells than was human IFN-α. The Daudi cell line, on the other hand, which exhibits a strong anticellular effect in response to treatment with human IFN-α, was found to be relatively insensitive to the anticellular effect of human IFN-γ, as were a variety of other lymphoid cell lines. The inability of the IFN-γ to inhibit the growth of the Daudi cells is paralleled by its inability to induce the synthesis of proteins observed to be synthesized in these cells in response to IFN-α.

The surface projections (peplomers) of avian infectious bronchitis virus (IBV) strain M41 have been separated from the nucleocapsid (N) and matrix (M) proteins by sedimentation in a sucrose gradient after virus disruption by the non-ionic detergent Nonidet P40. The peplomers comprised two glycopolypeptides of mol. wt. 90 × 103 (90K; S1) and 84K (S2), shown by analysis of differentially radiolabelled virus to be present in equimolar proportions. Polypeptides of 75K and 110K, which were detected by Coomassie Brilliant Blue staining in similar amounts to S1 and S2 in some unlabelled virus preparations, were absent from peplomer preparations and are probably host cell polypeptides. The S1:S2:N:M polypeptide molar ratio for IBV-M41 was approximately 1:1:6:15.

The phosphoproteins of vesicular stomatitis virus released from infected Drosophila melanogaster cells were examined. The membrane (M) protein was more phosphorylated than after multiplication in chicken embryo cells, even in Drosophila cell cytoplasm before its association with cellular membranes. Analysis of phosphopeptides generated after partial proteolysis and of phosphoamino acids obtained after complete acid hydrolysis showed that M phosphorylation was quantitatively and qualitatively changed, while NS protein phosphorylation was only slightly modified.

Measles virus matrix (M) proteins were compared by competitive monoclonal antibody-binding studies. Three strains of measles and of subacute sclerosing panencephalitis viruses were found to be identical in this way. The matrix protein formed by Edmonston strain virus during a persistent infection could be distinguished from that made in the lytic virus infection. It is concluded that structural alterations in the M peptide can occur during persistence.

The possibility that ferret lung macrophages may be one factor operating in vivo to prevent infection of susceptible alveolar cells (as demonstrated by organ cultures) by both virulent and attenuated strains of influenza virus has been investigated. Phagocytosis of four strains of influenza virus [A/PR/8/34 (H1N1) and clone 64d (attenuated for ferrets) and clones 64c and 7a (virulent for ferrets) of the recombinant virus A/PR/8/34-A/England/939/69 (H3N2)] by ferret alveolar macrophages in vitro showed that all strains, whether virulent or attenuated, attached equally well (72 to 93%). Recoveries of virus were similar (17 to 44%) whether phagocytosis occurred at the normal temperature of the ferret (39°C) or at pyrexial temperatures induced during infection (40°C for A/PR/8/34 and clone 64d; 41°C for clones 7a and 64c). Thus, alveolar macrophages probably contribute to the lack of alveolar infection observed in vivo.

KpnI and SstI fragments representing 96% of the varicella-zoster virus genome, including the termini, were cloned in plasmid vector pAT153. The clones were used to derive maps of virion DNA for SstI, KpnI, XhoI, PvuII, EcoRI and SalI by molecular hybridization and restriction endonuclease digestion.

A modified human interferon-α 2 was produced in Escherichia coli cells infected with phage M13 mp7 containing an interferon-α gene. After purification by immunochromatography with the monoclonal antibody NK2, the N-terminal amino acid sequence was determined. The N-terminal methionine was absent but an additional sequence of 18 amino acids at the N-terminus was retained. The modified interferon-α 2 was indistinguishable from authentic interferon-α 2 in its ability to activate natural killer cells, to slow the growth of Daudi cells, and to confer virus resistance on heterologous cells.