Brian W.J. Mahy

The synthesis of Sendai virus polypeptides in infected cells

Abstract The synthesis of virus-specific proteins in several cell types infected with Sendai virus have been analyzed by high resolution polyacrylamide-gel electrophoresis. Eight structural proteins of the virion have been identified in infected cells and the kinetics of their synthesis examined. In addition, two nonstructural proteins with molecular weights (MW) of ∼ 36,000 and ∼ 22,000 have been found. The results of pulse and pulse-chase experiments suggest that large, polyprotein precursors are not involved in Sendai virus replication, and that polypeptides are synthesized from monocistronic messenger RNA species. Throughout the replicative cycle, the virion polypeptides are synthesized in approximately the same unequal proportions at which they are present in the virus particle, except for a relatively greater amount of the P polypeptide and a smaller amount of the M polypeptide in the cells, indicating that polypeptide synthesis is strictly controlled. Results of a double-isotopic label difference analysis showed that host-cell protein synthesis declined by 8% only over 12 hr and that viral protein synthesis was superimposed upon cellular synthesis with total protein synthesis in infected cells reaching 205% of that in uninfected cells. The available evidence suggests that one of the viral polypeptides (No. 7, MW ∼ 42,000) is a cellular polypeptide and that infection selectively reduces the synthesis of this polypeptide.

Polypeptides specified by the influenza virus genome: I. Evidence for eight distinct gene products specified by fowl plague virus

The structural polypeptides of fowl plague virus (influenza A) and those synthesized in fowl plague virus-infected chick embryo fibroblasts have been analyzed by high resolution polyacrylamide gel electrophoresis. We detected eight distinct virus gene products: three polymerase-associated polypeptides (P1, P2, P3), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), membrane polypeptide (M), and a nonstructural polypeptide (NS). The molecular weights of these polypeptides correlate closely with the molecular weights of the eight genome RNA species found in fowl plague virus. The three high molecular weight polypeptides, P1, P2, and P3, were detected both in virions and infected cells, and their separate identity established by a two-dimensional tryptic peptide mapping procedure. An active RNA polymerase enzyme complex isolated from virions and virus-infected cells contained all three P proteins together with the NP protein. The nonstructural polypeptide (NS), together with the P proteins and the NP, appeared early in the infectious cycle, while the M protein and HA protein appeared later in infection. The NS and M polypeptides, which have similar molecular weights, were separated on SDS-polyacrylamide gels and shown to be distinct by tryptic peptide mapping.

Transcription and replication of influenza virus RNA

Abstract Methods are described for measuring the amounts of virus-specific polyadenylated (A+) cRNA, nonpolyadenylated (A−) cRNA, and virus genome RNA (vRNA) in nucleus and cytoplasm of cells infected with influenza virus. The amounts of A(+) cRNA to individual virus genes accumulated during infection at different rates, not related to gene molecular weight, indicating transcriptional control. The pattern of accumulation of each gene transcript was the same in the nucleus as in the cytoplasm. Addition of cycloheximide during infection abolished the transcriptional control and resulted in linear accumulation of each gene transcript. In cells treated with 0.1 μg/ml actinomycin D, which blocked late but not early virus-specific protein synthesis, there was a significant accumulation of all gene transcripts in the nucleus and a relative decrease in transcripts of genes coding for late proteins (M and HA) in the cytoplasm. Analysis of A(−) cRNA and vRNA accumulations in infected cells showed that the A(−) cRNA synthesis precedes vRNA synthesis and net accumulation of A(−) cRNA ceased by 3 hr postinfection (pi). Significant amounts of vRNA in excess of input vRNA were detected by about 2 hr pi and continued to accumulate in both nucleus and cytoplasm at least up to 6 hr pi. Protein synthesis was required for A(−) cRNA production, since in the presence of cycloheximide very little A(−) cRNA, and no vRNA, could be detected. Actinomycin D (0.1 μg/ml) reduced the amount of A(−) cRNA, and completely inhibited the increase in vRNA which occurred from 2 hr pi in normal infection.

The Molecular Biology of the Morbilliviruses

The morbilliviruses are a small, antigenically related genus within the family Paramyxoviridae. They are distinguished from the genus Paramyxovirus by their lack of neuraminidase activity. The group includes an important human virus, measles virus (MV), and three animal viruses, canine distemper virus (CDV), which causes disease in dogs and Mustelidae, rinderpest virus (RPV), causing disease in cattle and other large ruminants, and peste des petits ruminants virus (PPRV), causing disease in sheep, goats, and other small ruminants. Although closely related antigenically, the viruses can be distinguished quite easily by differential neutralization using homologous and heterologous viruses and sera. Their host range is restricted, as indicated above, but they can infect other hosts, although then they do not generally cause disease. Thus, rinderpest viruses cause disease in large ruminants, but cause a subclinical infection in small ruminants. The latter can, however, act as carriers of the disease in some areas of the world and this is of great epidemiological importance. Each virus is antigenically stable, but different strains can be isolated which show widely differing pathogenicity in the host species. In addition to the acute disease, two members of the group, measles and canine distemper viruses, are known to persist and cause chronic neurological disease in a small proportion of infected individuals (see Chapter 12).

Polypeptides specified by the influenza virus genome 2. Assignment of protein coding functions to individual genome segments by in vitro translation

Abstract Cytoplasmic RNA extracted from chick embryo fibroblasts infected with influenza A (fowl plague) virus (FPV) was translated in a wheat germ cell-free protein-synthesizing system. Polypeptides which comigrated during SDS-polyacrylamide gel electrophoresis with marker virus-specific polypeptides P 1 , P 2 , P3, NP, M, and NS were synthesized in vitro . The NP, M, and NS polypeptides were positively identified by tryptic peptide mapping. The polypeptide component of the virus glycoprotein HA was also synthesized in vitro , and was identified by tryptic peptide mapping. RNA extracted from purified FPV (vRNA) did not direct the synthesis of any recognizable virus-specific polypeptides in vitro , either as a total preparation, or as individual RNA genome segments. The protein coding functions of the vRNA segments were identified by hybridization of individual segments to a preparation of infected cell cytoplasmic RNA. On subsequent translation of the RNA in vitro , synthesis of the virus-specific polypeptide corresponding to the hybridized vRNA segment was specifically reduced. We conclude that, for FPV, virion RNA segments 1–3 code for the three P polypeptides and segments 4, 5, 6, 7, and 8 code for polypeptides HA, NP, NA, M, and NS, respectively.

Migration of influenza virus-specific polypeptides from cytoplasm to nucleus of infected cells

Abstract Previous studies have shown that various influenza virus polypeptides are associated with the infected cell nucleus. To clarify the pattern of distribution of influenza virus-specific polypeptides between nucleus and cytoplasm in infected cells and to gain further information concerning functions of influenza virus replication occurring in the host cell nucleus, we have used high-resolution pulse-labeling experiments and detailed ultrastructural examination of purified preparations of nuclei. In addition to NP and NS 1 , both P 1 and P 2 associate preferentially with the nucleus, while P 3 and NS 2 remain in the cytoplasm. The viral M protein is seen in nuclear fractions only when these fractions are significantly contaminated by plasma membrane. The preferential association of P 1 and P 2 with nuclei suggests that these proteins, in conjunction with NP and NS 1 , are involved in specific functions which may include viral transcription, while P 3 and NS 2 are involved in a distinct role in the cytoplasm.

Polypeptides specified by the influenza virus genome 3. Control of synthesis in infected cells

Abstract Influenza A (fowl plague) virus polypeptide synthesis in infected chick embryo fibroblasts occurs in an early (up to 2.5 hr pi) and a late stage. RNA extracted from infected cells at early and late times after infection directed the synthesis in vitro of virus polypeptides in similar proportions to those made in vivo at the corresponding times, suggesting that this temporal control operates at the level of virus mRNA synthesis. However, the pattern of polypeptides synthesised in vitro and in vivo in response to RNA formed during infection in the presence of the protein synthesis inhibitor cycloheximide indicated that the predominant synthesis of certain virus mRNAs up to 2.5 hr pi was not due to selective transcription of the infecting virus genome by the virion polymerase. It is suggested that virus-specific transcription occurs in three stages: first, unselective transcription of all the virus genes by the virion polymerase; second, amplification of synthesis of the mRNAs encoding the early polypeptides; and third, amplification of late mRNA synthesis. The transition between each of these stages appears to be dependent on the synthesis of new and presumably virus-specified polypeptides.

Evidence for the involvement of influenza A (fowl plague Rostock) virus protein P2 in ApG and mRNA primed in vitro RNA synthesis.

Eleven temperature-sensitive (ts) mutants of influenza A (fowl plague, Rostock) virus were analysed for in vitro RNA transcriptase activity in reactions primed by ApG or globin mRNA at 31 degrees C or at 40.5 degrees C, the restrictive temperature for ts mutant growth. Only those ts mutants studied which were defective in RNA segment 1, coding for the virion P2 protein, were defective in RNA transcriptase activity when compared to wild-type virus. Mutants having a defect in the P2 protein had no significant RNA transcriptase activity in reactions at 40.5 degrees C primed by globin mRNA. However, one mutant showed RNA transcriptase activity similar to wild-type virus at 40.5 degrees C when ApG (0.3 mM) was used as primer. The results suggest that influenza (fowl plague, Rostock) P2 protein is directly involved in the mRNA priming reaction, as well as in the RNA transcription reaction in vitro.

Nucleotide Sequence of Fowl Plague Virus RNA Segment 7

Nucleotide sequence analysis of a recombinant DNA clone of RNA segment 7 from FPV/Rostock/34 has shown it to be highly conserved in comparison with RNA segment 7 from two human strains (Allen et al., 1980; Winter & Fields, 1980; Lamb & Lai, 1981). FPV RNA segment 7 contains the coding capacity for two polypeptide chains. The sequence homology between RNA segment 7 of avian and human viruses was greater than 90%, and most of the changes did not result in amino acid substitutions.

Block to influenza virus replication in cells preirradiated with ultraviolet light

Ultraviolet (uv) irradiation of CEF cells immediately before infection with influenza A (fowl plague) virus inhibited virus growth; no inhibition of the growth of a parainfluenza virus (Newcastle disease virus) could be detected in irradiated cells. The kinetics of inhibition after various doses of uv irradiation were multihit, with an extrapolation number of two. When irradiated cells were allowed to photoreactivate by exposure to visible light for 16 hr their capacity to support influenza virus replication was largely restored; this process was sensitive to caffeine, suggesting that it required DNA repair. In CEF cells exposed to 360 ergs/mm(2) of uv radiation the rate of synthesis of host cellular RNA was reduced by more than 90%, and that of host cellular protein by 40-50%, as judged by incorporation of precursor molecules into an acid-insoluble form. When such irradiated cells were infected with influenza virus all the genome RNA segments were transcribed, but the overall concentration of virus-specific poly (A)-containing cRNA was reduced about 50-fold. Within this population of cRNA molecules, the RNAs coding for late proteins (HA, NA, and M) were reduced in amount relative to the other segments. The rates of synthesis of the M and HA proteins were specifically reduced in uv-irradiated cells, but the rates of synthesis of the P, NP, and NS proteins were only slightly reduced compared to normal cells. Immunofluorescent studies showed that, in uv-irradiated cells, NP migrated into the nucleus early after infection and later migrated out into the cytoplasm, as in normal cells. In contrast to normal cells, no specific immunofluorescence associated with M protein could be observed in uv-irradiated cells. It is concluded that uv-induced damage to host cellular DNA alters the pattern of RNA transcription in CEF cells infected with influenza virus, and that this results in a block to late protein synthesis which stops virus production.