Host range ( hr ) and temperature-dependent host range ( td CE) mutants

Viruses are obligate intracellular parasites with host cell factors intrinsic to their lifecycles. These host factors can therefore determine the outcome of an infection in a cell. Essential host factors for CHPV are largely unknown. Section 1.3 and Table 1.3 gives details of host cell proteins involved in VSV replication. The critical

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involvement of host factors in the replication of vesiculoviruses was highlighted by the isolation of VSV Indiana, VSV New Jersey and CHPV temperature-dependent host range (tdCE) mutants by mutagenesis using 50-200 µg/ml 5-fluorouracil (Gadkari & Pringle, 1980; Pringle, 1978; Rasool & Pringle, 1986). These mutants are characterised by their ability to grow at both 31ºC (the permissive temperature) and the restrictive temperature of 39ºC in BSC-1 or BHK-21 cells (the permissive host) to comparable titres to wild-type virus yet display temperature sensitivity in primary avian cells (chick embryonic fibroblasts, CE). The tdCE mutant phenotype has been defined as viruses with an efficiency of plating (E.O.P.) in BSC-1 cells of <0.7 and in CE cells of >2, where E.O.P. is calculated as log10 PFU39ºC subtracted from log10 PFU31ºC. These E.O.P. values were selected arbitrarily to reflect

significant growth impairment in CE cells compared to BSC-1 cells. This phenotype demonstrates a dependence of these vesiculoviruses on undetermined host factors (Gadkari & Pringle, 1980; Rasool & Pringle, 1986). All the CHPV tdCE mutants isolated and a summary of important characteristics are given in Table 1.4.

34 Mutant Additional host restriction(s) Efficiency of plating Transcriptase efficiency at

39°C determined by the plateau method (%of wt) (Rasool and Pringle, 1986)

Transcriptase efficiency at 39°C determined by the initial rate

method (%of wt)

(Rasool and Pringle, 1986)

BSC-1 CE CH42 ND 0.67 3.18 15 48 CH632 (re-cloned from CH859) ND 0.36 2.09 28 53 CH722 (re-cloned from CH565) ND 0.08 2.60 72 93 CH902 (re-cloned from CH530) ND 0.09 2.15 110 109 CH1122 (re-cloned from CH514) ND 0.57 5.20 14 51 CH1512 ND 0.00 2.91 11 41 CH1572 ND 0.54 3.54 56 67 CH1942 ND 0.12 3.45 91 113 CH2452 ND 0.04 3.77 70 85 CH2562 ND 0.15 4.37 37 29 CH3542 ND 0.00 3.30 83 62 CH3802 ND 0.06 3.05 23 40 CH5141 None 0.40 3.18 X X CH5161 None 0.70 3.70 X X CH5251 None 0.78 5.00 X X

CH5301 HE, RE, FEA, BEK, NIH/3T3, L929

1.18 5.00 X X

CH5651 BEK 1.18 3.00 X X

CH8591 RE, BEK, NIH/3T3, L929 0.60 3.18 X X

Table 1.4| Characteristics of CHPV tdCE mutants 1

Denotes mutants isolated by Gadkari and Pringle (1980) and 2 isolated by Rasool and Pringle (1986). Mutants highlighted in blue have been selected to analyse the tdCE phenotype in this study. Impaired growth at the restrictive temperature in chick embryo (CE), hamster embryo (HE), rat embryo (RE), feline embryo (FEA), bovine embryo kidney (BEK), mouse embryo (NIH/3T3), mouse (L929) cells are noted for several of the mutants. ND denotes not

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Two out of four of the VSV New Jersey tdCE mutants studied synthesised RNA to comparable levels as the wild type virus at 31°C and 39°C whereas the RNA transcriptases of the remaining mutants were shown to be severely disabled at the restrictive temperature in BHK-21 and CE cells in in vitro transcription assays (Szilagyi & Pringle, 1975). Experiments in which transcribing nucleoprotein complexes (TNP) of the wild type and mutant viruses were dissociated and then reconstituted in various combinations suggested that the L protein contained the host range lesion (Szilagyi et al., 1977). It was postulated that a host cell factor is

required to counteract the thermoliability of the viral polymerase in tdCE mutants found to have depressed RNA transcriptase activity whereas mutants displaying normal RNA synthesis have a defect at a different (later) stage in the viral life cycle (Szilagyi & Pringle, 1975).

Mutants that were unable to grow in CE and bovine kidney (MDBK) cells at any temperature, designated host restricted (hrCE) mutants, were also isolated from VSV New Jersey but not VSV Indiana or CHPV (Pringle, 1978; Rasool & Pringle, 1986). These mutants have provided a powerful tool for dissecting vesiculovirus replication. For hrCE and tdCE mutants of VSV, increasing the incubation temperature from 31°C to 39°C after two hours post-inoculation was shown to have limited effect on VSV viral titre, whereas raising the temperature at an earlier time point severely disrupted virus replication, implying host restriction occurs at an early stage in viral replication (Pringle, 1978). However, involvement of the attachment protein was discounted when pseudotyped virions expressing the G protein from the tdCE or

hrCE VSV mutants grew to titres comparable with the wild type virus in CE cells. Together, these results added weight to the hypothesis that the host range phenotype

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is caused by a genetic lesion in the virus polymerase which affects its association with host factor(s) that are not present in CE cells and are required to hold the polymerase in an active conformation at 39°C (Pringle, 1978).

As well as differences in mRNA synthesis, mutants also differed in their host restriction (Pringle, 1978). This is thought to highlight their unique characteristics and is suggestive of a range of mutational events and the involvement of more than one host cell factor. CHPV mutants with additional host cell restrictions are detailed in Table 1.4. A study of VSV tdCEmutants found no link between certain cell types and properties, for example morphology, and conditional growth mutants, except that embryonic cells appeared to be more conducive to temperature-sensitivity and

restrictiveness of murine embryonal carcinoma cells decreased after differentiation (Pringle, 1978).

Upon further investigation, the tdCE mutants of VSV and CHPV were shown to have important intrinsic differences regarding the in vitro heat sensitivity of their viral RNA transcriptase. Reduced RNA transcriptase activity in vitro at 39ºC resulting in significantly lower amounts of viral RNA synthesised was observed in 10 out of 12 of CHPV tdCE mutants compared to wild-type virus and a gradient of this activity ranging from 11% of the wild-type to greater than wild-type were determined across the mutants studied (Rasool & Pringle, 1986). This is in contrast with the severe impairment or wild type level RNA synthesis reported in the case of VSV tdCE mutants. Moreover, viral protein synthesis was also found to be either considerably depressed or non-existent in all the transcriptionally deficient CHPV tdCE mutants grown in CE cells at the restrictive temperature, leading to the postulation that the

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growth restriction is caused by diminished RNA synthesis which in turn results in decreased production of viral polypeptides (Rasool & Pringle, 1986). In support of this, intracellular viral protein synthesis increased to wild-type levels when tdCE mutants reverted to a non-temperature sensitive phenotype (Rasool & Pringle, 1986). Furthermore, in vitro RNA methylation activity of the virus RNA dependent RNA polymerase and heat shock protein induction were found to occur at similar levels as the wild type virus in all tdCE mutants (Rasool & Pringle, 1986).

VSV Indiana dual ts/hr mutants have also been isolated (Simpson & Obijeski, 1974). These mutants are restricted in many human cell lines including Hep-2 and display temperature sensitivity in CE and BHK cells. One of these mutants, hr1 contains a D1671V amino acid substitution in the MTase domain VI of the VSV L protein (within a predicted S-adenosyl-L-methionine-binding motif) rendering the mutant completely incapable of catalysing the methylation of 5’ mRNA caps at both guanine-N7 and 2’-O-adenosine (Grdzelishvili et al., 2005; 2006; Hercyk et al., 1988; Horikami & Moyer, 1982). In contrast to hr1 mutant hr8 retained low levels of MTase activity at 2’-O-adenosine (but not at the guanine-N7 position). This phenotype was shown to be the consequence of a G1481R amino acid substitution located between domains V and VI in the L protein in a region of 31 amino acids subsequently identified as essential for VSV MTase activities (Grdzelishvili et al., 2006).

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1.5. Vesiculovirus disease: infection of the central nervous system