TABLE 2.
Summary of genetic and biochemical properties of the D5 mutant proteins
| D5 | Affected residue(s) | Protein status at 39.7°Ca | ATP bindingb | ATPase activityc | Multimerizationd | Viral DNA replicatione |
|---|---|---|---|---|---|---|
| wt | Stable | + | + | + | + | |
| Cts17 | S161F | Unstable | + | + | + | − |
| Cts24 | T143I | Unstable | + | + | + | − |
| Ets69 | P682S | Stable, soluble | + | + | + | − |
| Dts6389 | S161F,A283T | Insoluble | + | + | + | − |
| Dts12 | M116I | Insoluble | + | + | + | − |
| Dts56 | V212A | Insoluble | + | + | + | − |
| Walker A | K509A | ND | Reduced | − | + | − |
| Walker B | E557Q | ND | + | − | +/− | − |
| SFIII motif C | N605D | ND | + | − | + | − |
| AAA+ motif | R619A,R620A | ND | + | − | +/− | − |
| 413-D5 | Δ1-412 | ND | + | − | − | − |
| 301-D5 | Δ1-300 | ND | + | + | + | − |
Whole-cell lysates or PNS were prepared from infected BSC40 cells at 8 hpi and analyzed using an anti-D5 antiserum. The D5 protein is referred to as “unstable” if less D5 was detected in whole-cell lysate prepared from nonpermissive infections than wt infection at permissive temperature; the “insoluble” moniker was given to those D5 proteins that were not detected in the PNS fraction from nonpermissive infections. D5 stability is host cell specific (Fig. 4), since all but one (Dts6389) of the tsD5 proteins are thermolabile during nonpermissive infections in L929 cells. ND, not determined.
+, ATP binding was observed at ≥65% of wt activity. The K509A mutation in the Walker A domain diminished ATP binding to ∼30% of wt activity.
+, ATPase activity was ≥65% of wt activity; −, ATPase activity was <10% of wt activity.
BSC40 cells were programmed to overexpress untagged full-length wtD5 along with 3XFLAG-tagged versions of the various D5 proteins. Affinity purification of the tagged protein and any associated untagged D5 was performed and examined by silver staining. +, the 3XFLAG protein associated with the untagged protein at near-stoichiometric levels; +/−, reduction in relative pull-down efficiency; −, complete inability of the tagged protein to retrieve untagged D5.
BSC40 or L929 cells infected with the ts viruses failed to accumulate viral DNA at high temperature as determined by Southern dot blot hybridization on cytoplasmic viral DNA harvested at various times postinfection. When introduced by transfection, plasmids encoding the site-directed or truncated D5 mutants were unable to support ongoing viral DNA synthesis (determined at 8 and 24 hpi) when permissive Cts24 infections were shifted to nonpermissive temperature in the midst of DNA replication (5 hpi).