N
B
NB t 102-
LLI 10" 10" 1 ’ ' '■ '” T i[— I 11 rfrtfj-10^ lOl 102 1 03 io4 10^ lOl 102 103
EGFP EGFP
Figure 5.10: C om petition assay profiles for In t.l
F A C S p ro files illu stratin g the effect o f tra n sd u c in g In t.l into N IH -3 T 3 and B A L B -3T 3 ex p ressin g endog en o u s F v l" and F v l^ respectively. E G F P e x p ressio n is show n on the x- axis, E Y F P on the y-axis. P e rcen tag es g iven in d icate the p ro p o rtio n o f E Y FP positive c ells in the E G F P n e g a tiv e and the E G F P p o sitiv e su b p o p u la tio n . T op row , N -tro p ic; m iddle row , B -tropic; bottom row , N B -tropic tester virus.
M utant Restricts
Competes
N-tropic MLV B-tropic MLV NIH-3T3 BALB-3T3
W ild-type F v l" _ +
n/a
V
W ild-type Fvl^ + ( - )V
n/a
Fvl"A 411-440 ___X
X
Fvl*’A411-459 ___ —X
X
F vl"A l-32 ___ +n/a
V
F vl"A l-50 ___ (+ )n/a
V
Fvl^’A l-32 +V
n/a
Fvl^Al-SO + —V
n/a
N -half ___ ___X
X
nC-half ___ —X
X
bC-half ___ —X
X
N -half + nC-half ___ ___X
X
N -half + bC-half ___ ___X
X
In t.l — —V
V
T able 5.1: Competition assay with F v l mutants
Restriction o f N- and B-tropic MLV by each mutant, and the effect o f expressing each mutant in NIH-3T3 and BALB-3T3 is shown. + indicates restriction; - indicates no restriction; V indicates out-competes endogenous F v l; X indicates no competition with endogenous F v l; n/a indicates the mutant and endogenous F v l restrict the same class o f MLV.
5.8 Summary
The deletion mutants identified several regions o f F v l that were unnecessary for restriction. 50 amino acids could be removed from the N-terminus o f F v l" without losing activity, unless the three residues at the C-terminus specific to the product o f the n-allele had also been deleted. Full restriction was retained if these residues were removed alone, implying redundancy between the termini. 50 amino acids could also be deleted from the N-terminus o f F v l’’ without losing the primary activity o f N-tropic MLV restriction. In this case, truncating the N-terminal deletion mutant to residue 437 only caused a slight decrease in restriction. However, the secondary activities o f F v l’’, on B- and NB-tropic MLV, were affected by changes to the C-terminus o f the protein. Addition o f TKL, the C-terminal residues o f Fvl", greatly enhanced restriction, while the presence o f the F v l’’ C-terminal amino acids generally decreased activity. Restriction o f B-tropic MLV was the weakest, and most sensitive to N-terminal deletions o f F v l’’. The different activity patterns o f Fvl"A 438-440 and F v l’’A438-459 confirmed residues 358 and 399 are important for specificity.
The internal deletion mutants determined residues 123-251 were also unnecessary for restriction. Residues 109 to 119 appeared to have an important, but unknown function. Deletion o f these residues abolished restriction, but allowed competition o f endogenously expressed F vl" and F v l’’. This mutant, In t.l, was the only inactive mutant o f any kind to show competition.
To test whether internal deletions could be combined with other mutations, the N- terminus was removed from two large internal deletion mutants. Sequence coding for the first 50 amino acids was deleted from constructs coding for Int.2-5 and Int.5-7 using PCR, and the mutants tested for activity (data not shown). Int.2-5A1-50 had the same pattern as Fvl"A l-50; partial restriction o f B-tropic MLV, but interestingly Int.5-7A l-50 had only 50% activity against B-tropic MLV (data not shown). Together with the fact that the largest internal deletion only had 50% activity, this implied that removing too many
interactions with MLV. Until a binding assay is available this question cannot be addressed.
Although two "domains" o f F v l could be identified, expressing the two halves as separate proteins did not cause restriction. Each half was distributed outside the nucleus, but there appeared to be some difficulty in detecting the C-terminus o f F v l using the anti- F vl antibody available, which needs further investigation.
To study the localisation o f F vl further, immunofluorescence was performed on other F vl mutants, in conjunction with M. Yap. The C-terminal deletion mutants Fvl"A411- 440 and Fvl^A 411-459, five N-terminal deletion mutants o f Fvl": F vl"A l-32, F v l"A l- 61, F v l "A 1-201, F vl "A 1-263 and F vl "A 1-335, and three o f the inactive internal deletion mutants: In t.l, Int.9, and Int. 10 were analysed. M. dunni cells were transduced with virus to achieve 30-50% infection. Immunofluorescence was performed as above. O f the N- terminal deletions, only Fvl"A l-263 and Fvl"A l-335 could not be detected. The other N- terminal deletion mutants gave the same localisation pattern as wild-type F vl", although the signal from F v l "A 1-201 was faint and some F v l appeared to be detected in the nucleus (M. Yap, unpublished data). This seemed to confirm the low level o f antigenic sites on the C-terminal half o f F v l, although it was still possible these mutants were not being expressed. Western blot analysis was also unable to detect F vl "A 1-263 and Fvl"A l-335, but could detect the other N-terminal deletion mutants, including F v l"A l- 201 (Figure 5.5 and data not shown). The C-terminal and internal deletion mutants could all be detected by immunofluorescence. Interestingly, these mutants, all inactive, gave slightly different staining patterns to wild-type F v l. The mutant F v l proteins were still perinuclear, but the punctate pattern was replaced with a somewhat fibrous one. The Int. 1 mutant was most interesting, as it had a localisation pattern in-between the punctate pattern o f wild-type F v l and the fibrous pattern o f Int.9, Int. 10 and the C-terminal deletion mutants.
Fv l is 50kDa, and this may be too large to pass through the nuclear pores. This may account for the lack o f nuclear F v l, particularly with the inactive mutants. When studying the distribution o f these mutants it may be better to refer to the punctate verses fibrous
patterns. Observing the localisation o f the N-terminal half more closely shows it may have a fibrous rather than wild-type distribution. This will be discussed further in chapter
8.
Fvl activity can obviously be broken down into various parts, all o f which are needed for restriction. All the active Fvl variants have a punctate distribution, but the inactive EGFP/Fvl "C-terminal half fusion also has a similar distribution, implying this is not sufficient for restriction. Similarly, active mutants can compete with endogenous F v l, but as inactive Int. 1 also competes, activity is not required for competition. Certain mutants seem to have lost the ability to perform particular functions, whilst retaining others.