The effect of point mutations within the N-terminal domain of Mason-Pfizer monkey virus capsid protein on virus core assembly and infectivity
Authors: Eric Hunter
Marcela Wildová
Ivana Krízová
Iva Pichová
Michaela Rumlová
Tomás Ruml
Jitka Stokrová
Romana Hadravová
Journal: Virology (2008)
Discipline: MICROBIOLOGY
Keywords: Retroviral capsid protein; assembly; immature viral particles; mature virions; N-terminal domains
Affiliations: Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

Abstract:  Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a beta-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50-Asp57 plays an important role in stabilization of the beta-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA. 

Retracted: No

PubPeer comments:
 Concern about Figure 1A: * Red boxes: Lanes 5 (P1Y) and 6 (D50A) appear to look very similar. ![file] , 
 Concern about Figure 1B. * Blue boxes: Lanes 6 (D50A) and 7 (T54A) appear to look very similar over most of the lanes length, with differences at the 40, 35, and 27 kDa regions. * Green boxes: Repetitive areas appear to be visible around 35-40 kDa. ![file] Forensically agrees: ![file] , 
 Concern about Figure 1C. * Boxes of the same color highlight regions within and across lanes that look unexpectedly similar. ![file] , 
 Concerns about Figure 2A (image made a bit darker to bring out details): * Boxes of the same color highlight regions within and across lanes that look unexpectedly similar. * Rectangular areas of different background patterns appear to be visible in lanes 6 and 7 ![file] , 
 Figures 1A-C:These are pulse-chase experiments, during which the cells are treated with radioactively labelled S$35$Met, Cys medium and then immunoprecipitated by a polyclonal antibody. Then, the individual samples were loaded onto SDS PAGE and developed autoradiographically using Typhoon 9410. We show below the original uncropped gels present in Figure 1. As all the samples were treated the same way, the similarity among individual samples (i.e. lanes) mainly for pulse experiment was highly expected, because they represent the transfected cells producing the same polyprotein precursors. Figure 2A is an imunoanalysis of individual viral samples of various mutants: The polyclonal antibody anti M-PMV capsid proteins was used to identify the efficiency of Gag polyprotein cleavage. We show here another western blots of two biological replicates of the same experiments shown in Fig. 2A, clearly supporting the results displayed in Fig. 2A. There was no intentional adjustments corrections of the results provided in the original figures ![file] ![file] ![file] ![file] ![file] , 
 Figures 1A-C:These are pulse-chase experiments, during which the cells are treated with radioactively labelled S35Met, Cys medium and then immunoprecipitated by a polyclonal antibody. Then, the individual samples were loaded onto SDS PAGE and developed autoradiographically using Typhoon 9410. We show below the original uncropped gels present in Figure 1. As all the samples were treated the same way, the similarity among individual samples (i.e. lanes) mainly for pulse experiment was highly expected, because they represent the transfected cells producing the same polyprotein precursors. ![file] ![file] ![file] Figure 2A is an imunoanalysis of individual viral samples of various mutants: The polyclonal antibody anti M-PMV capsid proteins was used to identify the efficiency of Gag polyprotein cleavage. We show here another western blots of two biological replicates of the same experiments shown in Fig. 2A, clearly supporting the results displayed in Fig. 2A. There was no intentional adjustments corrections of the results provided in the original figures ![file] ![file] , 
 Dear Dr. Rumlovu00e1, thank you for your reply and for providing the originals blots and some replicates. Unfortunately, these higher resolution originals still appear to contain duplicated parts, often down to the pixel, as demonstrated here below with Forensically analysis. Even the last replicate blot provided by you appears to contain regions of very high similarity. ![file] ![file] ![file] ![file] , 
 I agree with Dr. Bik: Here is a false color image of Figure 1B where you can see the multiple areas of similarities in the two lanes (5 and 6) she highlighted. I do not believe these similarities could occur by chance. *** ![file] , 
 Concern about Figure 1B: I shifted a copy of the image by one lane and took the difference. You can see from the black lane that the pixels are identical between lanes 5 and 6 except for five exactly rectangular bands that do not match. The bottom non-match is an exact match if shifted by 1 pixel vertically. Method: the image was loaded into the open-source GIMP graphics editor, shifted, and the difference operation applied. -- Ken Shirriff, Ph.D. ![Image showing difference between the lanes] https: twitter.com kenshirriff status 1487213158653595649 ,