Effect of tRNA on the Maturation of [631800]

Effect of tRNA on the Maturation of
HIV-1 Reverse Transcriptase
Tatiana V. Ilina1,2, Ryan L. Slack1, John H. Elder3, Stefan G. Sarafianos4,
Michael A. Parniak2,†and Rieko Ishima1
1 – Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, United States
2 – Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219,
United States
3 – Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, United States
4 – Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322,
United States
Correspondence to Rieko Ishima: Department of Structural Biology, University of Pittsburgh School of Medicine, Room
1037, Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15260, United States. [anonimizat]
https://doi.org/10.1016/j.jmb.2018.02.027
Edited by M.F. Summers
Abstract
The mature HIV-1 reverse transcriptase is a heterodimer that comprises 66 kDa (p66) and 51 kDa (p51)
subunits. The latter is formed by HIV-1 protease-catalyzed removal of a C-terminal ribonuclease H domain from a
p66 subunit. This proteolytic processing is a critical step in virus maturation and essential for viral infectivity. Here,we report that tRNA significantly enhances in vitro processing even at a substoichiometric tRNA:p66/p66 ratio.
Other double-stranded RNAs have considerably less pronounced effect. Our data support a model whereinteraction of p66/p66 with tRNA introduces conformational asymmetry in the two subunits, permitting specificproteolytic processing of one p66 to provide the mature RT p66/p51 heterodimer.
© 2018 Elsevier Ltd. All rights reserved.
Introduction
HIV-1 reverse transcriptase (RT) is a multifunctional
enzyme with both DNA polymerase and ribonuclease
H (RNH) activities and is essential for HIV-1 replication
[1]. While the gene for RT encodes a 66-kDa protein,
RT is initially translated as part of the 160-kDa Gag-Pol
precursor polyprotein, which is proteolytically proc-
essed by HIV-1 protease (PR) during virion assembly
and maturation. The mature RT is a heterodimercomprising 66-kDa (p66) and 51-kDa (p51) subunits;
the p51 subunit is generated upon proteolytic cleavage
of the p66 subunit between residues 440 and 441,thereby removing most of the RNH domain from this
p66 subunit [1–5]. This cleavage event is essential for
viral infectivity [6,7].
Formation of the mature p66/p51 heterodimer is
generally thought to proceed viaa p66/p66 homo-
dimer intermediate (Sequential model in Fig. 1a),
rather than generation of p51 followed by p66/p51
heterodimer formation (Concerted model in Fig. 1a)
[8–10]. However, data in support of the Sequentialmodel are primarily based on studies that introduce
mutations leading to dissociation of subunits in p66/
p66in vitro or at a viral level [8–10]. Although lack of
p66/p51 production using the mutants may support
the requirement of the homodimer formation for the
RT maturation (i.e., Sequential model), the mutantswere originally known to affect p66/p51 heterodimerformation [12–15]. Therefore, such experiments do
not consider whether the mutations themselvesdiminish maturation of p66/p66 or whether theycause p66/p51 dissociation which in turn results in
decreased RT detection. Furthermore, structures of
both p66/p51 ( Fig. 1 b) and the isolated RNH domain
indicate that the p51-RNH cleavage site is buried
within the core β-sheet of the folded RNH domain
(Fig. 1c) and likely inaccessible to the protease
[16–21]. Despite efforts to determine the structure of
the RNH domain in the immature RT p66/p66homodimer, a detailed structure of p66/p66 is not
available [22–25].
Our previous NMR studies suggested that the p51-
RNH cleavage sites in both subunits of p66/p66 were
0022-2836/© 2018 Elsevier Ltd. All rights reserved. J Mol Biol (2018) 430, 1891 –1900Communication

buried and thus poorly accessible to protease [25].
We therefore postulated that some virion-associated
factor may play a role in promoting the RT maturationprocess. HIV-1 virions are known to contain substantial
amounts of cellular tRNA [26–30],a n dR Tc a nb i n d
such tRNAs with nanomolar affinity [31].H e r e ,w e
describe biochemical experiments to assess theimpact of tRNA on the in vitro HIV-1 PR-catalyzed
cleavage of p66/p66 to mature RT p66/p51.
We show that processing of p66/p66 is slow and
results in numerous aberrant products in the absence
of tRNA. Surprisingly, in the presence of even a sub-stoichiometric tRNA:p66/p66 ratio, processing of p66/p66 by HIV-1 PR is greatly accelerated and with few or
no aberrant cuts. Our data, obtained using two different
p66 concentrations, show that the mature p66/p51arises from proteolytic processing of the p66/p66, and
not from processing of p66 monomers, in agreement
with previously published data [8,9,32,33]. Although a
certain processing enhancement was observed in the
presence of ds-RNA, based on the fact that HIV-1
virions are known to contain substantial amounts ofcellular tRNA [26–30], we propose a model where
virion-encapsidated tRNA facilitates RT maturation top66/p51 that is essential for HIV-1 replication.Results and Discussion
p66/p66 homodimer formation is essential for
efficient RT proteolytic processing
We evaluated the kinetics of in vitro proteolytic
processing by HIV-1 PR at different concentrations of
RT p66 in the absence and in the presence of tRNA(Fig. 2 ).
With 8 μM p66, over 50% of RT is predicted to be the
p66/p66 homodimer, based on a p66/p66 dissociationconstant of ~4 μM[34–36]. In the absence of tRNA,
minimal formation ( b10%) of p51 is noted after 1 h
(Fig. 2a and b). In contrast, with stoichiometric amounts
of tRNA, essentially complete processing to equivalentamounts of p66 and p51 (the RT p66/p51 heterodimer)
is seen within 30 –40 min. Under the same conditions,
enhancement of processing is noted even with sub-
stoichiometric levels of tRNA (0.5 μM). Surprisingly, the
extent of processing under these sub-stoichiometricconditions, after 20 min, was more than 50% of thatobserved for the 8 μM tRNA samples. This suggests
that the tRNA may act as a catalytic factor in theprocessing event.
Fig. 1. (a) Concerted and Sequential Models for RT maturation [5,8– 10]. (b) Location of the RNH domain in the known
p66/p51 RT structure. (c) An expanded view of the RNH domain. In panels a –c, the p51 and the RNH domains in the p66
subunit are shown in cyan and orange, respectively, while the p51 subunit is shown in lime color. In panels b and c, the
p51-RNH cleavage site (F440-Y441) is shown as a red ribbon. The images were generated using PDB code 1DLO [11].1892 Maturation of HIV-1 Reverse Transcriptase

In contrast, at low p66 concentrations (1 μM) where
p66/p66 homodimer formation is minimal ( b40%)
[34–36], very little p51 was formed even after 1-hincubation, either in the absence of tRNA or in the
presence of excess tRNA (2 μM) (Fig. 2 ca n dd ) .
Significant aberrant processing was also evident with
Fig. 2. Time dependence of p66 processing by HIV-1 PR in 20 mM sodium acetate buffer at pH 5.2 and 37 °C, monitored
by SDS-PAGE (a and b) at a high concentration of p66 (8 μMa sap 6 6m o n o m e r ,4 μM as a dimer) proteolytically processed
by 1 μM HIV-1 PR in the presence of 0, 0.5, 8 μM tRNA concentrations, (c and d) at a low concentration of p66 (1 μMp 6 6
monomer concentration, 0.5 μM as a dimer) processed by 0.25 μM HIV-1 PR in the presence of 0, 0.1, and 2 μMt R N A
concentrations, and (e and f) at a high concentration of p66 (8 μM as a p66 monomer, 4 μM as a dimer) proteolytically
processed by 1 μM HIV-1 PR in the presence of 0.5 μM ds-RNA (40/22 nt), ss-RNA (40 nt), and ss-RNA (22 nt). In panels b,
d, and f, p66/p51 fractions, determined from the gel images in panels a, c, and e, are shown, respectively. Average data points
were used to fit a curve with one standard deviation as an uncertainty of each data point. Since p66/p51 production occurs in
parallel with p66 degradation by PR, the build-up curves do not necessarily reach a plateau. Because of these multiple factors,normalized χ2,χn2, values for the curve fits were between 0.2 and 10.7.1893 Maturation of HIV-1 Reverse Transcriptase

low p66 and excess tRNA (Fig. S1). Because of such
p66 degradation, p66/p51 production did not neces-
sarily saturate as a function of time ( Fig. 2 ). These
results suggest that p66/p66 formation is important for
proper maturation of RT to the p66/p51 heterodimer.
To determine whether maturation enhancement
occurs in the presence of other RNAs, a similar set ofexperiments was performed using some other smallRNA molecules: ds-RNA (40/22 nt), ss-RNA (40 nt),
and ss-RNA (22 nt) ( Fig. 2 ea n df ) .W ec h o s et h e sub-
stoichiometric nucleic acid concentration, 0.5 μM, to
see the effect of RNA on the RT maturation but not theeffect of stabilizing the matured RT. The positive
control, 0.5 μM tRNA, showed similar, although slightly
lower, activity compared to that obtained in our first setof experiments (see Fig. 2a and b), presumably due to
slightly lower activity of PR used in the experiments inFig. 2e. The efficiency of RT maturation in the presenceof ds-RNA was about half, ~47%, of that obtained in the
presence of tRNA ( Fig. 2 e and f). In the presence of the
ss-RNAs, the efficiency was even lower, 29%. The
results demonstrate that ds-RNA, in addition to tRNA,
also enhances RT maturation, although to a lesser
degree for those tested in our study.
Other factors potentially impacting tRNA-mediated
p66 processing
The data presented in Fig. 2 show that tRNA
facilitates the formation of RT p66/p51 heterodimers.Confirmation was obtained using dose-dependencyexperiments, in which p66/p51 production by PR was
assessed at different tRNA concentrations ( Fig. 3 a).
Since both p66/p66 dimer formation and PR activity
may be influenced by ionic strength [37,38] , and tRNA
may act as a polyanion [39–41], we investigated the
impact of different salt concentrations on p66 process-ing. In the absence of tRNA, at physiological NaCl
concentrations (50– 150 mM), aberrant non-specific
cleavage products ( Fig. 3b, arrows), with molecular
sizes between those of p51 and p66, were seen;
significant p66/p51 formation became evident only
above 400 mM NaCl ( Fig. 3b). In contrast, even at low
levels of tRNA, substantial RT p66/p51 formation was
noted in the presence of 100 mM NaCl ( Fig. 3c).
Heparin did not enhance p66/p66 maturation (Fig. 3 d),
and PR activity in the assay with fluorescent HIVprotease substrate 1 (Sigma) was actually less in the
presence of 0.5, 1.0, or 5.0 μM tRNA (concentrations at
which we observed enhanced p66 processing efficien-
cy) compared to its activity without tRNA or at 10 μM
tRNA (Fig. S2), indicating that a simple PR activity
enhancement by polyanions does not explain our
results [41]. These data show that while higher ionic
strength may enhance proteolytic activity, as well as
dimer formation, it does not impact the total production
of p66/p51 ( Fig. 3 f).
Kinetic experiments of p66 processing in a buffer that
contained 100 mM NaCl ( Fig. 3 e and g and Fig. S3)also showed a pattern of the p66/p51 production similarto that observed in a buffer lacking 100 mM NaCl
(Fig. 2a), that is, tRNA impacts p66/p51 production. Of
note, the reaction was performed at 20 °C instead of
37 °C because the PR catalytic rate increases in thepresence of 100 mM NaCl, compared to the absence
of NaCl (37 °C, in Fig. S3). Overall, our data, obtained
with different NaCl conditions ( Figs.
2 and 3 ), consis-
tently show that tRNA influences the selectivity ofcleavage at the processing site . Although there are
reports that tRNA may act as a polyanion [39–41],
tRNA clearly increases selectivity of the processing sitefor p66/p51 production in the presented RT maturation
experiments.
p66/p66 interaction with tRNA
Our biochemical in vitro proteolytic processing
studies strongly suggest that RT p66/p66 homodi-
mer is the substrate for HIV-1 PR processing to
mature RT p66/p51 heterodimer. To further evaluatethis, we carried out biophysical analysis of the
RT species using size-exclusion chromatography
(Fig. 4 ).
In the absence of tRNA, both monomer and dimer
peaks of p66 were observed, consistent withprevious data ( Fig. 4a) [25]. tRNA alone eluted at a
slightly greater volume than p66 monomer alone,based on both UV and the fluorescence detection
(Fig. 4b). In the mixture of p66 and tRNA, two
additional peak signals, compared to p66 alone,
were noted ( Fig. 4c); these stem from p66/p66 –tRNA
and p66 –tRNA complex species, as confirmed by
the fluorescence emission of Cy3-labeled tRNA. Theloaded p66 and tRNA concentrations, 40 and 5 μM,
respectively, are empirically estimated to be 4 μM
p66 and 0.5 μM tRNA on a column, which are similar
to the conditions used in the processing experiments
(Fig. 2 a).
The tRNA interaction with p66 protein was quanti-
fied by recording changes in fluorescence emission of
Cy3-labeled tRNA at varying p66 concentrations in
the fluorescence spectroscopic analysis ( Fig. 4 d). The
emission changes at 560 nm could not be described
by a single-site binding model ( Fig. 4 e, dashed line)
and were better explained with a two-binding modemodel ( Fig. 4 e, solid line) that gave two K
Dvalues:
65.8 ± 26.9 nM and 2.38 ± 0.69 μM. When consid-
ered in the context of the known concentrations ofp66, we conclude that these dissociation constantsreflect tRNA dissociation from p66/p66 (dimer) andfrom p66 (monomer), respectively. Competitive gel
mobility shift assay of a solution containing tRNA, p66,
and PR suggests that tRNA more strongly interactswith p66 than PR (Fig. S4). Taken together, we
confirmed the p66/p66 –tRNA interaction. In addition,
observation of p66 –tRNA species explains why p66
processing at low p66 concentration showed tRNAdependence ( Fig. 2 c and d).1894 Maturation of HIV-1 Reverse Transcriptase

A proposed model for RT maturation
Here, we demonstrate that tRNA interacts with p66/
p66 homodimer and facilitates selective cleavage at the
p51-RNH site by HIV-1 PR ( Fig. 2 ). We also
demonstrate that the effect of tRNA on the selectivity
of p66 processing is distinct from the effect of ionic
strength ( Fig. 3 and S2). Consistent with the processing
experiments, both tRNA –p66/p66 and tRNA –p66
forms were observed. Although tRNA can bind p66/p51 tightly ( K
D=3–50 nM [5,31,42] ), tRNA interactionwith p66/p66 is the same as, or weaker than, the p66/
p51 binding ( Fig. 4 e). Because of this moderate binding
and observation of the significant tRNA effect at a sub-stoichiometric concentration, it is possible that transient
interaction of tRNA with p66/p66 mediates the selective
processing at the p51-RNH site, with tRNA likely beingreleased before heterodimer formation is complete
[5,36,38]. Such gain of RT maturation at the sub-
stoichiometric concentration suggests that the tRNAmay serve as a catalytic molecule in RT maturation,
rather than just stabilizing the matured RT in solution.
Fig. 3. Effects of varying amounts of (a) tRNA, (b) NaCl, (c) tRNA in the presence of 100 mM NaCl, and (d) heparin on p66
processing by HIV-1 PR for 20 min at 37 °C, and (e) the time dependence of p66 processing by HIV-1 PR in the presence of
100 mM NaCl at 20 °C. In all the experiments, reactions were initiated by the addition of 1 μM HIV-1 PR to p66 at a high
concentration of p66 (8 μM as a monomer, 4 μM as a dimer) and monitored by SDS-PAGE, and the buffer contains 20 mM
sodium acetate at pH 5.2. In panel e, experiments were carried out in the presence of 0, 0.5, and 8 μMt R N A .( f )P l o t so f
intensity changes and the fit curves, χn2values 2.7 and 4.5, are shown for panels a and c, respectively. (g) Plots of intensity
changes and the fit curves, χn2values from 0.88 to 2.2, are shown for panel f. In panel b), aberrant non-specific cleavage
products are shown by arrows.1895 Maturation of HIV-1 Reverse Transcriptase

Ourin vitro data suggest that ds-RNA, in addition
to tRNA, also enhances RT maturation, although to a
lesser degree for those tested in our study.Considering this in vitro data, we cannot conclude
which RNA mediates the RT maturation in virio .
However, these data are consistent with previouslyobserved changes in enzymatic activities of p66
upon tRNA interaction [43,44] . The proposed model
also explains why p66/p51 formation in cells occurs
efficiently within 1 h [45,46] , whereas in vitro , in the
absence of nucleic acid, RT heterodimer formationtakes significantly longer and with lower yield. HIV-1is known to contain numerous copies of multipletRNA species in addition to the essential primer
tRNA [26–30]; thus, these other tRNA species may
play a role in directing appropriate proteolytic matu-ration of HIV polyproteins, especially the conversion of
the RT p66/p66 homodimer to the mature RT p66/p51heterodimer.
Conclusions
Our data show that the RT p66/p66 can interact with
tRNA and that this interaction facilitates selective
cleavage at the p51-RNH site by HIV PR. Importantly,
we also show that this selective cleavage is indepen-dent of ionic strength, but dependent on the concen-
tration of RT p66, a factor directly related to the RT p66
subunit dissociation strength. Facilitation of theselective cleavage at the p51-RNH site by HIV-1 PR
is significant even at a sub-stoichiometric tRNA
Fig. 4. tRNA binding to p66 protein, monitored by (a –c) SEC elution profiles of (a) p66 protein solution, (b) tRNA solution, and
(c) that mixed with tRNA, and (d, e) change of fluorescence emiss ion of Cy3-labeled tRNA at varying p66 concentrations obtained
by spectrofluorometry. In panels a –c, UV absorbance at 254 nm (gray line) and 280 nm (black line), and fluorescence emission at
560 nm (dashed line) are shown. In panel d, changes in fluorescence of total 1 μM tRNA, containing 40 nM Cy3-labeled tRNA, as
a function of p66 concentration were recorded. In panel e, fluor escence intensity changes at 560 nm (d) are plotted. The dash line
is a fit curve calculated with a single binding mode model ( χn2= 6.5), and the solid line indicates a fit-curve calculated with a two-
binding mode model ( χn2= 0.8). The null hypothesis was rejected with p= 0.00036.1896 Maturation of HIV-1 Reverse Transcriptase

concentration. We propose a model in which interac-
tion of the p66/p66 homodimer with tRNA introduces
conformational asymmetry in the two subunits,
permitting specific proteolytic processing of one ofthe p66 subunits leading to formation of the maturep66/p51 RT.
Materials and Methods
Protein expression and purification
p66/p51 HIV-1 RT was prepared using the p6HRT-
PROT plasmid [47]as previously described [48,49] .
The p66 sequence from p6HRT-PROT used for p66/
p51 expression [47]was cloned into the pPSG-IBA3
vector using the StarGate cloning system (IBA
Solutions for Life Sciences, Göttingen, Germany).
p66 protein was expressed in BL21 (DE3) Escherichia
colic e l l sa n dp u r i f i e du s i n gaS t r e p – T a c t i ng r a v i t y
flow column (IBA Solutions). Protein concentration
(calculated as p66 monomer) was determined by
measuring absorbance at 280 nm with an extinction
coefficient of 137,405 M−1cm−1. Purified p66 and
p66/p51 were stored in 25 mM sodium phosphate
(pH 7.0), 250 mM NaCl, and 50% v/v glycerol at
−80 °C. HIV-1 PR was expressed and purified as
previously described [50–52].
HIV-1 PR-catalyzed processing of p66/p66
Proteolytic processing of p66 protein was evalu-
ated using kinetic (time-course) experiments that
determined the rate of processing and fixed-time
experiments that assessed the impact of differencesin tRNA:protein ratio on extent of processing. Since amonomer:dimer ratio of p66 depends on protein
concentration and tRNA concentration, all p66
concentrations are reported as monomer proteinconcentration for the Materials and Methods pur-
pose. Processing experiments were carried out in
20 mM sodium acetate buffer (pH 5.2) at 37 °C,
unless otherwise noted.
Kinetic experiments were conducted at two p66
concentrations: “high p66 concentration ”(8μM),
where RT is predominantly in the p66/p66 homodi-mer form and processed by 1 μM PR in the presence
of 0, 0.5, or 8 μM synthetized tRNA
Lys3of human
sequence (TriLink BioTechnologies LLC, San Diego,CA), and “low p66 concentration ”(1μM), where RT
is predominantly in the p66 monomer form and
processed using 0.25 μM PR in the presence of 0,
0.1, or 2 μM tRNA. Additional kinetic experiments at
high p66 (8 μM) were conducted in the sodium
acetate buffer containing 100 mM NaCl at 20 °C.
Fixed-time experiments were conducted using highp66 (8 μM) conditions only, at different concentra-
tions of tRNA or NaCl or heparin (Sigma-Aldrich. StLouis, MO), which were added to p66 protein prior tostarting the reaction, incubated at room temperature
for 5 min, and allowed to react at 37 °C for 10 –
20 min. The fixed-time experiments at different tRNA
concentrations were performed in sodium acetatebuffer with/without 100 mM NaCl.
The kinetic (time-course) modes of p66 proteolytic
processing were performed at 8 μMp 6 6a n d1 μMP R ,
in the presence of 0.5 μM ds-RNA (40/22 nt), ss-RNA
(40 nt), and ss-RNA (20 nt), of which 40- and 20-ntsequences are 5 ′-AGGUGAGUGAGAUGAUAACAA
AUUUGCGAGCCCCAGAUG and 5 ′-GCAUCUGGG
GCUCGCAAAUUUG, respectiv ely (TriLink BioTech-
nologies LLC).
In all the processing experiments, reactions were
stopped by addition of Tricine sample loading buffer
(Bio-Rad Laboratories, Berkeley, CA) and denatur-
ation at 95 °C for 5 min. Samples were loaded ontoprecast 4 –15% Tris-glycine gels (Bio-Rad) and
stained with Bio-safe Coomassie stain (Bio-Rad).Band intensities were quantified using an OdysseyCLX gel imaging system by Image Studio software
(Li-Cor Biosciences, Lincoln, NE) or an Amersham
Imager 600 (GE Healthcare Life Sciences). Forquantification, these gel experiments were repeated
at least three times. Production of RT p66/p51
heterodimer was determined based on the ratio ofp66 to p51 band intensities in the following way: theratio of p66 to p51 band intensities of a reference
heterodimer was first quantified in the same gel, and
production of the heterodimer against the initial p66intensity was determined using the p51 band intensity
normalized by the reference p66/p51 intensity ratio.
An average of the three quantified p66/p51 productionwas plotted with the standard deviation as an error
bar. Trends of intensity changes were shown by fit
curves using Igor (Wavemetrics, Inc., Lake Oswego,OR).
Analytical size exclusion chromatography to
monitor p66/p66 –tRNA interaction
All size exclusion chromatography (SEC) experi-
ments used a 24-ml analytical Superdex 200 Increase10/300 GL column (GE Healthcare), mobile phase of
25 mM Bis –Tris buffer, pH 7.0, containing 100 mM
NaCl with 0.02% sodium azide at a flow rate of
0.5 ml/min. Injection volume was 50 μl, and protein
elution was monitored by UV absorbance at 254 and280 nm. Elution profiles of 40 μM p66 RT were
evaluated in the absence of tRNA, or followingpreincubation with 5 μM tRNA containing tracer tRNA
3′-end labeled with pCp-Cy3 (Jena Bioscience, Jena,
Germany). As a control, the labeled tRNA was injectedwithout mixing with p66. With the SEC experiments that
contain labeled tRNA, in addition to UV, the fluores-
cence emission at 560 nm (excitation 485 at nm) wasalso measured using an in-line Shimadzu RF-10AXL
Fluorescence Detector.1897 Maturation of HIV-1 Reverse Transcriptase

Fluorescence spectroscopic analysis of RT
p66/p66–tRNA interaction
The interaction of tRNA with RT p66 was
evaluated in 25 mM Bis –Tris (pH 7.0) and 100 mM
NaCl. RT p66 protein in various concentrations was
incubated for 30 min with 1 μM total tRNA containing
tracer Cy3-labeled tRNA. Emission spectra were
collected using a FluoroMax-4 (Horiba Scientific,
Edison, NJ) with excitation at 485 nm. All experi-
ments were carried out at least three separate timesto determine an average and a standard deviation of
the data. The change in fluorescence at 560 nm was
plotted at different protein concentrations 0–20 μM,
with error bars representing one standard deviation.The tRNA dissociation constant, K
D, was determined
assuming two models, one with a single KDand the
other with two independent KDs, using χ2-minimization
routine in Matlab (MathWorks, Natick, MA), and
evaluated using the Ftest.
Acknowledgments
This study was supported by grants from the
National Institutes of Health (R01GM105401 to R.I.and R.L.S.; R01GM109767 to R.I.; R01AI00890 to T.
I., S.G.S., and M.A.P.; P50GM103368 to J.H.E., S.G.S., and M.A.P.). We thank Michel Guerrero for his
technical assistance, Michael Tsang at the University
of Pittsburgh for use of Odyssey CLX gel imagingsystem, and Teresa Brosenitsch for reading themanuscript.
Appendix A. Supplementary Information
Fig. S1: A set of the entire SDS gels showing
degradation bands other than p51 and p66 duringthe processing experiment in Fig. 2. Fig. S2: PRactivity changes in the presence of tRNA, in the
absence (a) and the presence (b) of 100 mM NaCl,
recorded using fluorescence substrate-1 (Sigma-Aldrich). Fig. S3: Time dependence of 8 μM p66
processing in a buffer containing 100 mM NaCl at37 °C. Fig. S4: Experiments to characterize molec-ular interactions among PR, p66, and tRNA at
different p66 concentrations (a) and PR concentra-
tions (b). Supplementary data to this article can befound online at https://doi.org/10.1016/j.jmb.2018.
02.027 .
Received 22 December 2017;
Received in revised form 21 February 2018;
Accepted 22 February 2018
Available online 8 May 2018Keywords:
HIV-1;
proteolysis;
maturation;
tRNA;
reverse transcriptase;
RNase H
†Deceased.
Abbreviations used:
RT, reverse transcriptase; RNH, ribonuclease H; SEC,
size exclusion chromatography.
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