viruses
ArticleCis-Allosteric Regulation of HIV-1 Reverse Transcriptase
by Integrase
Takao Masuda 1, *, Osamu Kotani 2, *, Masaru Yokoyama 2 , Yuya Abe 1 , Gota Kawai 3
1

2

3

*

and Hironori Sato 2

Department of Immunotherapeutics, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University (TMDU), Yushima, 1-5-45 Bunkyo-ku, Tokyo 113-8519, Japan
Laboratory of Viral Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Gakuen,
4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan
Department of Life Science, Faculty of Advanced Engineering, Chiba Institute of Technology,
2-17-1 Tsudanuma, Narashino-shi, Chiba 275-0016, Japan
Correspondence: tmasu.impt@tmd.ac.jp (T.M.); konioo@niid.go.jp (O.K.)

Abstract: Reverse transcriptase (RT) and integrase (IN) are encoded tandemly in the pol genes ofretroviruses. We reported recently that HIV-1 RT and IN need to be supplied as the pol precursor
intermediates, in which RT and IN are in fusion form (RTIN) to exert efficient reverse transcription in
the context of HIV-1 replication. The mechanism underlying RTIN’s effect, however, remains to beelucidated. In this study, we examined the effect of IN fusion on RT during reverse transcription by
an in vitro cell-free assay, using recombinant HIV-1 RTIN (rRTIN). We found that, compared to recombinant RT (rRT), rRTIN generated significantly higher cDNAs under physiological concentrations ofdNTPs (less than 10 µM), suggesting increased affinity of RTIN to dNTPs. Importantly, the cleavageof RTIN with HIV-1 protease reduced cDNA levels at a low dose of dNTPs. Similarly, sensitivities
against RT inhibitors were significantly altered in RTIN form. Finally, analysis of molecular dynamics
simulations of RT and RTIN suggested that IN can influence the structural dynamics of the RT
active center and the inhibitor binding pockets in cis. Thus, we demonstrated, for the first time, thecis-allosteric regulatory roles of IN in RT structure and enzymatic activity.

Citation: Masuda, T.; Kotani, O.;

Keywords: HIV-1; reverse transcriptase; integration; integrase; pol; protease; deoxyribonucleoside
triphosphates; molecular dynamics

Yokoyama, M.; Abe, Y.; Kawai, G.;
Sato, H. Cis-Allosteric Regulation of
HIV-1 Reverse Transcriptase byIntegrase. Viruses 2023, 15, 31.
https://doi.org/10.3390/v15010031
Academic Editor: Luis
Menéndez-Arias
Received: 11 November 2022
Revised: 19 December 2022
Accepted: 19 December 2022
Published: 21 December 2022

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Reverse transcriptase (RT) and integrase (IN) are essential enzymes encoded in the polgene for retroviruses to establish proviral DNA, in which viral genomic RNA (gRNA) isreverse transcribed into DNA followed by its integration into the host cell chromosome [1].
Reverse transcription and the integration of a virus genome are catalyzed by RT [1] andIN [2], respectively. Human immunodeficiency virus type 1 (HIV-1) infection depends
on the activation status of target cells, CD4+ T cells [3] and monocytes/macrophages [4].
SAMHD1 has been identified as a host restriction factor against HIV-1 infection by reducing
cellular levels of deoxyribonucleoside triphosphates (dNTPs) [5]. Reverse transcription
is therefore a critical step depending on cellular status, especially on the concentrationof dNTPs.
Retrovirus RT contains two independent catalytic sites for RNA-dependent DNA
polymerase (RDDP) and RNase H [6] activities to generate DNA copies of vRNA [1]. IN
has one catalytic site for 30 -end processing and a strand-transfer of viral DNA ends tointegrate viral DNA into the host chromosome [2]. Meanwhile, the additional roles of IN
at steps prior to integration, including viral maturation and reverse transcription, havebeen reported for HIV-1 [4,7–12], which has become a target for the development of a
new class of IN inhibitors known as allosteric inhibitors (ALLINIs) [13,14]. Interactionof IN with virus genomic RNA (vRNA) has been reported to be crucial for HIV-1 virion

Viruses 2023, 15, 31. https://doi.org/10.3390/v15010031

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morphogenesis [15–17], providing a molecular base for pleiotropic effects of IN mutations
and/or ALLINIs [12].
HIV-1 RT and IN are originally expressed as parts of Gag-Pol precursor polyproteinsfor assembly into viral particles, followed by protease-mediated cleavage into individual
RT and IN during or after virus release [18,19]. Our previous trans-complementation assay
in the context of virus replication showed that HIV-1 IN might exert a crucial role during
reverse transcription through the Pol precursor intermediate, in which RT and IN were
kept in fusion form (RTIN) [20]. However, the mechanism underlying the critical role(s) of
RTIN during reverse transcription remains to be elucidated.
In this study, to delineate the impact of HIV-1 IN fusion to RT during reverse transcription, we prepared recombinant RTIN protein (rRTIN) and evaluated the impact of HIV-1 IN
fusion to RT during reverse transcription using an in vitro assay [21,22]. We also conducted
MD simulations of RT and RTIN proteins. The results provided direct evidence of a novel
cis-regulatory role of IN during HIV-1 reverse transcription through the RTIN fusion form.
2. Materials and Methods
2.1. Construction of Expression Vectors
For His-tagged recombinant protein expression in the E. coli, the entire RTIN region
0of HIV-1 was amplified with primer sets of 5 GGA CCC GGG CCC ATT AGT CCT ATT000
GAG ACT GTAC3 and 5 AGC CTC GAG TTA ATC CTC ATC CTG TCT ACT3 using
a pNL4-3luc∆env vector [8] as a template. Fragments of RT carrying IN NTD (residues
1-55) or NTD and CCD (residues 1-212) were similarly generated, using primer sets with00
a reverse primer of 5 AGC CTC GAG TTA GTC TAC TTG TCC ATG CAT or 5 AGC CTC
GAG TTA TTC TTT AGT TTG TAT GTC, respectively. The amplified fragment was cloned
into a pET-47b (+) vector (Novagen, Madison, WI, USA) at Sma I and Xho I sites. Entire
RTIN regions were verified by DNA sequence analysis.
2.2. Preparation of Recombinant RTs
A His-tagged form of HIV-1 RTIN or RT(p66) was expressed in the E. coli BL21
derivative strain of Rosetta (DE3) (Invitrogen/Thermo Fisher Scientific, Waltham, MA,
USA) by incubation with 0.1 mM IPTG for 16 hr at 18 ◦ C. Cells were incubated in lysis buffer(BugBuster® Protein Extraction Reagent; Merck, Darmstadt, Germany) with lysosome(FUJIFILM-Wako, Osaka, Japan) and nuclease (Benzonase, Merck/Millipore, Burlington,
MA, USA) for 1 hr. After cell lysis, the soluble fraction was obtained by centrifugation
at 10,000× g for 30 min. His-tagged RTIN or RT in the soluble fraction was purified
through a Ni-affinity column (Ni-NTA agarose, Qiagen, Hilden, Germany). The His-tag
was then removed by treatment with HRV3C protease (Turbo3C, Nacalai-Tesque, Kyoto,
Japan), followed by glutathione-Sepharose column purification (Glutathione Sepharose™
4B, Cytiva Life Science, Marlborough, MA, USA) to remove the HRV3C. The flow-throughfraction was then subjected to cation-exchange chromatography (SP-HP, GE Healthcare,
Chicago, IL, USA). Fractions eluted by SP buffer [20 mM HEPES-NaOH (pH 7.0), 0.1 mM
EDTA, 0.01% TritonX-100, 10% glycerol, and 1 mM DTT] containing 250–500 mM NaCl
were pooled. The purified recombinant HIV-1 RT IN (rRTIN) was concentrated througha concentrator column with a filtration cutoff of 50 kDa (VIVAspin 50K, GE Healthcare)and stored in SP buffer containing 300 mM NaCl at −80 ◦ C. To estimate the purity and/or
oligomer status of rRTIN, an aliquot of purified rRTIN was subjected to size-exclusion
chromatography (SEC) analysis using a Superdex 75 or 200 Increase 100/300 GL column
operated by the AKTA go system (Cytiva Life Science, Marlborough, MA, USA). The elution
profile was analyzed by UNICORN ver. 7.4 software (Cytiva Life Science, Marlborough,MA, USA). SEC fractions were subjected to SDS-PAGE followed by staining with Coomassie
brilliant blue.

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2.3. Proteolysis of RTIN by HIV-1 Protease
Purified rRTIN was treated with HIV-1 protease (PROSPEC, Rehovot, Israel) in PRbuffer [20 mM Tris-HCl (pH7.0) and 1 M NaCl] for 30 min at 37 ◦ C. Proteolysis of rRTIN
was verified by SDS-PAGE analysis and Western blot analyses using anti-HIV-1 RT or an
IN antibody (Abcam, Cambridge, UK).
2.4. In Vitro Reverse Transcription Assay
An in vitro reverse transcription assay was performed as described. Briefly, 100 ng
of synthetic HIV-1 RNA (corresponding to 0.1 pmole) and 10 pmole of pbs-sRNA primers(Sigma-Aldrich Japan Genosis, Tokyo, Japan) were annealed by heating at 70 ◦ C for
10 min, followed by cooling on ice. Then, vRNA/pbs-sRNA mixtures were incubatedwith 20 pmoles of synthetic NCs (Peptide Institute, Osaka, Japan) for 15 min. A serial dilution of vRNA/pbs-sRNA/NC was then inoculated in the reaction buffer [50 mM Tris-HCl(pH8.3), 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 0.1–100 µM dNTPs and 8 pmol of rRTor rRTIN]. The reaction was initiated by incubation at 42 ◦ C to avoid mis-annealing ofvRNA to facilitate efficient cDNA synthesis and correct 1st strand-transfer as described
before [22]. At 30–300 min, the reaction was terminated by heating at 75 ◦ C for 10 min.
Following dilution with TE buffer (10 mM Tris-HCl [pH8.0], 1 mM EDTA), the number ofcDNA spices was determined by qPCR assay using primer sets specific to the R/u5, U3/u5
or U3/pbs region. For velocity analysis, the copy number of cDNAs generated during
60 min incubation was determined by q-PCR using the R/u5 primer set. The efficiency of f
strand transfer of -sscDNA (1st strand-transfer) was estimated by calculating the amountof the U3/u5 relative to that of R/u5 products as described previously [22].
2.5. Reagents
Efavirenz and Nevirapine (Tokyo Chemical Industry, Tokyo, Japan) were resolved indimethyl sulfoxide (DMSO). 3’-Azido-2’,3’-dideoxythymidine-5’-triphosphate (AZT-TP,
Jena Bioscience, Jena, Germany) was resolved in nuclease-free distilled water. Raltegravir(Cayman Chemical, Ann Arbor, MI, USA) was resolved in DMSO.
2.6. In Silico Analysis of HIV-1 RT and RTIN
Molecular dynamics (MD) simulations: A three-dimensional model of RTIN of HIV-1
was constructed by the AlphaFold 2 program [23] using the reported amino acid sequenceof the HIV-1 NL4-3 infectious molecular clone (GenBank accession no. AAK08484) [24]. The
obtained RTIN model gave a per-residue confidence score (pLDDT) of 83.3, which is in the“Confident” range [23]. An RT model was constructed from the RTIN model by deleting
the IN region using the Molecular Operating Environment (MOE) (Chemical Computing
Group, Montreal, Quebec, Canada). The accuracy of the RTIN model was evaluated by
the root-mean-square deviation (RMSD) score between the predicted RTIN model and thereported X-ray crystal structure of the RT or IN (PDB code: RT; 3HVT [25], IN; 1K6Y [26]and 1EX4 [27]) using the “Structure Superposition” tool in MOE. Each domain of RTIN
model was similar to the reported RT or IN models (RMSD score: RT; 2.161 Å, IN-NTD;
1.931 Å, IN-CCD; 1.81 Å, IN-CTD; 0.785 Å). The RTIN and RT models were subjected to
MD simulations as described for HIV-1 capsid protein [28] and other viral proteins [29–31].
Briefly, the simulations were performed using the pmemd.cuda.MPI module in the Amber
16 program [32] with the ff14SB force field for protein simulation [33]. The RTIN and
RT models were solvated in a truncated octahedral box of TIP3P water molecules with adistance of at least 9 Å around the models [34]. A non-bonded cut-off of 10 Å was used.
Bond lengths involving hydrogen were constrained with SHAKE, a constraint algorithm
that satisfies Newtonian motion [35]. The time step for all MD simulations was set to 2 fs.
After heating calculations were performed for 20 ps up to 310 K using the NVT ensemble,
simulations were executed using the NPT ensemble at 1 atm, at 310 K and in 150 mM NaCl
for a total of 500 ns.

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Root-mean-square deviation (RMSD): The trajectory files during MD simulations wereused to calculate RMSD. RMSDs between the heavy atoms of the initial complex structure
and the structure at given time points during the MD simulation were calculated to monitor
the overall structural changes as described previously [29–31]. Calculations of RMSDs weredone by the cpptraj module in AmberTools 16, a trajectory analysis tool [32].
Molecular surface area: The trajectory files during MD simulations were used to
calculate the molecular surface area of the RT and RTIN using the linear combinations ofpairwise overlaps algorithm [36] in cpptraj operated in AmberTools 16 [32].
Hydrogen bond: A hydrogen bond in a protein was identified by geometric criteria, inwhich the bond is defined by the distance and angle between an acceptor heavy atom anda donor heavy atom in a given amino acid pair. The trajectory files during MD simulations
were used to calculate the number of hydrogen bonds between the fingers and thumbsubdomains in the RT region using the cpptraj module in AmberTools 16 [32].
Root-mean-square fluctuation (RMSF): The trajectory files during the last 100 ns of
MD simulations (n = 50,000) were used to calculate RMSF. The RMSF of the Cα atoms ofamino acid residues was calculated to obtain information about the atomic fluctuations ofindividual amino acid residues of RTIN protein during MD simulations, using the cpptraj
module in AmberTools 16 [32].
3. Results
3.1. Preparation of Recombinant RTIN
To evaluate the direct contributions of IN through its fusion with RT (RTIN) during
reverse transcription in vitro, recombinant HIV-1 RTIN (rRTIN) was prepared by using an
E. coli expression system based on the protocol for recombinant RT (rRT) [22] (Figure 1a).
However, we found that a majority of rRTIN was in insoluble form and precipitated in
the inclusion body during the induction protocol with 1 mM IPTG at 37 ◦ C, the protocol
for the rRT preparation. To reduce the aggregation of rRTIN in the inclusion body, mild
induction to express rRTIN was carried out with 0.1 mM IPTG at 18 ◦ C for 20 hrs. Using
this modified induction protocol, we successfully obtained rRTIN in soluble form. Sizeexclusion column (SEC) analysis and SDS-PAGE analysis revealed that a majority (~80%)of the rRTIN may have been formed from a hexamer or a higher multimer (Figure S1).
Moreover, the proteolysis profile of rRTIN by HIV-1 protease was examined. As expected,
HIV-1 protease cleaved rRTIN into RT subunits (p66 and p51) and IN (p32), which werereacted to anti-RT or IN antibodies, respectively (Figure 1b), thus verifying the successful
preparation of the rRTIN. The faint bands for p66 and p51 detected in rRTIN preparation
without HIV-1 protease (Figure 1b, Prot-) may be generated most probably by proteolysis
with E. coli-derived protease(s) during the induction phase of rRTIN.
3.2. Evaluation of rRTIN Enzymatic Activities during Reverse Transcription
The enzymatic activity of rRTIN was addressed by an in vitro reverse transcription
assay as described previously [21,22]. The levels of cDNA intermediates were monitoredby q-PCR using a specific primer set to amplify the R/u5, U3/u5, or U3/pbs region of theHIV-1 genome. Amplified products of R/u5, U3/u5, or U3/pbs reflect cDNA intermediatesof minus-strand strong-stop cDNA (−sscDNA), strand-transfer products of -sscDNA (1ststrand-transfer), or plus-strand strong-stop cDNA (+sscDNA), respectively [22]. Levels ofthese cDNA products could be used to monitor the RT enzyme activities of RNA-dependent
DNA polymerase (RDDP), RNase H, and DNA-dependent DNA (DDDP) polymerase
activities (Figure 2a). The level of -sscDNA products (R/u5) was efficiently generated
and accumulated linearly from 30 min to 60 min of incubation, followed by sequential
accumulation of the 1st strand-transfer products (U3/u5) and +sscDNA products (U3/pbs)(Figure 2b). These results demonstrated that purified rRTIN possesses all RDDP, RNase H,
and DDDP activities.

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Figure1.1.Preparation
Preparationof recombinant
protein.
(His) -tagged form of HIV-1 RTIN
Figureof recombinant
HIV-1HIV-1
RTIN RTIN
protein.
(a) (His)(a)6-tagged 6form of HIV-1 RTIN◦
wasexpressed
in
E.
coli
Rosetta(DE3)
byincubation
with
0.1
mM
20 °C.
hrs(His)at 18
was expressed in E. coli Rosetta (DE3) by incubation with 0.1 mM IPTG forIPTG
20 hrsfor
at 18
6- C. (His)6 tagged
RTIN
in
the
solublefractionwaspurifiedthroughaNi-affinity
column.
The
(His)
6
-tag
was
tagged RTIN in the soluble fraction was purified through a Ni-affinity column. The (His)6 -tagthen removed by treatment with HRV3C protease followed by glutathione-sepharose column chrowas then removed by treatment with HRV3C protease followed by glutathione-sepharose column
matography to remove the HRV3C. The flow-through fraction was then subjected to cation exchromatography
to removetheeluted
HRV3C.
Thebufferflow-throughfraction was(pHthensubjectedchangechromatography.
Fractionsby 1xSP[20 mM HEPES-NaOH
7.0),
0.1 mM to cationexchange
chromatography.
Fractionselutedby
1
×SP
buffer[20mM
HEPES-NaOH(pH 7.0), 0.1 mM
EDTA, 0.01% TritonX-100, 10% glycerol, and 1 mM DTT] containing 250–500 mM NaCl were pooled.
The
purifiedrecombinant
RTIN(rRTIN)was concentrated
througha concentrator
spinmM
column
with
EDTA,
0.01%
TritonX-100,10%glycerol,and 1 mM DTT]
containing
250–500
NaCl
were pooled.
aThefiltration
cutoff
of
50
kDa
(MCW
50) and stored in 1xSP buffer containing 300 mM NaCl at −80 °C.
purified recombinant RTIN (rRTIN) was concentrated through a concentrator spin6column
Viruses 2022, 14, x FOR PEER REVIEW
of 19 with(b). Purified rRTIN with (+) or without (−) HIV-1 protease treatment was subjected to Western blotafiltration
cutoff
of
50
kDa
(MCW
)
and
storedin
1
×SP
buffercontaining
300
mM
NaCl
at −80 ◦ C.
50
analysis using an anti-RT (left) or anti-IN (right) antibody.
(b). Purified rRTIN with (+) or without (−) HIV-1 protease treatment was subjected to Western blot
analysisusingofan
anti-RT(left) or Activities
anti-IN (right)
3.2.
EvaluationrRTIN
Enzymatic
duringantibody.
Reverse Transcription
The enzymatic activity of rRTIN was addressed by an in vitro reverse transcription
assay as described previously [21,22]. The levels of cDNA intermediates were monitoredby q-PCR using a specific primer set to amplify the R/u5, U3/u5, or U3/pbs region of theHIV-1 genome. Amplified products of R/u5, U3/u5, or U3/pbs reflect cDNA intermediatesof minus-strand strong-stop cDNA (-sscDNA), strand-transfer products of -sscDNA (1ststrand-transfer), or plus-strand strong-stop cDNA (+sscDNA), respectively [22]. Levels ofthese cDNA products could be used to monitor the RT enzyme activities of RNA-dependent DNA polymerase (RDDP), RNase H, and DNA-dependent DNA (DDDP) polymerase
activities (Figure 2a). The level of -sscDNA products (R/u5) was efficiently generated andaccumulated linearly from 30 min to 60 min of incubation, followed by sequential accumulation of the 1st strand-transfer products (U3/u5) and +sscDNA products (U3/pbs) (Figure 2b). These results demonstrated that purified rRTIN possesses all RDDP, RNase H,
and DDDP activities.

Figure 2. In vitro RT assay using rRTIN. (a) rRTIN was subjected to in vitro reverse transcription assay
Figure2. In vitro
RT assay[22].
using
rRTIN.
rRTIN
wasstopsubjected
to in vitro
reverse transcription
as described
previously
cDNAs
of (a)
minus
strong(−sscDNA),
strand-transfer
of -sscDNA (1stassay as described previously [22]. cDNAs of minus strong stop (-sscDNA), strand-transfer of sscDNA (1st strand-transfer) products, and plus strong stop (+sscDNA), generated by RNA-dependent DNA polymerase (RDDP), RNase H and DNA-dependent DNA polymerase (DDDP) activities, are schematically depicted. (b). At 30, 60, or 120 min after reaction, the levels of cDNA intermediates were determined by using the primer set for the R/u5, U3/u5, or U3/pbs region to mon-

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strand-transfer) products, and plus strong stop (+sscDNA), generated by RNA-dependent DNA
polymerase (RDDP), RNase H and DNA-dependent DNA polymerase (DDDP) activities, are schematically depicted. (b). At 30, 60, or 120 min after reaction, the levels of cDNA intermediates were
determined by using the primer set for the R/u5, U3/u5, or U3/pbs region to monitor RDDP, RNaseH
and DDDP activities. Each value shown is a copy number of a cDNA in 1 µL of the reaction mixture
at each time point.

3.3. Impact of IN Fusion on RT Functions
We directly compared RT activities of rRTIN and rRT in modified assay conditions
in which the amounts of the reaction substrates were varied to address differences between these proteins in detail. As the critical role of IN during HIV-1 virion morphogenesis through interaction with vRNA has been reported [15–17], we first compared-sscDNA (R/u5) levels generated by rRTIN or rRT under different concentrations of synthetic vRNA [22] (Figure 3a). In this experiment, the velocity of -sscDNA synthesis during
60 min of the reaction was measured; data are shown as values relative to the velocity at
100 ng vRNA as 1.0. From the velocity plot results, we estimated the affinity of rRTIN orrRT for vRNA (Km for vRNA) by calculating the vRNA concentration to give a relative
velocity of 0.5. The estimated Km value for vRNA was not significantly different betweenrRTIN and rRT (Figure 3b). We also found that rRTIN possessed the first strand-transfer(Figure3c) and +sscDNA synthesis (Figure 3d) efficiencies equivalent to those of rRT.7These
Viruses 2022, 14, x FOR PEER REVIEW
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results demonstrated that rRTIN possessed RDDP, RNase H and DDDP activities that were
almost equivalent to those of RTp66 under these experimental conditions.

Figure 3.
of of
RTRT
with
vRNA.
(a) Anreversetranscription
assay
Figure3. Impact
Impact ofofIN
INfusion
fusionononaffinity
affinity
withvRNA.
(a) inAnvitroin vitro
reversetranscription
was performedin the presenceof different
doses ofdoses
vRNA
1, 10,
1001,ng).
The ng).
R/u5
copy
numberassay
was performedin the presenceof differentof(0.1,vRNA(0.1,
10, 100
The
R/u5
copy
generated
during 60during
min incubation
was measured
and the value
each
doseatwas
plottedas a plotted
relative
numbergenerated
60 min incubation
was measured
andatthevalue
eachdose wasasa relative
reactionwith 100
vRNA
The
value
at eachvRNAvelocity
to thevelocity
reactiontooftherRTINwith of
100rRTIN
ng of vRNA
as ng
1.0.ofThe
valueasat1.0.
each
vRNA
dose
is shown
as
dose
± for
SD vRNA(n = 3).was(b)estimated
Km for vRNA
was estimatedcalculating
velocity atmeansis±shown
SD (n =as3).means(b) Kmby calculating
velocity byat vRNA(100 ng/reaction)vRNA (100 ng/reaction) as the maximum velocity in the assay condition. Student’s t analysis
showed no significant differences between RTIN and RTp66 (ns, p > 0.05). (c) The efficiency of the
1st strand-transfer was determined by calculating the relative (%) amount of the U3/u5 to that ofeach R/u5 product (% of [U3/u5]/[R/u5]) in the reaction with 100 ng vRNA). d. The efficiency of the+sscDNA synthesis was determined by calculating the relative (%) amount of the U3/pbs to that of

Viruses 2023, 15, 31

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as the maximum velocity in the assay condition. Student’s t analysis showed no significant differences between RTIN and RTp66 (ns, p > 0.05). (c) The efficiency of the 1st strand-transfer wasdetermined by calculating the relative (%) amount of the U3/u5 to that of each R/u5 product (% of[U3/u5]/[R/u5]) in the reaction with 100 ng vRNA). d. The efficiency of the +sscDNA synthesis
was determined by calculating the relative (%) amount of the U3/pbs to that of each U3/u5 product(% of [U3/pbs]/[U3/u5]) in the reaction with 100 ng vRNA. (c,d) Student’s t analysis showed nosignificant differences (ns) between RTIN and RTp66 (n = 3, p > 0.05).

3.4. Impact of IN Fusion on Affinity of RT to dNTPs
Cellular levels of deoxyribonucleoside triphosphates (dNTPs) are critical determinants
Viruses 2022, 14, x FOR PEER REVIEWfor retrovirus reverse transcription and infection [5]. We next examined -sscDNA generation
8 of 19

under different concentration of dNTPs with a fixed amount of vRNA (100 ng per reaction).
The velocity of R/u5 (-sscDNA) synthesis during 60 min of the reaction was measured, anddataalso
are examinedshown as values
relativeto thevelocity
100 µMwith
dNTPs
as 1.0.
Here, wenoticedwasin parallel.
When
rRTIN
wasattreatedHIV-1
protease,
efficient
that
rRTIN
produced
R/u5
products
withsignificantly
higherefficiency
at
low
doses
synthesis of -sscDNA at low doses of dNTPs (1 or 10 μM) was severely abrogated (FigureofdNTPs(1 or 10 µM)
with4a). We also
examinedtoa theheterodimer
4c).
The estimatedKmcomparedvalues forthe rRTp66
dNTPs (Figure
were increased
significantly,
level of
form
of
rRT
(p66/p51)
inparallel.
Fromthevelocityplot
results,
weestimatedthethatKmrRT (p66/p51), by protease treatment of RTIN (Figure 4d). These data demonstrated
value
of
rRTIN
or
rRTs
for
dNTPs
by
calculatingaconcentration
of
dNTPs
to
give
a
relative
the fusion of IN to RT is critical to the exertion of a stimulatory effect on RDDP activity at
velocityof 0.5.
It should be noted that the Km value of rRTIN was more than ~6-fold lower
lower
doses
of dNTPs.
than that of rRTp66 or rRTp66/p51, suggesting that rRTIN possesses a significantly higher
affinity to dNTPs than rRTs (Figure 4b).

Figure4.4.Impact
ImpactofofIN
INfusion
fusiononon affinity
affinity of
inin
thethepresence
Figureof RT
RT to
todNTPs.
dNTPs.(a)(a)AAreaction
reactionwas
wasperformed
performed
presofdifferent
doses
of
dNTPs
(0.1,
1,
10,
100
µM)
with
a
fixed
vRNA
dose
(100
ng/reaction)andence of different doses of dNTPs (0.1, 1, 10, 100 μM) with a fixed vRNA dose (100 ng/reaction) RTIN
andor RTs.
copy copy
numbergenerated
duringduring
60 min60incubation
was measured,and theand
value
RTIN
or TheRTs.R/u5
The R/u5
numbergenerated
min incubation
was measured,
theat
value
each
dose
was asplottedas a relative
velocitytorelative
to thewith
reactionwith
μMas
of1.0.
dNTPs
as 1.0.at
each atdose
wasplotteda velocity
the reaction
100 µMof 100dNTPs
The value
The value at each dNTP dose is shown as means ± SD (n = 3). (b) The Km for dNTPs was estimatedby calculating velocity at 100 μM dNTPs as the maximum velocity in the assay condition. The Km
value of each protein is indicated on the top of each bar. Student’s t analysis showed significant
differences between RTIN and either RTp66 or RTp66/p51 (** p > 0.01, n = 4). (c) Effect of proteasecleavage of RTIN. rRTIN with or without digestion with HIV-1 protease was subjected to reaction,and data are plotted as described for a. (d) The Km for dNTPs was estimated by calculating velocity
at 100 μM dNTPs as the maximum of velocity in the assay condition. The Km value of each protein

Viruses 2023, 15, 31

8 of 18

each dNTP dose is shown as means ± SD (n = 3). (b) The Km for dNTPs was estimated by calculating
velocity at 100 µM dNTPs as the maximum velocity in the assay condition. The Km value of eachprotein is indicated on the top of each bar. Student’s t analysis showed significant differences between
RTIN and either RTp66 or RTp66/p51 (** p > 0.01, n = 4). (c) Effect of protease cleavage of RTIN.
rRTIN with or without digestion with HIV-1 protease was subjected to reaction, and data are plotted
as described for a. (d) The Km for dNTPs was estimated by calculating velocity at 100 µM dNTPs as
the maximum of velocity in the assay condition. The Km value of each protein is indicated on the
top of each bar. Student’s t analysis showed significant differences between rRTIN with and without
protease treatment (** p > 0.01, n = 4).

To address the crucial role of IN fusion to RT, the effect of proteolytic cleavage of
RTIN by HIV-1 protease was addressed. Since HIV-1 protease produced a heterodimeric
form of RT composed of p66 and p51subunits, rRT in the heterodimeric form (p66/p51)
was also examined in parallel. When rRTIN was treated with HIV-1 protease, efficientsynthesis of -sscDNA at low doses of dNTPs (1 or 10 µM) was severely abrogated (Figure 4c).
The estimated Km values for the dNTPs were increased significantly, to the level of rRT(p66/p51), by protease treatment of RTIN (Figure 4d). These data demonstrated that thefusion of IN to RT is critical to the exertion of a stimulatory effect on RDDP activity at lower
doses of dNTPs.
3.5. Effects of RTIN on RT and IN Inhibitors
The experiments above demonstrated that RT RDDP activity is stimulated in RTIN
form, most probably by inducing a conformational change of RT that might confer increased
affinity to dNTPs. We next examined the drug sensitivity of RTIN against an RT or IN
inhibitor by measuring -sscDNA generation under different concentrations of efavirenz(EFV) or raltegravir (RAL). The levels of R/u5 products were measured and are shownas values relative to the level with solvent (DMSO) only as 1.0 (Figure 5a). In comparison
with RTp66, we found that RTIN showed lower sensitivity to inhibition by EFV. At 1 nM of
EFV, where more than 90% of RDDP was inhibited for RTp66, no apparent inhibitory effectwas detected for RTIN. The effective concentration 50 (EC50 ) of EFV to RTIN or RTp66
was calculated to be 12.0 ± 3.16 nM or 0.507 ± 0.01 nM, respectively (Figure 5b). This
result demonstrated that the drug sensitivity of EFV on RTIN was reduced significantly, by
23.7-fold, compared to RTp66. In contrast, no apparent inhibition by RAL was detected
for RTIN and RTp66 up to 100 µM, indicating that the catalytic activity of IN might not be
involved in the RDDP activity of RTIN or insufficient interaction of RAL with RTIN. We
also examined another non-nucleoside RT inhibitor, nevirapine (NEV), and a nucleoside RT
inhibitor, 3’-Azido-2’,3’-dideoxythymidine-5’-triphosphate (AZT-TP). The inhibitory effects
of NEV and AZT-TP on RTIN or RTp66 were similarly examined (Figure 5b). Although
the magnitudes of the effect of RTIN compared to RTp66 were diverse, RTIN was again
found to significantly affect the EC50 of EFV and AZT-TP. The differences in the degrees ofeffect among RTIs reflect that the RT conformational change introduced by IN fusion might
induce local changes near the RT catalytic center. As noticed regarding the effect of RTIN
on affinity to dNTPs (Figure 4), we observed that protease treatment of rRTIN significantly
increased sensitivity to EFV (Figure 5c), demonstrating, here again, an allosteric regulationof HIV-1 RT conformation through RTIN form.

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9 of 18

Figure5.5.Effect
Effectof IN
fusionon sensitivity
RT inhibitors.
An inreverse
vitro reverse
transcription
Figureof IN
fusionon sensitivity
to RTtoinhibitors.
(a) An(a)in vitrotranscription
assay
assay
was
performedinthepresenceofdifferent
concentrationsofefavirenz(EFV)or
raltegravir
was performed in the presence of different concentrations of efavirenz (EFV) or raltegravir(RAL).
(RAL).ofLevelsof R/u5 products
were measured
and arevaluestorelative
to withthe level
withLevelsR/u5 products
were measured
and are shownasshown
values as
relative
the levelsolvent
solvent(DMSO)
only
as
1.0.
(b)
The
effective
concentration
50
(EC
)
of
EFV,
nevirapine(NEV),(DMSO) only as 1.0. (b) The effective concentration 50 (EC50) of EFV, nevirapine(NEV), or 3‘-Azido-or
50002′,3‘-dideoxythymidine-5′-triphosphate(AZT-TP)
for
RTIN
or
RTp66
was
determinedby calculat-by
3‘-Azido-2 ,3‘-dideoxythymidine-5 -triphosphate (AZT-TP) for RTIN or RTp66 was determined
ing
the
drug
concentration
toinhibit50%
ofRDDP
activity
relativetocontrol.
Student’s
t analysis t
calculating the drug concentration to inhibit 50% of RDDP activity relative to control. Student’s
showedsignificant
differences
between
rRTIN
and
RTp66
(**
p
>
0.01,
*
p
>
0.05,
n
=
3).
(c)
The
analysis showed significant differences between rRTIN and RTp66 (** p > 0.01, * p > 0.05, n = 3). effect(c) Theof cleavage by HIV-1 protease on the EFV sensitivity of RTIN was examined as described in a. The
effect of cleavage by HIV-1 protease on the EFV sensitivity of RTIN was examined as described in a.
EC50 of EFV for rRTIN with or without pretreatment with HIV-1 protease is shown. Student’s t analThe EC50 of EFV for rRTIN with or without pretreatment with HIV-1 protease is shown. Student’s
ysis showedthat rRTIN had significant effect on EC50 by pretreatment with HIV-1 protease (** p >t analysis
0.01,
n = 3). showed that rRTIN had significant effect on EC50 by pretreatment with HIV-1 protease(** p > 0.01, n = 3).

3.6. MD Simulations of RT and RTIN Proteins
HIV-1 IN contains three structural domains: NTD, CCD, and CTD [37]. To address the
To gain structural
insights
impacts ofIN fusionon thebiochemical
activities
contributionof IN domains
tointothe thestimulatory
effectsof RDDP
activity,rRT carrying
IN
ofNTD
RT, weconducted
MD
simulations
of
RT
and
RTIN
proteins.
MolecularmodelsRTIN(residues 1–55, rRTIN55 ) or NTD and CCD (residues 1–212, rRTIN212 ) were ofpreparedfusionproteinandtheir
RT RDDPproteinactivityof the levels
HIV-1were
NL4-3 clone were constructed as described in(FigureS2a) andevaluated under different concentrations
Materials
andMethods(Figure6a)andsubjected
MDrRTINsimulations
using Amber 16 [32].
of dNTPs (Figure S2b). Although both rRTIN55toand
212 retained a certain abilityThe
MD simulation
is aatcomputational
methodtoKm
delineate
behaviorsof biologto generate-sscDNA
1 or 10 µM dNTPs,
thevaluesdynamic
for dNTPs
were significantly
ical macromolecules under thermal motions of atoms and molecular collisions in solution.

Viruses 2023, 15, 31

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Viruses 2022, 14, x FOR PEER REVIEW

11 of 19higher than those for rRTIN. Similarly, RTIN55 and rRTIN212 showed increased sensitivity
to EFV with lower EC50 values compared to rRTIN (Figure S2c). These results suggest thatthe full-length
form
of appliedIN with tothreedomains wasnecessary
to induce
sufficientofallostericThis techniquethus hasbeencharacterize
in silicothe physicalproperties
effectsonRT
enzymatic
functions.
biomolecules near the physiological conditions [38].
Structural dynamics during the simulations were evaluated with RMSD between the
3.6. MD Simulations of RT and RTIN Proteins
initial model structure and the structures at given time points. The RMSD of RT reached
Toimmediately
gain structural
insights
intoofthe
impacts ofwhereas
IN fusion
onofthebiochemicala near plateau
after
the start
simulation,
that
RTIN
reached aactivities
of RT,after
we conductedMD
simulationsof RT6b).
andThese
RTINresults
proteins.
Molecularmodelsnear plateau
200 ns of thesimulation(Figureindicatethat theRTINof RTIN
fusionproteinandRT
protein
of
the
HIV-1
clone
were
constructed
as
in
NL4-3
fusion protein requires more time to fold as a thermodynamically stable conformationdescribedandMaterials
andMethods(Figure6a)andsubjected
toMD
simulations
using
Amber
16
[32].
suggest that both RT and RTIN structures reached a state of thermodynamic equilibrium
Theconditions
MD simulation
is a200
computational
method to delineate
dynamic
behaviorswithof biologicalin solution
aroundns of MD simulations.
These results
are consistentmacromolecules
underthermalmotions
of
atoms
and
molecular
collisions
in solution.
an analysis of the areas of exposed molecular surfaces of RT and RTIN in solution, whereThis
technique
thus
has
been
appliedtocharacterizeinsilicothephysicalproperties
ofonly the exposed surface of RTIN gradually decreased during MD simulations (Figure 6c).
biomolecules near the physiological conditions [38].

6. MD simulations
RT andRTIN proteinsof theHIV-1clone.
The
aminoacid sequence
NL4-3
Figure 6.Figure
MD simulationsof RT andofRTIN
proteinsof the HIV-1
NL4-3
clone.
The
amino
acid
sequenceof theRTINofportion
the Polof themolecularclone[24]towas
used toof the RTIN
portionthe Polof
regionof region
the HIV-1
NL4-3HIV-1
molecular[24] wasusedconstructanconstruct
NL4-3 clone
RTIN modelby theAlphaFold2[23].program
The obtainedmodel gavea per-residue
an RTINmodel
by the program
AlphaFold2[23]. RTIN
The obtained
RTIN
model gaveconfia per-residue
dence score(pLDDT)of 83.3,
in theof“Confident”[23]. AnRT modelwastheconfidence
score(pLDDT)
83.3, in the range“Confident”
range[23]. An
RTconstructed
model was withconstructedwith
RT domain
of
the
RTIN
model
by
deletingtheINdomainusing
MOE(Chemical
ComputingGroup,
the RT domain of the RTIN model by deleting the IN domain using MOE (Chemical Computing
Montreal,Group,
Quebec,Canada).
The RTCanada).
and RTIN
subjected
to MD
simulations
using
Montreal,
Quebec,Themodels
RT andwere
RTIN
models were
subjected
to MD
simulations
modules in Amber 16 [32] as described previously [28,29,31]. (a) Initial models of RT and RTIN
using modules in Amber 16 [32] as described previously [28,29,31]. (a) Initial models of RT and RTIN
fusion protein before MD simulations. (b) Structural dynamics of RT and RTIN fusion protein durfusion protein before MD simulations. (b) Structural dynamics of RT and RTIN fusion protein during
ing MD simulations. RMSDs between the structure of the initial model and those at given time
RMSDs
betweenthe structureof theinitial modeland during
those atsimulations.
given time points ofpoints ofMD
MDsimulations.
simulation wereused
to monitor
the overall
structuralchangesMDofsimulation
were used
to monitor
simulations. (c) Analysis(c) Analysis
areas of exposed
surfacesof RTtheandoverall
RTIN structural
in solutionchanges
during during
MD simulations.
of areas of exposed surfaces of RT and RTIN in solution during MD simulations.

3.7. Structural Impacts of IN Fusion on RT Active Center
The RT and RTIN structures at 500 ns of MD simulations were used to characterize
the structural impacts of IN fusion on the RT active center (Figure 7a). Notably, the fingers
and thumb subdomains of RT were positioned more closely in RTIN than RT via

Viruses 2023, 15, 31

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Structural dynamics during the simulations were evaluated with RMSD between theinitial model structure and the structures at given time points. The RMSD of RT reacheda near plateau immediately after the start of simulation, whereas that of RTIN reached anear plateau after 200 ns of the simulation (Figure 6b). These results indicate that the RTIN
fusion protein requires more time to fold as a thermodynamically stable conformation andsuggest that both RT and RTIN structures reached a state of thermodynamic equilibrium insolution conditions around 200 ns of MD simulations. These results are consistent with ananalysis of the areas of exposed molecular surfaces of RT and RTIN in solution, where only
the exposed surface of RTIN gradually decreased during MD simulations (Figure 6c).
3.7. Structural Impacts of IN Fusion on RT Active Center
The RT and RTIN structures at 500 ns of MD simulations were used to characterize
the structural impacts of IN fusion on the RT active center (Figure 7a). Notably, thefingers and thumb subdomains of RT were positioned more closely in RTIN than RT via
configurational changes in the thumb subdomain (Figure 7b). Consequently, noncovalentattractive interactions, such as hydrogen bonds, formed frequently between the thumb andfingers subdomains during MD simulations only with the RTIN fusion protein (Figure 7c).
Such a hydrogen bond formation was not detected in the RT monomer lacking IN (Figure 7c,
left panel). The intimate interactions between the thumb and fingers subdomains in RTIN
were not permanent, and completely abolished once during 500 ns of MD simulations(Figure 7c, RTIN arrowhead). These results suggest that IN fusion into the C-terminal endof RT can allosterically influence the dynamic aspect of intramolecular interactions between
the thumb and fingers subdomains of RT in cis.
Structural fluctuations of biological molecules in solution play crucial roles in molecular interactions [39–41] and thus in the biological phenotypes of viruses [28]. To gain
insights into the possible impacts of IN fusion on fluctuations in the interaction surfacesof RT, we compared structural fluctuations of individual amino acid residues in the RT
domain between RT and RTIN models by using RMSF as a quantitative indicator. The
RMSF has been used to clarify cis-allosteric effects of genetic mutations [28,42–44] andeffects of molecular interactions [30,31] on the physical properties of the interaction surfaces
on the viral biomolecules. Basically, the magnitudes of the fluctuations were augmented
in most of the amino acid residues in RTIN as compared with those in RT (Figure 8a, redline). Importantly, sites heavily influenced by IN fusion, with RMSF increases above 1.4 Å,
contained 113D and 114A residues for dNTP binding [45] and NRTI binding [46] on thepalm subdomain, and contained the β7/β8 loop on the fingers subdomain (136N, 137N,
138E, 139T, 140P and 141G) (Figure 8a). Fluctuations in NNRTI binding pockets on the palm
subdomain [46,47] were also augmented with an RMSF increase of 0.8-1.0 Å (Figure 8a).
On the other hand, changes in the RMSF of RNA template binding regions, composed of
the fingers, thumb, and palm subdomains, were mild and increased between 0.1–0.4 Å(Figure 8b). Figure 8b summarizes the three-dimensional locations of the influenced sites
with an RMSF increase above 1.4 Å (red circle) along with those of amino acid residues
involved in binding to natural substrate and inhibitors. These results suggest that INfusion into RT can influence the structural dynamics of the polymerization active centerand inhibitor binding sites of RT in cis.

(Figure 7c, left panel). The intimate interactions between the thumb and fingers subdomains in RTIN were not permanent, and completely abolished once during 500 ns of MD
simulations (Figure 7c, RTIN arrowhead). These results suggest that IN fusion into the Cterminal end of RT can allosterically influence the dynamic aspect of intramolecular interactions between the thumb and fingers subdomains of RT in cis.
Viruses 2023, 15, 31

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Figure 7. Effects of IN fusion on conformation of RT protein. Structures obtained from the MD
simulations
6 wereused to characterize
impactsof RTINobtained
fusion on
RT. the(a) MD
Comparison
Figure
7. Effects inof FigureIN fusion
on conformationof RT protein.
Structuresfromsimbetween
overall6 structures
and RTIN fusion
proteinat 500ns ofonMD(b) Enlarged
ulations
in Figurewere usedoftoRT
characterize
impactsof RTIN
fusionRT.simulations.
(a) Comparison
beviewof active
center of RT
RTRTINand RTIN
protein500
of MD
simulations.
(c) Numbers
tween
overallstructures
RT in
and
fusionfusion
proteinat 500atns
ofns
MD
simulations.
(b) Enlarged
viewofofhydrogen
active centerof RT
in RTbetween
and RTIN
at 500subdomains
ns of MD simulations.
(c) Numbers
bonds
formedthefusion
thumbprotein
and fingers
of RT during
500 ns of MD
of hydrogenbonds
formedbetween
the
thumb
and
fingers
subdomainsofRT
during500
nsof MD
simulations. The trajectory files during 500 ns of MD simulations were used to calculate thenumbersimulations.
The
trajectoryfilesduring500
nsofMD
simulations
were
used
to
calculatethenumberof hydrogen bonds between the fingers and thumb subdomains in the RT region using the cpptraj
module in AmberTools 16 [32].

Viruses 2022, 14, x FOR PEER REVIEW
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13 of 18

Figure 8. Effects of IN fusion on fluctuations of RT protein. RMSF values, which indicate the atomic
Figure 8. Effects of IN fusion on fluctuations of RT protein. RMSF values, which indicate the atomic
fluctuationsofofthethemainmainchainschainsofofindividual
individualamino
aminoacids
acidsduring
duringMD
MDsimulations,
simulations,werewerecalculated
calculated
fluctuations
using
50,000snapshots
intheequilibrium
statesduring
the
last
100
ns
of
MD
simulations
using 50,000 snapshots in the equilibrium states during the last 100 ns of MD simulations usingusingthethe cpptraj
modulein AmberToolsa trajectory
analysistool. [32].
(a) Distributionsof RMSF
cpptrajmodulein AmberTools
16, a 16,
trajectory
analysistool [32](a) Distributionsof RMSFin RT.in
RT. Numbers
onhorizontal
the horizontal
axes indicate
positionsthe matureof HIV-1
NL4-3(GenBank
Numbers
on theaxes indicate
positionsin theinmature
RT ofRT
HIV-1
NL4-3(GenBank
accession
no. no.
AAK08484)
[24].
P1 P1
andand
P2,P2,
palm
subdomain;
F1F1
and
F2,F2,
fingers
subdomain;
accession
AAK08484)
[24].
palm
subdomain;andfingers
subdomain;T,T,thumb
thumb
subdomain;
subdomain;C,C,connection
connectionsubdomain.
subdomain.(b)(b)Overall
Overallview
viewofofRT
RTactive
activecenter.
center.The
Theredredshadedshadedareas
areas
indicateregions
where
RMSF
increases
to
above
1.4
Å
by
IN
fusion.
Lightyellow;
YMDD
indicate regions where RMSF increases to above 1.4 Å by IN fusion. Light yellow; YMDDmotif
motifofof
polymerase. Light. Lightpolymeraseactive
activecenter
center[45,48][45,48].
Lightblue;
blue;region
regioninvolved
involvedinindNTP
dNTPbinding
binding[45][45].
Lightgreen;green;
region. Light
regionofofRNA
RNAtemplate
templatebinding
binding[45][45].
Lightpurple;
purple;region
regionof
ofNNRTI
NNRTIresistanceresistance [45][45]..

Discussion
4.4.Discussion
Ourprevious
previousfindings
findingssuggested
suggestedthat
thatHIV-1
HIV-1RT
RTand
andIN
INneedneedtotobe
besupplied
suppliedininthe
the
Our
RTIN
fusionform
forefficient
reversetranscription
tooccur
within
cells
[20],
which
in
turn
RTIN fusion form for efficient reverse transcription to occur within cells [20], which insuggested
the criticalrole of
INof
forIN
HIV-1
reversereverse
transcription
throughthe RTIN
turnsuggested
the criticalrole
for HIV-1
transcription
through
theform.
RTINInthepresent
study,
wedemonstrated
theimpactsofINfusiononRTactivities
andsensitivity
form. In the present study, we demonstrated the impacts of IN fusion on RT activities andto RT inhibitors, and provided the first evidence of the cis-regulation of IN, which occurs
most probably by inducing a conformational change near RT catalytic sites.

Viruses 2023, 15, 31

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Using in vitro reverse transcription assay, we demonstrated that IN fusion at the Cterminal end of RT could markedly alter the biochemical properties of RT, i.e., the dNTP
concentration dependence of reverse transcription (Figure 4) and the sensitivity to RT
inhibitors targeting the polymerization active center (Figure 5). These results strongly
suggest that IN fusion can regulate the biochemical phenotype of HIV-1 RT. Previous
studies have suggested the presence of trans-acting roles of IN in RT enzymatic functions bystimulating RT processivity [49,50]. Although impact of RTIN on RT processivity remains
to be determined, this is the first evidence that HIV-1 IN can regulate RT activity in cis.
Concentrations of dNTPs vary depending on cell type and proliferation status [51]. For
human primary T cells, major targets of HIV-1 infection, the concentration of dNTPs wasestimated to be 1–5 µM in activated status [52]. In monocyte-derived macrophages, anothertarget of HIV-1 infection, a much lower concentration was estimated. HIV-1 infection
depends on the activation status of T cells [3] or macrophages [4]. Comparative analysis ofrRTIN, rRTs, and protease-cleaved rRTIN revealed that the fusion of IN to RT significantly
stimulated cDNA generation at low concentrations of dNTPs, i.e., less than 10 µM, whichcorresponds to the concentrations in activated T cells. Therefore, the superior RDDP
activity of rRTIN at lower concentrations of dNTPs might have physiological significancefor reverse transcription in the context of HIV-1 infection. Analysis of domain deletionmutants of RTIN showed that all three domains of IN are required for the full allostericeffect on RT (Figure S2). We noticed, however, that cDNA synthesis at low concentrationsof dNTPs was partly retained in rRTIN55 or rRTIN212, which, respectively, contain only
IN NTD or NTD-CCD.
Our in vitro experimental data indicate that IN-fusion on RT can remotely influence
the structural properties of the RT active center consisting of binding pockets to dNTP and
RT inhibitors. To address this issue, we constructed RT and RTIN monomer models using
AlphaFold2 [23] and conducted MD simulations. The in silico studies indeed revealed
two types of marked structural impacts on the RT active center via IN fusion on RT:
configurational and dynamics effects. First, our MD simulations suggested that IN fusion
into RT can alter the configuration of the RT thumb subdomain, leading to the creationof weak de novo interactions between the thumb and fingers subdomains (Figure 7). In
combination with the palm subdomain, these subdomains constitute a polymerase active
center and participate in binding to the RNA template, dNTP and NRTI. Therefore, it
is possible that the IN-induced conformational changes in these subdomains primarily
alter the selectivity of substrate/NRTI and the efficiency of RT translocation, and thereby
eventually lead to changes in its polymerization activity and/or in the NRTI susceptibilityof RT. Our MD simulations also suggest that IN fusion can alter the fluctuation profilesof amino acid residues involved in binding to dNTP and NNRTI (Figure 8). It should
be noted that the structural fluctuations of the interaction surfaces of biomolecules insolution are critical for the molecular interactions and thus for the overall function of thebiomolecules [28,30,31,39–41,43,53,54]. Therefore, as indicated in this study, it is possiblethat the alterations in the magnitudes of fluctuations in the RT active center influence thebinding efficiencies of the substrate and NNRTI to RT and contribute to changes in thebiochemical phenotype of RT.
Our MD simulations suggest that IN fusion to RT induced the augmentation ofamino acid fluctuations at both the dNTP and NNRTI binding sites of RT (Figure 8).
This seems puzzling because IN fusion was likely to induce the opposite effects on thepolymerization and NNRTI sensitivities of RT (Figures 4 and 5). These data suggest thatchanges in fluctuations at the substrate/inhibitor binding sites alone are insufficient to
induce IN fusion effects, and may imply that coordinated changes in other structuralproperties are necessary. Further study is necessary to clarify how the above two and/or
other undescribed structural impacts coordinately regulate biochemical phenotypes of RT.
Previous studies have shown that the interaction of HIV-1 RT and IN involves RT residue
Ile178 [55,56]. However, at least in our MD analyses, we did not detect possible interactionof RT residue Ile178 with IN. This discrepancy might be explained by different effects ofViruses 2023, 15, 31

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IN through cis and trans modes on RT structures. In addition, it remains to be clarified
whether IN fusion can influence the RT structure in the context of an RTIN multimer, whichmight be the functional entity for the polymerization in vivo. What is clear at present is that
IN fusion into the C-terminal end of the HIV-1 RT p66 subunit can allosterically regulate
the structural properties of the polymerization active center and inhibitor binding site at
the monomer level.
Within the HIV-1 virus particle, some portions of RT and IN were kept in fused (RTIN)
form, although the bulk of the Pol precursor was cleaved into individual forms by viral protease [20]. Indeed, rRTIN was proteolyzed by HIV-1 protease quite efficiently (Figure 1b).
Currently, the underlying mechanism that keeps the RTIN form by avoiding complete
proteolysis by HIV-1 protease remains to be elucidated. One intriguing possibility is thatsome portion of intermediate products of the Pol precursor might associate with vRNA,
which makes the Pol precursor resistant to cleavage by HIV-1 protease. In some retroviruses (α-retrovirus), RT enzyme is composed of a heterodimer of RT (α) and incompletely
processed RTIN (β) subunits in the avian leukosis viruses [57]. Interestingly, the β subunitof the α-retrovirus RT has many allosteric properties, including specific binding to tRNAtrp
primer and controlling IN-mediated DNA endonuclease activity [58]. Our present data
might reflect allosteric regulation of HIV-1 RT by IN, which has been noticed for the βsubunit of the α-retrovirus RT.
Although our present data demonstrated that RTIN has enzymatic superiority over RT
in RDDP activity in low dNTPs, the cleaved form of RT (p66/p51) might have critical roles
in completing reverse transcription where higher concentrations of dNTPs are available,
such as in nuclear compartments. Since integration catalyzed by IN is also a critical step toretrovirus infection following reverse transcription of a retrovirus genome, IN enzymatic
activity in RTIN form is another important issue to address. However, we could not detect
any significant integrase activity of rRTIN. In this regard, it seems likely that IN that isalready cleaved in a virus particle might play a main role in integration. Alternatively,
RTIN might be cleaved into IN by cellular protease during the early events of HIV-1
replication. We favor the latter possibility, since our previous study showed that transcomplementation of RT and IN separately did not rescue HIV-1 lacking RT and IN [20],suggesting that retroviral reverse transcription and integration might be tightly connectedto each other. Recent studies using imaging and biochemical assays to track HIV-1 capsids
and virus gRNA suggested that uncoating and reverse transcription may occur in nuclear
compartments [59–61]. These studies led us to revisit our current understanding of HIV-1
early replication events [62]. Reverse transcription and integration might be connected toeach other in a heretofore unknown manner. The RTIN functions elucidated in the present
study would serve as a platform upon which to understand HIV-1 early replication eventsand develop a novel anti-retrovirus strategy.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v15010031/s1, Figure S1: SEC profiles of rRTIN; Figure S2:Contribution of IN domains on RTIN functions.
Author Contributions: Conceptualization, T.M. and H.S.; methodology, T.M, Y.A, G.K. and O.K.;
software, O.K. and M.Y.; validation, T.M., O.K., M.Y., Y.A., G.K. and H.S.; formal analysis, T.M., O.K.
and Y.A.; investigation, T.M., O.K. and Y.A.; resources, T.M., O.K. and Y.A.; data curation, T.M., O.K.,
M.Y., G.K. and H.S.; writing—original draft preparation, T.M., O.K. and H.S.; writing—review andediting, T.M., O.K., M.Y., Y.A., G.K. and H.S.; visualization, T.M., O.K. and H.S.; supervision, T.M.,
G.K. and H.S.; project administration, T.M.; funding acquisition, T.M., O.K. and G.K. All authors haveread and agreed to the published version of the manuscript.
Funding: Pl This work was supported by grants from the Japan Agency for Medical Research andDevelopment, AMED (Research Program on HIV/AIDS: JP21fk0410041 and JP22fk0410041 to T.M.,
O.K. and G.K.) and from JSPS KAKENHI (Grant Number JP20K07525 to T.M.).
Institutional Review Board Statement: Not applicable.

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Informed Consent Statement: Not applicable.
Data Availability Statement: Please contact the corresponding authors (T.M. and O.K.) for data on
the present study.
Acknowledgments: We thank M. Hara and K. Katagiri for their technical assistance. We also thanks
Y. Gu, S. Yamaoka and Y. Fujiyoshi for supporting our use of the experimental facilities of TMDU.
The MD simulations were carried out partly with the TSUBAME3.0 supercomputer at the Tokyo
Institute of Technology.
Conflicts of Interest: The authors declare no conflict of interest.

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