STRUCTURE AND ASSEMBLY

crossmAllosteric Regulation of HIV-1 Capsid Structure for Gag
Assembly, Virion Production, and Viral Infectivity by a
Disordered Interdomain Linker
Takaaki Koma,a Osamu Kotani,b Kei Miyakawa,c Akihide Ryo,c Masaru Yokoyama,b Naoya Doi,a Akio Adachi,d Hironori Sato,bMasako NomaguchiaaDepartment of Microbiology, Tokushima University Graduate School of Medical Science, Tokushima, Tokushima, Japanb

Laboratory of Viral Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japanc

Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Kanagawa, Japand

Department of Microbiology, Kansai Medical University, Hirakata, Osaka, Japan

ABSTRACT The retroviral Gag capsid (Gag-CA) interdomain linker is an unstructured
peptide segment connecting structured N-terminal and C-terminal domains. Although the region is reported to play roles in virion morphogenesis and infectivity,
underlying molecular mechanisms remain unexplored. To address this issue, we determined biological and molecular phenotypes of HIV-1 CA linker mutants by experimental and in silico approaches. Among the nine linker mutants tested, eight exhibited attenuation of viral particle production to various extents mostly in parallel witha reduction in viral infectivity. Sucrose density gradient, confocal microscopy, andlive-cell protein interaction analyses indicated that the defect is accompanied by attenuation of Gag-Gag interactions following Gag plasma membrane targeting in thecells. In silico analyses revealed distinct distributions of interaction-prone hydrophobic patches between immature and mature CA proteins. Molecular dynamics simulations predicted that the linker mutations can allosterically alter structural ﬂuctuations, including the interaction surfaces apart from the mutation sites in both theimmature and mature CA proteins. These results suggest that the HIV-1 CA interdomain linker is a cis-modulator of the CA interaction surfaces to optimize efﬁciency of
Gag assembly, virion production, and viral infectivity.
IMPORTANCE HIV-1 particle production and infection are highly ordered processes.

Viral Gag proteins play a central role in the assembly and disassembly of viral molecules. Of these, capsid protein (CA) is a major contributor to the Gag-Gag interactions. CA consists of two structured domains, i.e., N-terminal (NTD) and C-terminal(CTD) domains, connected by an unstructured domain named the interdomain
linker. While multiple regions in the NTD and CTD are reported to play roles in virion morphogenesis and infectivity, the roles of the linker region in Gag assemblyand virus particle formation remain elusive. In this study, we showed by biologicaland molecular analyses that the linker region functions as an intramolecular modulator to tune Gag assembly, virion production, and viral infectivity. Our study thus illustrates a hitherto-unrecognized mechanism, an allosteric regulation of CA structureby the disordered protein element, for HIV-1 replication.
KEYWORDS Gag-CA, HIV-1, assembly, interdomain linker, virus particle production

Citation Koma T, Kotani O, Miyakawa K, Ryo A,
Yokoyama M, Doi N, Adachi A, Sato H,
Nomaguchi M. 2019. Allosteric regulation of
HIV-1 capsid structure for Gag assembly, virion
production, and viral infectivity by a disorderedinterdomain linker. J Virol 93:e00381-19.
https://doi.org/10.1128/JVI.00381-19.
Editor Viviana Simon, Icahn School of
Medicine at Mount Sinai
Copyright © 2019 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Hironori Sato,hirosato@nih.go.jp, or Masako Nomaguchi,
nomaguchi@tokushima-u.ac.jp.
T.K. and O.K. contributed equally to this article.

G

ag precursor protein of retroviruses, including human immunodeﬁciency virus
type 1 (HIV-1), is a major viral structural protein and plays pivotal roles in both earlyand late phases of replication (1, 2). Correct assembly of immature viral particles by Gag
proteins, followed by the steps of budding and maturation, leads to proper formation

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Received 3 March 2019
Accepted 4 June 2019
Accepted manuscript posted online 12
June 2019
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Journal of Virologyof the viral core, which is essential for infectious virion production and virus replication.
HIV-1 Gag protein consists of four major domains, matrix (Gag-MA), capsid (Gag-CA),
nucleocapsid (Gag-NC), and p6 (Gag-p6), and two spacer peptides, SP1 and SP2. Eachdomain of HIV-1 Gag protein has an independent role during HIV-1 particle assembly:
Gag-MA anchors Gag proteins to plasma membrane (PM) through its N-terminal
myristoylation; Gag-NC is involved in packaging of viral genome RNA (gRNA), and theinteraction of Gag-NC with gRNA also has been suggested to be important for Gag
oligomerization; and Gag-p6 participates in the release of viral particles by utilizing
host’s ESCRT (endosomal sorting complex required for transport) machinery. Of the fourGag domains, Gag-CA is a main driver of Gag-Gag interactions required for HIV-1
particle assembly (1–7).
HIV-1 Gag-CA is composed of two independent structured domains, the N-terminal
domain (CA-NTD; residues 1 to 145) and the C-terminal domain (CA-CTD; residues 151
to 231), and a short ﬂexible peptide (interdomain linker; residues 146 to 150) that links
these domains (Fig. 1A) (1, 5, 7). The HIV-1 CA-NTD and CA-CTD contain seven and ﬁve
␣-helices (H1 to H7 and H8 to H12), respectively. Another helix, designated 310 helix(310H; residues 150 to 152), is located downstream of the linker region (Fig. 1B) (8, 9).
Immediately after 310H, a major homology region (MHR; residues 153 to 172) has been
mapped; this region is highly conserved among retroviruses and has been shown to be
critical for the assembly of retroviruses, including HIV-1 (Fig. 1) (10–13). Many studies
showed that mutations in Gag-CA have pleiotropic effects on various viral replication
steps (14–19). The Gag-CA mutants that decrease virion production or form aberrant
core displayed simultaneous drastic reduction in viral infectivity as determined bysingle-cycle infection assays using various indicator cell lines. The mutations that cause
defects in virion production were identiﬁed in H4 to H6 of the CA-NTD and in MHR and
H9 to H10 of the CA-CTD (19–22). Recent structural analyses of immature retroviralcapsids of HIV-1 and Mason-Pﬁzer monkey virus (M-PMV) by cryo-electron microscopy(cryo-EM) revealed the residues that are predicted to be involved in interhexameric
NTD-NTD contacts, interhexameric CTD-CTD contacts, and intrahexameric CTD-CTD
contacts formed by Gag-CA (23–25). Interestingly, virion production-defective mutations described above were located within and adjacent to the Gag-CA contact sites
revealed by structural analyses, suggesting that the residues/regions in both the
CA-NTD and CA-CTD are required for Gag assembly and subsequent virus particle
production (Fig. 1) (21).
Cryo-EM analyses have provided a highly ordered structural model for the Gag-CA
layer in retroviral immature particles that shows various interactions between the
CA-NTD and CA-CTD (24, 25). However, the cryo-EM density corresponding to the CA
linker region is partly visible for immature M-PMV capsid and can be seen only at lower
isosurface thresholds for immature HIV-1 particles. These results have suggested an
unstructured nature for the domain, and the observed ﬂexibility appears to work forensuring the correct arrangement of the CA-NTD and CA-CTD in HIV-1 mature capsids(9, 26, 27). Because the linker region of HIV-1 Gag-CA is inherently unstructured andﬂexible and is not located at the capsid surface, studies on its signiﬁcance in Gag-Gag
interactions and HIV-1 particle production have been limited. A previous study has
shown that although mutants of the HIV-1 Gag-CA linker region exhibit wild-type Gag
expression, processing, and progeny production, they are noninfectious, having defects
in conical core assembly and viral DNA synthesis (28). Based on these results, the linker
region of the HIV-1 Gag-CA has been concluded to be critical for proper core assemblyand stability (28). In addition, one of the serine residues within the domain (at position
149) has been shown to be a major phosphorylation site in HIV-1 Gag-CA (29–32). The
alanine substitution of S149 led to aberrant core assembly or impaired core stability (28,29, 31, 32), whereas it did not appear to affect viral particle production (29, 30). In
contrast, for Moloney murine leukemia virus, insertion of amino acid residues into the
Gag-CA linker region, or fusion with HIV-1 sequence at the linker region, reduced viralparticle production (33). Thus, it remains unclear how mutations in the retroviral
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FIG 1 Structure and amino acid sequence of HIV-1 Gag-CA illustrating the positions of the interdomain linker
region and residues used for mutagenesis. (A) Three-dimensional (3D) locations of amino acid residues analyzed
in the present mutational study. The Gag-CA monomeric structure is derived from PDB code 4USN (25). A red
ribbon indicates ﬁve residues in the linker region (residues 146 to 150). Residues E75/E76 in helix 4 (H4) andK158/Y164 in the MHR, which are known to be important for Gag assembly (11, 19, 20), are also shown. These sites
were used as references for the present mutational analyses. (B) Alignment of HIV-1 and SIVmac Gag-CA sequences.
Amino acid sequences in Gag-CA of HIV-1NL4-3 (GenBank accession no. AF324493) and SIVmacMA239 (GenBank
accession no. M33262) were aligned by Genetyx version 14. Locations of helices (H) in this alignment are as
described previously (21), and the CypA binding loop, interdomain linker, 310 helix (310H) (8, 9), and MHR are
indicated. Amino acid sites to which mutations were introduced in this study are indicated by red letters.

Gag-CA linker region, including that of HIV-1, inﬂuence Gag assembly and progeny
production.
In this study, we mainly focused on deﬁning the role of the HIV-1 Gag-CA linker
region in Gag assembly and viral particle production at the late viral replication phase.
To better understand molecular events associated with the interdomain linker, we
performed in vitro experimental and in silico structural analyses. Here we provide
evidence for critical involvement of the HIV-1 Gag-CA linker region in Gag-Gag interaction/Gag assembly, virus particle production, and viral infectivity.
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TABLE 1 Characterization of HIV-1 CA mutants in this study
Parent and mutant
clones
NL4-3
EE75/76AA
K158A
Y164A
S146A
P147A
T148A
S149A
I150A
S146N
S149N
S149D
S149K

Location ofsubstitutions
None
NTD helix 4
CTD MHRCTD MHR
LinkerLinkerLinkerLinkerLinkerLinkerLinkerLinkerLinkerGrowth in
H9 cellsa,b
⫹⫹
–
–
–
⫹
⫹
⫹
–
–
–
⫹
–
–

Infectivity in
TZM-bl cellsb,c1.00 ⫾ 0.00
0.08 ⫾ 0.04
0.07 ⫾ 0.04
0.07 ⫾ 0.04
0.67 ⫾ 0.03
0.66 ⫾ 0.06
0.62 ⫾ 0.07
0.06 ⫾ 0.01
0.07 ⫾ 0.04
0.13 ⫾ 0.03
0.99 ⫾ 0.09
0.00 ⫾ 0.00
0.00 ⫾ 0.00

Virus particle productionin H9 cellsb,c1.00 ⫾ 0.00
0.10 ⫾ 0.06
0.12 ⫾ 0.02
0.04 ⫾ 0.02
1.02 ⫾ 0.10
0.45 ⫾ 0.08
0.79 ⫾ 0.02
0.12 ⫾ 0.01
0.09 ⫾ 0.02
0.53 ⫾ 0.09
0.46 ⫾ 0.05
0.09 ⫾ 0.01
0.31 ⫾ 0.08

aVirus growth properties are summarized based on the results of repeated infection experiments. ⫺, nodetectable virus growth in the test period (15 days); ⫹, retarded virus growth relative to NL4-3; ⫹⫹,positive-control NL4-3.
bInput virus amounts were normalized by the virion-associated RT activity produced from 293T cells
transfected with the indicated proviral clones. For the particle production assays, H9 cells were transfectedwith 2 ␮g of proviral DNA clones and cultured for 2 days in the presence of CXCR4 antagonist AMD3100.
cValues relative to those for NL4-3 are shown. Data presented are means ⫾ standard errors of three
independent experiments.

RESULTS
Mutations in HIV-1 Gag-CA linker region can affect both early and late replication phases. To elucidate how the HIV-1 CA linker region acts at early and latereplication stages, we conducted site-directed mutagenesis of the linker region by
regular alanine substitutions as previously described (28) (Table 1). A CA-NTD mutant(EE75/76AA) and two MHR mutants (K158A and Y164A), which were reported to display
a marked reduction in virus particle production and viral infectivity, were constructed
and used as controls (19, 21, 22). Viral growth ability overall and viral replication abilities
at early and late phases were monitored by multicycle replication ability in human T cellline H9, single-cycle infectivity in reporter cell line TZM-bl (early), and virus production
assay in transfected H9 cells (late), respectively. For infection experiments, input viruses
were normalized by virion-associated reverse transcriptase (RT) activity (RT units) toallow comparison of relative infectivity.
As shown in Table 1, all control mutants (EE75/76AA, K158A, and Y164A) exhibited
an undetectable growth ability in H9 cells, a drastic decrease in both infectivity (meanvalues: 0.07 to 0.08) and particle production (mean values: 0.04 to 0.12), as expectedfrom previous reports (19, 21, 22). Concurrent with the replication defects, thesemutants (EE75/76AA and K158A) have been shown to display an aberrant viral morphology including enlarged and irregularly shaped cores (19). Among ﬁve mutants
having single alanine substitutions in the linker region, the S149A and I150A mutants
exhibited a remarkably decreased viral infectivity and virus particle production andwere resultantly found to be noninfectious for H9 cells. The S146A mutant grew morepoorly than NL4-3 but the virus production level was similar to that of NL4-3, indicating
its only defect at the early replication phase. The P147A and T148A mutants displayed
a reduced viral replication potential in H9 cells, and both viral infectivity and virus
particle production were moderately decreased, indicating relatively mild mutational
effects at these sites. The above-described results for the linker mutants (S146A, T148A,
S149A, and I150A) were in line with the electron microscopic data previously described(28). Upon transfection, viral populations generated by infectious S146A and T148A
mutants contained viruses with conical cores at percentages similar to that observed
for the wild-type clone, whereas noninfectious S149A and I150A mutants produced
progenies with conical cores at a severely reduced ratio (28). Our results for the P147A
mutant were different from those previously reported (28), for unknown reasons. While
the P147A mutant was reported to be unable to produce virus particles as judged by
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RT assays, we readily detected RT activity in the supernatant ﬂuids of transfected cells,
and the supernatants were infectious for H9 cells, albeit poorly. The results describedabove indicate that the effects of single amino acid substitutions are dependent on thesite in the linker region.
To address effects of amino acid types to be substituted, we constructed four
additional mutants that have single substitutions speciﬁc to the Gag-CA protein ofsimian immunodeﬁciency virus (SIV) clone SIVmacMA239 (S146N and S149N) (Fig. 1B) or
those reported previously (S149D and S149K) (28–32). Unlike the S146A mutant, theS146N mutant displayed a striking reduction in viral infectivity and growth ability withmoderately decreased particle production (Table 1). Furthermore, unlike the S149A
mutant, the S149N mutant was found to display a moderately impaired phenotype,retaining viral infectivity comparable to that of HIV-1NL4-3 (Table 1). Meanwhile, theS149D and S149K mutants showed a marked reduction in virus particle production andinfectivity, as seen for the S149A mutant (Table 1). These results are consistent withprevious reports on the phenotypes of the S149A, S149D, and S149K mutants (28–32)
and indicate that the type of amino acid residues in the linker region is critical todetermine HIV-1 replication phenotypes. In sum, our results demonstrate that singleamino acid substitutions within the linker region of HIV-1 Gag-CA diminish virus particle
production in a site- and property-dependent manner.
Amino acid sequence in the Gag-CA interdomain linker is highly conserved
among HIV-1 subtype B viruses except for the T148 site. Amino acid variations in
the Gag-CA protein of HIV-1 subtype B were examined using the public HIV Sequence
Database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html) (n ⫽ 14,120)
with Shannon entropy scores as the indicator of amino acid variations at individual
positions (34–36). Scores obtained for individual sites ranged from 0.007 to 1.683 bits,with an average score of 0.16 bit, indicating that Gag-CA diversity in the subtype B
population is relatively small, as seen in many viral enzymes (36–38). This is in contrast
to virion surface proteins, for which entropy scores often exceeded 2.0 bits, reachingclose to a maximum value of 4.4 bits in the antigenic sites (34, 39, 40).
The entropy scores for amino acid residues at positions 146, 147, 149, and 150 in the
Gag-CA linker region were 0.041, 0.012, 0.106, and 0.012 bits, respectively (Fig. 2A). The
scores were as low as those for residues at positions 75 (0.194), 76 (0.025), 158 (0.012),
and 164 (0.011), which are critical for virion production (Table 1) (11, 19–22). The
entropy analysis clearly demonstrated that the interdomain linker is highly conserved
among HIV-1 subtype B strains and suggests the presence of strong constraints againstchanges in these amino acid residues during HIV-1 subtype B circulation and maintenance in nature. Notably, however, subtype B was found to accept variation at position
148, with an entropy score of 1.446 (Fig. 2A), and various residues, including T, V, A, G,
I, L, P, and S, were detected at this position (Fig. 2B, upper row). It is worth mentioning
here that T148 is important for optimal virus growth (Table 1), and the most dominantamino acid residues found at position 148 were different between subtype B alone andHIV-1 sequence populations, including group M (n ⫽ 25,222) (Fig. 2B, lower row). Our
results may imply that this position is variable to optimize growth ability of viruses
under the different CA backbone sequences. In total, amino acid residues in the linker
region were conserved or functionally important in the HIV-1 subtype B population,suggesting a signiﬁcant role for the interdomain linker in viral replication.
Mutations in the linker region (S149A and I150A) impede accumulation ofassembly intermediates formed by multimerized Gag proteins at the PM. It has
been reported that mutations in the amino acid residues affecting immature capsid
assembly have little effect on Gag expression itself (19, 21, 22). We assumed that the
reduction in virus particle production observed for the mutants of the Gag-CA linker
region may be associated with their Gag assembly defects. For veriﬁcation, we performed velocity sedimentation assays previously established (21, 22) to monitor the
intracellular assembly process of mutant Gag proteins. With this assay, we can distinguish several assembly intermediates as 10S (sedimentation value), 80S, 150S, 500S,
and 750S. The 10S intermediate contains newly synthesized Gag precursor proteins.
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FIG 2 Variation around the interdomain linker in HIV-1 CA. (A) Shannon entropy scores at individual aminoacid positions. Scores were calculated (34–36) using a total of 14,120 full-length capsid amino acid sequences ofHIV-1 subtype B from different geographic regions in the world (http://www.hiv.lanl.gov/content/sequence/HIV/
mainpage.html). The majority consensus sequence around the linker region is shown on the x axis along withcorresponding Shannon entropy scores on the y axis. An entropy score of 0 indicates absolute conservation,
whereas a score of 4.4 indicates complete randomness. (B) Types and frequencies of amino acid residues detectedin the linker region. Full-length capsid amino acid sequences of HIV-1 subtype B (n ⫽ 14,120) and group M(n ⫽ 25,222) from the HIV Sequence Database were used to analyze the types and frequencies of amino acidresidues at indicated positions in the linker region.

The 80S intermediate is a complex of Gag oligomer and gRNA, which targets the PM.
The 150S intermediate appears to be highly transient. Following binding of the 80S/
150S intermediates to the PM, 500S and subsequent 750S intermediates are formed byfurther Gag multimerization there. When cells expressing Gag mutant proteins withsome defects in the assembly process are examined by these sedimentation assays,
intermediates that precede the step at which assembly is blocked are readily detectable. For example, mutants with defects in 500S intermediate formation (i.e., failure in
Gag multimerization at PM) accumulate 10S to 150S intermediates (21, 22).
To analyze effects of the linker mutations on Gag assembly, we chose two alanine
substitution mutants (S149A and I150A) that exhibit remarkably reduced virus particle
production (Table 1). MHR K158A and Y164A mutants, which have been shown to bearrested at 80S intermediates in velocity sedimentation assays (22), were used ascontrols. To facilitate detection and quantiﬁcation of the immature Gag precursor
protein, we generated HIV-1NL4-3-derived constructs without Pol-protease and Env.
Parental (NL4-3ΔPro/ΔEnv) and mutant (S149AΔPro/ΔEnv, I150AΔPro/ΔEnv, K158AΔPro/
ΔEnv, and Y164AΔPro/ΔEnv) clones were transfected into HeLa cells, and at 24 h posttransfection, cells were lysed for velocity sedimentation assays (Fig. 3A). Steady-state
expression levels of Gag proteins in total cell lysates were similar among test samples.
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FIG 3 Effects of linker mutations on Gag assembly process as revealed by velocity sedimentation analysis.
Cell lysates prepared from transfected HeLa cells were ultracentrifuged through sucrose gradients for
sedimentation analysis (22), and fractions (F) were collected stepwise from centrifuge tubes (top tobottom) for Gag monitoring by the Western blotting method. Sedimentation coefﬁcients (S values) werealso determined. Results of three independent experiments with representative immunoblotting data areshown for comparison among the linker and MHR mutants (A) and for comparison between the linker
and NTD mutants (B). Immunoblotting data for Gag proteins in total cell lysates are presented at the topof each panel.

Parental NL4-3ΔPro/ΔEnv showed a small amount of intermediates at early Gag assembly steps (10S to 80S), and 500S/750S intermediates were readily detected. Consistent with a previous report (22), MHR mutants (K158AΔPro/ΔEnv and Y164AΔPro/ΔEnv)
showed defects in Gag multimerization on PM. While 150S to 750S intermediates wereundetectable, a much higher level of 10S to 80S intermediates accumulated for the
MHR mutants than for NL4-3ΔPro/ΔEnv. The linker mutants (S149AΔPro/ΔEnv and
I150AΔPro/ΔEnv) exhibited lower and higher accumulations of early 10S to 80S and late
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500S/750S intermediates, respectively, than did the MHR mutants. The linker mutants,
however, clearly displayed increased 10S to 80S intermediates, comparable 80S/150S
intermediates, and decreased 500S/750S intermediates relative to those of NL4-3ΔPro/
ΔEnv. These results strongly suggest that the mutations in the interdomain linker
impede Gag multimerization after the binding of 80S intermediates to the PM.
It has been shown that the assembly process of a CA-NTD mutant (EE75/76AA) isarrested at the 500S intermediates prior to 750S intermediates (21). Thus, in anotherseries of experiments, the linker and CA-NTD mutants were comparatively analyzed forformation of 500S/750S intermediates to see if there is any substantial difference
between the mutants. Velocity sedimentation assays were performed as describedabove using parental NL4-3ΔPro/ΔEnv and the mutants (S149AΔPro/ΔEnv and EE75/
76AAΔPro/ΔEnv). Since the Gag expression level for the EE75/76AAΔPro/ΔEnv mutant
in transfected cells was always higher than those for NL4-3ΔPro/ΔEnv and S149AΔPro/
ΔEnv (Fig. 3B), sample amounts for the sedimentation assays were adjusted to containequal Gag proteins. As shown in Fig. 3B, while Gag proteins of NL4-3ΔPro/ΔEnv at 24 h
posttransfection were predominantly detected in the 500S/750S fractions, the 500S
intermediates for the EE75/76AAΔPro/ΔEnv mutant were not observed (Fig. 3B). The
result for the EE75/76AAΔPro/ΔEnv mutant appeared to be different from that previously described (21) in that 500S intermediates were undetectable under this condition.
Robinson et al. (21) have reported that the EE75/76AA mutant displays a pattern of Gag
assembly intermediates similar to that of the wild type and that it exhibits inefﬁcient
progression of Gag assembly following 500S intermediate formation. When samples
prepared at 36 h posttransfection were examined, the 500S intermediates became
detectable in our system (Fig. 3B) as reported previously. The EE75/76AAΔPro/ΔEnv
mutant prepared at this timing exhibited accumulated 500S intermediates and substantially reduced 750S intermediates relative to those for NL4-3ΔPro/ΔEnv, suggesting
its arrest prior to 750S intermediates. Consistent with the previous report (21), thisresult showed that the EE75/76AAΔPro/ΔEnv mutant is impaired at the ﬁnal step of the
Gag assembly process, just before virus budding. It should be noted that an apparent
difference was also noted for the results for NL4-3ΔPro/ΔEnv (Fig. 3). The different
appearance of data obtained could be attributable to variations in Gag assembly
kinetics among experiments, possibly due to transfection efﬁciency, cell conditions, andso on. Nevertheless, in three independent experiments, amounts of the 10S/80S
intermediates were constantly increased in the linker mutants relative to those for
NL4-3ΔPro/ΔEnv, and those of the 500S/750S intermediates were always decreased in
the linker mutants relative to the wild type, as shown by the representative results (Fig.
3). These results suggested that the S149AΔPro/ΔEnv mutant, unlike the EE75/
76AAΔPro/ΔEnv mutant, is impeded in formation of 500S intermediates, i.e., Gag
multimerization on the PM, compared with NL4-3ΔPro/ΔEnv. It is conceivable that thelinker mutations affect the ability of Gag to assemble to a lesser extent than the MHR
mutations, because a relatively high level of 500S/750S intermediates was seen for the
S149AΔPro/ΔEnv and I150AΔPro/ΔEnv linker mutants than that for the K158AΔPro/
ΔEnv and Y164AΔPro/ΔEnv MHR mutants.
Linker mutations as well as MHR mutations strongly affect Gag multimerization ability at the PM but only moderately reduce membrane targeting of Gag. Of
virus production-defective mutants reported so far, mutations (e.g., G2A) in Gag-MA,
which is involved in anchoring Gag at the PM, have been shown to diminish markedly
the Gag expression level at the PM (less than 10% relative to the wild type) (19, 21, 22).
On the one hand, mutations of the residues which are located in the MHR and CA-CTD(e.g., K158A and WM184/185AA) affect only moderately the amount of Gag targeted to
the PM (40 to 70% relative to the wild type), yet they drastically reduce virus production(19, 21, 22). These results imply that the process(es) leading to virus particle production
from Gag targeting to the PM onward is severely attenuated for MHR and CA-CTD
mutants. In previous studies (21, 22), MHR and CA-CTD mutants with only a modest
reduction in PM-targeted Gag level were found to accumulate substantial amounts of
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FIG 4 Effects of linker mutations on Gag targeting to cell membranes as judged by membrane ﬂotation
analysis. HeLa cells were transfected with the indicated clones and lysed to prepare the postnuclear
supernatant for membrane ﬂotation analysis (22). The sample in a high concentration of sucrose solution
was placed at the bottom of a centrifugation tube and layered with lowered concentrations of sucrose
from the bottom to the top. After ultracentrifugation, fractions (F) were collected from centrifugation
tubes (top to bottom) for examination of Gag by the Western blotting method. Gag proteins inmembrane (MF) and in nonmembrane (non-MF) fractions are indicated. Representative immunoblotting
data from three independent experiments are shown in panel A. In panel B, the ratios of MF and non-MF
Gag proteins for various clones are presented (n ⫽ 3; mean ⫾ SE). Signiﬁcance relative to control NL4-3
was determined by Welch’s t test. **, P ⬍ 0.01; *, P ⬍ 0.05.

impaired ability to multimerize on the PM after reaching it. Our velocity sedimentation
assays of the particle production-defective linker mutants (S149AΔPro/ΔEnv and
I150AΔPro/ΔEnv) showed increased and decreased accumulations of 10S to 80S and
500S intermediates, respectively (Fig. 3). Thus, we predicted that the linker mutants
would also diminish Gag multimerization on the PM without severe reduction in Gag
amounts there. To examine mutational effects of the linker region on membranetargeting of Gag, we performed membrane ﬂotation assays (22) that can quantify
Gag accumulation at the PM. HeLa cells were transfected with NL4-3ΔPro/ΔEnv,
S149AΔPro/ΔEnv, I150AΔPro/ΔEnv, K158AΔPro/ΔEnv, or Y164AΔPro/ΔEnv, and at 24 h
posttransfection, postnuclear supernatants were prepared and mixed with a highconcentration of sucrose solution. Samples were then placed at the tube bottoms andfractionated by ultracentrifugation through sucrose gradients to ﬂoat the membranes(22). As shown in Fig. 4, most Gag proteins were located at membrane fractions (MF)rather than in non-MF for NL4-3ΔPro/ΔEnv. Mutant Gag proteins showed that asubstantial portion of Gag proteins was present at MF, although their relative amounts
in non-MF were increased for linker mutants (S149AΔPro/ΔEnv and I150AΔPro/ΔEnv)
and MHR mutants (K158AΔPro/ΔEnv and Y164AΔPro/ΔEnv). Gag amounts in MF,
relative to that for NL4-3ΔPro/ΔEnv, were estimated to be 80% for the S149AΔPro/ΔEnv
mutant, 78% for the I150AΔPro/ΔEnv mutant, 65% for the K158AΔPro/ΔEnv mutant,and 61% for the Y164AΔPro/ΔEnv mutant. Although statistical difference was noted
between the data for wild-type and MHR mutant clones, it can be summarized from theresults in Fig. 4 that mutant (linker and MHR) Gag proteins on the PM were only
moderately reduced.
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FIG 5 Effects of linker mutations on Gag subcellular localization as revealed by confocal microscopy
analysis. HeLa cells were transfected with pNL4-3Gag-EGFPΔEnv or derivative Gag-CA mutant clones by
Lipofectamine 2000, ﬁxed with paraformaldehyde at 10 h posttransfection, and processed for confocal
microscopy as previously described (63, 66). At least 100 EGFP-positive cells were examined for eachsample to determine the subcellular localization pattern of Gag-EGFP. Typical distribution patterns ofGag (EGFP) in cells are shown in panel A. aPM, plasma membrane Gag accumulation; Diff, diffused Gag
localization; Int, intracellular Gag accumulation. Bar ⫽ 10 ␮m. In panel B, Gag distribution patterns arecompared among clones. Patterns aPM⫹Diff and aPM⫹Int⫹Diff are considered plasma membrane
distribution, whereas pattern Diff⫹Int is considered internal distribution, as indicated in panel A. Resultsof three independent experiments are shown (means ⫾ SE). Signiﬁcance relative to the positive control,
NL4-3, was determined by Welch’s t test. **, P ⬍ 0.01.

To conﬁrm biochemical results shown in Fig. 4, we examined intracellular
Gag distribution by confocal microscopy. A construct that expresses wild-type Gagenhanced green ﬂuorescent protein (EGFP) fusion protein (NL4-3Gag-EGFPΔEnv)
and its derivative mutants (S149AGag-EGFPΔEnv, I150AGag-EGFPΔEnv, K158AGagEGFPΔEnv, and Y164AGag-EGFPΔEnv) were transfected into HeLa cells. At 10 h posttransfection, cells were ﬁxed for confocal microscopic analyses. Expression patterns of
Gag-EGFP observed in transfected cells were grouped as follows: (i) widely diffuses incytoplasm (Diff), (ii) accumulates at the PM (aPM), or (iii) accumulates intracellularly andpresents dot-like structures in cytoplasm (Int) (Fig. 5). Based on these subcellular
localization patterns of Gag-EGFP, cells were divided into two categories: a PM distribution group (aPM⫹Int⫹Diff and aPM⫹Diff) and an internal distribution group(Diff⫹Int) (Fig. 5). As shown in Fig. 5, EGFP-positive cells transfected with parental
NL4-3Gag-EGFPΔEnv were classiﬁed predominantly in the PM distribution group, and aminor population belonged to the internal distribution group. These results were ingood agreement with those obtained by membrane ﬂotation assays (Fig. 4). For thelinker and MHR mutants, EGFP-expressing cells in the internal distribution and PM
distribution groups were signiﬁcantly increased and decreased, respectively, relative to
NL4-3Gag-EGFPΔEnv. These results for mutant Gag proteins were also consistent inessence with those obtained by membrane ﬂotation assays. Apparent differences in the
MF/non-MF ratio observed for mutants (Fig. 4 and 5) could be due to experimental
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FIG 6 Detection of Gag-Gag interactions by NanoBRET. HEK293T cells were cotransfected with expression vectors encoding Gag-HaloTag and its corresponding Gag-NanoLuc (43). At 48 h posttransfection,
NanoBRET signals were measured, and values relative to that for control NL4-3 are presented (n ⫽ 4;means ⫾ SE). NanoBRET data were normalized by subtracting the signal value for a Gag CA linker/CTD
deletion mutant (deletion of CA amino acids 146 to 231). Signiﬁcance relative to control NL4-3 wasdetermined by Welch’s t test. **, P ⬍ 0.01; *, P ⬍ 0.05. A black bar, red bars, and blue bars indicate NL4-3,
linker mutants, and MHR mutants, respectively.

conditions (timing for sample preparations posttransfection, etc.) and/or the usedmethod itself. Overall, results from two very different analyses show that a signiﬁcantamount of mutant (linker and MHR) Gag proteins accumulated at the PM (61 to 80%
relative to the wild type and 47 to 52% relative to the wild type as shown in Fig. 4 and
5, respectively), consistent with previous reports (21, 22). Meanwhile, these mutants(S149A, I150A, K158A, and Y164A) exhibited marked reduction in virus particle production (4 to 12% relative to the wild type [Table 1]). These results suggest that Gag-Gag
interactions after PM binding are important determinants for the attenuation in Gag
assembly of the mutants.
Following membrane targeting, Gag forms high-order oligomers at the PM (41). We
directly measured Gag-Gag interactions in living cells using nano-bioluminescenceresonance energy transfer (NanoBRET) technology (42, 43). We have previously demonstrated that the NanoBRET system can detect speciﬁc intracellular Gag-Gag interactions of HIV-1NL4-3 (43). For this NanoBRET analysis, we selected some linker mutants
that show normal (S146A), moderately impaired (S146N and S149N), and impaired(S149A, S149D, and I150A) phenotypes with respect to virus particle production (Table1). The K158A and Y164A MHR mutants were used for controls as virus particle
production-defective mutants. As shown in Fig. 6, while mutants with a normal ormoderately impaired phenotype (S146A, S146N, and S149N) exhibited signals comparable to those of wild-type NL4-3, those with an impaired phenotype (S149A, S149D,
and I150A) produced signals at signiﬁcantly reduced levels. MHR mutants showedfurther reduction in signal production. Thus, the ability of mutants to mediate intracellular Gag-Gag interactions was well correlated with their ability to produce virus
particles. However, relative reduction values obtained for the two markers of themutants (virus production levels in Table 1 and NanoBRET signals in Fig. 6) weredifferent. While virus particle production of the S149A, S149D, and I150A mutants
decreased to around 10% of that of NL4-3, their NanoBRET signals were approximately
25 to 50% of that of NL4-3. Similarly, the attenuation levels of the K158A and Y164A
mutants in the two assays were not the same. The differences in experimental values
may be accounted for by differences in transfection efﬁciency and/or protein expression level, which could result from the use of distinct cells (H9 versus HEK293T) andconstructs (ΔPro/ΔEnv proviral clones versus codon-optimized expression vectors). It isthus possible that NanoBRET assays gave relatively overestimated values for the
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Gag-Gag interaction ability of the mutants. A greater reduction in the Gag-Gag interaction signals observed for MHR mutants relative to the linker mutants (Fig. 6) was also
noteworthy. Considering that the linker mutants but not the MHR mutants could form
500S intermediates albeit slightly (Fig. 3), it is conceivable that the mutations in theinterdomain linker may affect Gag assembly more weakly than those in MHR. In sum,
while somewhat impaired in the membrane binding and PM localization (Fig. 4 and 5),
the mutants showed quantitatively more signiﬁcant effects on the Gag-Gag interactions
as revealed by NanoBRET assays (Fig. 6). Despite the presence of substantial amountsof Gag proteins, the reduced interaction ability of the mutants would severely impede
Gag multimerization on the PM in the HIV-1 assembly process. Overall, our results
suggest that the decrease in virus particle production observed for the linker and MHR
mutants could result mainly from their reduced capacities to drive Gag-Gag interactions.
Two types of hydrophobic patches for molecular interactions are elucidated on
the surface of HIV-1 immature CA protein. The above-described results strongly
suggested that the linker mutations except for S146A inﬂuence the structural property
of CA interaction surfaces primarily for Gag assembly. To gain structural insights into
the effects of linker mutations on Gag assembly, we conducted in silico analyses.
Because hydrophobic interplay is a major contributor for macromolecule interactions,
including protein oligomerization, we examined three-dimensional (3D) distributions ofhydrophobic patches (a minimal patch area of 50 Å2) on the surface of HIV-1NL4-3 CA
protein. A near-full-length immature CA model of HIV-1NL4-3 was constructed from thereported structure of HIV-1 immature capsid lattice in the virus particles (25) andsearched for hydrophobic patches for molecular interactions (44–47).
The analysis disclosed two types of hydrophobic patches (Fig. 7): those along the
CA-CA interfaces in an immature capsid lattice (25) (Fig. 7A, pink areas) and thoselocated outside the CA-CA interfaces (Fig. 7B, light green areas). The former included
the reported regions for direct CA-CA interactions in an immature CA lattice, such as H1,
H2, and H9 (25). The latter included reported regions important for formation of Gag
assembly intermediates, such as H4, H6, H8, H10, and H11 (21). Although its importancein the Gag assembly has not formally been described, H7 forms a hydrophobic patch
along the CA-CA interface (Fig. 7A) and was recently shown by us to play a critical rolein virus particle production (48). The signiﬁcance of the H3 patch (Fig. 7B) in the latestage of HIV-1 replication has not been reported. H5 did not participate in formation of
the hydrophobic patch (Fig. 7A and B). Hydrophobic patches were predominantly
formed along the helices, and the amino acid residues contributed to form the patches
were generally highly conserved among the global HIV-1 subtype B strains (Fig. 7C).
Together with the functional and structural data on the immature CA protein so far
reported (21, 25), the results suggest a critical role of the hydrophobic patches in thelate stage of HIV-1 replication. In sum, the CA helices seem to play important roles infolding, stabilization, and interactions of the HIV-1 immature CA protein as generally
believed.
MD simulations predict that the linker and MHR mutations remotely affectstructural ﬂuctuations of the immature CA interaction surfaces. Because the direct
CA interaction surfaces for Gag assembly have been suggested to be located in the
CA-CTD and CA-NTD rather than in the linker region (21, 25, 49) (Fig. 7A), the linker
mutations were predicted to inﬂuence the structures away from the mutation sites. To
address this issue, we performed molecular dynamics (MD) simulations of the HIV-1NL4-3
near-full-length CA protein with or without a single linker mutation, S146A, S149A, or
I150A. Among the linker mutants, the S149A and I150A mutants, but not the S146A
mutant, exhibited attenuation in viral particle production (Table 1) along with attenuations in Gag-Gag interactions (Fig. 6). We also performed MD simulations of an MHR
mutant, the Y164A mutant, whose assembly was more severely impaired than the linker
mutants (Fig. 3 to 6), and its mutation is located outside the direct CA-CA contact site
(Fig. 7A and C). Because structural ﬂuctuations in solution play key roles in molecularinteractions (50–53), we analyzed the root mean square ﬂuctuation (RMSF) of the C␣
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FIG 7 Elucidation of the hydrophobic patches for molecular interactions of HIV-1NL4-3 immature CA protein. An
HIV-1NL4-3 CA protein model was constructed by the homology modeling method from the reported immature CA
structure (PDB code 4USN) (25). Distribution of the hydrophobic-interaction-prone patches (a minimal patch areaof 50 Å2) on the surface of the CA model was assessed and visualized using the Protein Patch Analyzer tool in MOE(44–47). (A) Hydrophobic patches on the CA-CA interface. Patches that participate in the direct CA-CA interactionsin an immature CA lattice (25) are shown in pink. (B) Hydrophobic patches outside the CA-CA interface. Patches thatare not involved in the direct CA-CA interactions in an immature CA lattice (25) are shown in green. (C) Amino acidresidues involved in forming the hydrophobic patches and their variations. Residues involved in the formation ofhydrophobic patches are marked with colored circles: pink circles, residues for patches involved in direct CA-CA
interactions; green circles, residues for patches that are not involved in direct CA-CA interactions. Shannon entropy
scores calculated with the CA amino acid sequences of HIV-1 subtype B (n ⫽ 14,120) are shown above thecorresponding residues.

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atoms of the main chains (54) using 15,000 snapshots in the equilibrium states during
MD simulations.
Notably, all mutations tested induced changes in ﬂuctuations at various regions
away from the mutation sites, as indicated by changes in the RMSF (Fig. 8). These
remote effects of single mutations on the ﬂuctuation proﬁle of immature CA protein
were reproducible with repeated MD simulations, suggesting that the effects are
physicochemically inevitable. The changes were greatest with Y164A, and the ﬂuctuations were augmented throughout the CA protein containing various CA-CA interfaces(Fig. 8, upper portion). The results suggest a critical role for MHR in maintaining proper
CA dynamics for Gag assembly. Although less extensive, S149A mutation also induced
overall augmentation of the CA ﬂuctuations (Fig. 8, upper middle portion), suggestingthat the linker region has a similar function with MHR in controlling CA surface
dynamics. In contrast, effects of I150A and S146A mutations were limited in particularregions (Fig. 8, lower middle and bottom portions). I150A with marked attenuation inboth virus production and viral infectivity (Table 1) induced changes in H6 and its
downstream loop, H7 and its downstream linker region, and H10 and its downstream
loop (asterisks at the lower middle portion of Fig. 8). These regions are all involved in
the formation of the hydrophobic patches in an immature CA lattice (Fig. 7). Amongthem, H7 region is involved in the direct interactions between CA and CA in theimmature CA lattice (Fig. 7A). Meanwhile, S146A mutation with no detectable attenuation in virion production but with attenuation in viral infectivity (Table 1) induced apattern of changes distinct from that of I150A: the changes were seen in the H4/H5
loop (cyclophilin A [CypA] binding loop), H6 and its downstream loop, H8 and its
downstream loop, and H10 and its downstream loop (asterisks at the bottom portionof Fig. 8). Although the last three regions participate in the formation of hydrophobic
patches (Fig. 7), no changes in the regions for the CA-CA direct interactions weredetected. These results show that the effects of the linker mutations on the CA
dynamics are unique in individual mutations and suggest that the change in the H7
ﬂuctuation may be a causative factor for the I150A phenotype (Table 1). Alternatively,
the multiple changes on the CA surfaces rather than the change at the speciﬁc site may
generate the I150A phenotype.
In summary, our results suggest that the linker and MHR mutations could remotely
control the physical properties of regions outside the mutation sites. The MHR mutation
Y164A and the linker mutations S149A and I150A, which caused attenuation in the
Gag assembly and virus particle production, were predicted to induce distinct
ﬂuctuation changes in the CA-CA contact site(s), whereas linker mutation S146A,
which did not cause signiﬁcant effects on the virus particle production, did notcause such changes.
Distribution of the hydrophobic patches is distinct between mature and immature CA proteins of HIV-1. HIV-1 CA protein changes its structure upon virion
maturation (23, 25, 55). To gain structural insights into the effects of linker mutations
on the early stages of viral replication, we conducted hydrophobic patch analysis andMD simulations of the mature CA. A full-length mature CA model of HIV-1NL4-3 wasconstructed from the reported structure of the HIV-1 mature capsid from a tubular
HIV-1 CA assembly (55) and was searched for hydrophobic patches for molecularinteractions as described above (44–47).
The analysis showed marked differences in the 3D distribution of the hydrophobic
patches between the immature and the mature CA proteins, in parallel with thestructural changes (Fig. 7 and 9). The patches for the CA-CA direct interactions on themature CA lost the ordered parallelism along the CA lattice in the immature CA, being
positioned irregularly to allow more compact CA-CA interactions in the CTD (Fig. 7A
and 9A). Meanwhile, the patches outside the CA-CA contact sites on the mature CA
were primarily positioned in the NTD more widely than those in the immature CA (Fig.
7B and 9B). Notably, however, the amino acid residues responsible for the formation of
the patches in the mature CA were generally highly conserved, as was seen for thosein the immature CA (Fig. 7C and 9C). These results strongly suggest that the
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FIG 8 Effects of the linker and MHR mutations on the structural dynamics of HIV-1NL4-3 immature CA
protein. Gag-CA protein models of HIV-1NL4-3 and its mutants were constructed from the reported
structure of HIV-1 immature capsid (PDB code 4USN) (25). MD simulations of Gag-CA proteins of
HIV-1NL4-3 and its mutants were carried out at 1 atm, at 310 K, and in 150 mM NaCl for 200 ns using theAmber 16 program package (54). RMSF values, which indicate the atomic ﬂuctuations of the main chainsof individual amino acids during MD simulations, were calculated using 15,000 snapshots in theequilibrium states during MD simulations. Distributions of RMSF in the Gag-CA proteins and its mutants
are shown. Numbers on the horizontal axes indicate amino acid positions in the HIV-1NL4-3 Gag-CA
protein. Arrows indicate the positions of mutated residues. Blue asterisks for I150A and S146A indicate
sites where the ﬂuctuation proﬁles differ between the wild type and the mutants.

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FIG 9 Elucidation of the hydrophobic patches for molecular interactions of HIV-1NL4-3 mature CA protein. An
HIV-1NL4-3 mature CA protein model was constructed from the reported structure of HIV-1 mature capsid (PDB
code 3J34) (55). Hydrophobic patches were assessed and visualized as described for Fig. 7. Hydrophobic patcheson the CA-CA interfaces in the mature CA (A) and those outside the CA-CA interfaces (B) are shown in pink andgreen, respectively. In panel C, amino acid residues involved in the formation of hydrophobic patches and theirvariations are indicated. Pink circles, residues for patches involved in the direct CA-CA interactions; green circles,
residues for patches that are not involved in the direct CA-CA interactions. Shannon entropy scores calculatedwith the CA amino acid sequences of HIV-1 subtype B (n ⫽ 14,120) are shown above the corresponding residues.

hydrophobic-interaction-prone patches play distinct but conserved roles in HIV-1 Gag
assembly and disassembly.
MD simulations predict that the linker and MHR mutations remotely affectstructural ﬂuctuations of the mature CA interaction surfaces. Inﬂuences of theSeptember 2019 Volume 93 Issue 17 e00381-19

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linker mutations on the ﬂuctuations of the mature CA were examined using a panel ofCA mutants (Y164A, S146A, and S149N). The Y164A mutant exhibited marked attenuation in both virus production and viral infectivity (Table 1). The S146A mutant showedno detectable attenuation in virus production and moderate attenuation in the viral
infectivity, while the S149N mutant exhibited no detectable attenuation in the viral
infectivity and moderate attenuation in virus production (Table 1). Molecular models of
the mature CAs of these HIV-1NL4-3 mutants were constructed, and MD simulations
were performed as described above for the immature CA mutant models.
The mature CA of HIV-1NL4-3 tended to ﬂuctuate more greatly in the NTD than theimmature CA, especially at the CypA binding loop (Fig. 10A). This may increase theprobability of interactions with trans-acting factor(s) in the NTD during Gag disassembly. Notably, all mutations tested again induced changes in ﬂuctuations at regions apartfrom the mutation sites (Fig. 10B). Y164A mutation caused augmentation of the
ﬂuctuations primarily in the CTD, including a region near the critical CTD-CTD interface
on H9 for stabilizing mature core (WM184/185) (55) (asterisk in the upper portion of Fig.
10B). The S146A mutation also induced similar changes near the WM184/185 region(asterisk in the middle portion of Fig. 10B). These changes may decrease viral infectivity
via destabilization of the CA core. Consistently, such changes at the WM184/185 region
were not detected for S149N (asterisk in the bottom portion of Fig. 10B). Meanwhile, all
the mutations induced changes at the CypA binding loop (“#” in Fig. 10B) and aroundthe linker and MHR (Fig. 10B). These remote effects of single mutations on the structuraldynamics of mature CA protein were reproducible with repeated MD simulations.
Although it is unclear at present how the changes can inﬂuence viral infectivity,
multiple changes on the CA surfaces in addition to the changes at the speciﬁc sites may
be important to realize distinct viral phenotypes. In sum, these results suggest that
unoptimized structural ﬂuctuation around the WM184/185 region may be a factor todecrease viral infectivity and highlight the role of the interdomain linker in controlling
ﬂuctuations of HIV-1 CA interaction surfaces away from the mutation sites.
DISCUSSION
In this study, by a systemic mutational analysis, we aimed to elucidate how the
Gag-CA interdomain linker works for HIV-1 replication, with a special interest in the latephase. Virus particle production by some linker mutants (S149A, S149D, S149K, andI150A) was markedly reduced, and that by the other mutants (S146N, P147A, T148A,
and S149N) was moderately reduced (Table 1). Only the S146A mutant produced viralparticle at a wild-type level. Most mutants in Table 1 displayed a considerable or drastic
reduction in viral infectivity in TZM-bl cells. The S149N mutant is defective for viralparticle production only but not for its early infectivity. These results strongly suggestthat the interdomain linker functions in virus particle production. Biochemical andmicroscopic analyses of Gag-Gag assembly, which drives virus particle production,
revealed that the linker mutations (S149A and I150A) impede the progression of Gag
multimerization on the PM in spite of the accumulation of certain amounts ofmembrane-targeted Gag proteins as previously reported for MHR mutations (K158A
and Y164A) (Fig. 3 to 5) (19, 21, 22). NanoBRET assays directly showed that these four
mutations (S149A, I150A, K158A, and Y164A) signiﬁcantly diminish the ability of Gag todrive Gag-Gag interactions (Fig. 6). In sum, we conclude that the decreased ability of
the linker mutants for Gag-Gag interactions/multimerization on the PM rather thantheir reduced ability to target the PM leads to the decrease in virus particle production(Table 1). MD simulations showed that the linker mutations can remotely affect thestructural ﬂuctuations of interaction surfaces on the immature Gag-CA (Fig. 8). Taken
together, the results lead us to propose that the interdomain linker facilitates Gag
assembly by supporting the interactions of the CA-NTD and CA-CTD.
Although several mutational analyses on the linker region have been reported(28–30, 33), it remained unclear whether the linker acts on virus particle production. In
this study, we clearly showed that all mutations except for one in the linker region havenegative effects on virus particle production (Table 1). This may be due to the
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FIG 10 Effects of the linker and MHR mutations on the structural dynamics of HIV-1NL4-3 mature CA protein. Gag-CA
protein models of HIV-1NL4-3 and its mutants were constructed from the reported structure of HIV-1 mature capsid(PDB code 3J34) (55). MD simulations were carried out under the same conditions as described for Fig. 8. RMSF
values were calculated using 15,000 snapshots in the equilibrium states during MD simulations. Distribution of(Continued on next page)
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experimental systems used in this study. Cells used for transfections (lymphocyte line
H9) and details of the transfection experiments (DNA amount, incubation time after
transfection, and/or transfection methods) may have allowed us to obtain a positive
readout on the changes in virus particle production.
Our MD simulations suggest that linker mutations would cause changes in ﬂuctuations of amino acid residues away from the linker region (Fig. 8 and 10). These results
were reproducible with repeated MD simulations, suggesting that the phenomena are
inherent in the mutations. In this regard, a recent study has revealed that a disorderedelement of protein plays key roles in regulating overall structural dynamics and thus
activity of the protein (52). The dynamic aspect of the disordered region theoreticallyincreases entropy associated with the structure. Increasing evidence indicates that thestructural dynamics in solution is critical to executing its physiological function involving recognition and release of substrates (50–53). Therefore, it is possible that thedisordered linker region in the CA protein has similar regulatory functions for theoverall structural dynamics and molecular interactions of CA protein. It remains unsolved, however, how such a remote control of the dynamics of CA protein could be
attained. A possible explanation is that there exist networks of hydrophobic andhydrophilic interactions on the protein surface to remotely inﬂuence the mobility ofamino acid residues apart from mutation sites. Alternatively, structural ﬂuctuations may
have resonant features, as often seen in the physical phenomena, and proper synchronization of ﬂuctuations may be modulated by mutations. Whatever the physicochemical mechanisms are, what is clear in this study is that the linker region is a key site toregulate remotely structural dynamics and interaction potency of the CA domain.
Changes in ﬂuctuations by mutations were primarily seen in the potential interaction surfaces, including helices and loops (Fig. 7 to 10). The mutations that causedchanges in the ﬂuctuations of the CA-CA contact sites induced attenuation in the Gag
assembly and virus particle production, whereas S146A, which did not cause changes
in the CA-CA contact sites, did not induce signiﬁcant effects on the viral phenotypes.
Thus, proper ﬂuctuations in the CA-CA contact sites seem to be critical to optimize Gag
assembly and virus particle production, as expected from protein science (50–53).
Y164A and S149A, which induced augmentation of amino acid ﬂuctuations throughout
the protein (Fig. 8), caused greater attenuations in the Gag assembly and virus particle
production. The results suggest that overall amino acid ﬂuctuations of CA protein are
optimized in the wild type for Gag assembly and virus particle production. Meanwhile,the sites with changes in ﬂuctuations were differently distributed among the mutants.
The results may imply that overall structural dynamics in addition to the speciﬁcchanges in the CA-CA contact sites are also important for optimizing Gag assembly andvirus particle production.
On the one hand, virus particle production was differently affected by amino acidresidues altered and their locations within the linker region (e.g., S146A versus S149A
and S149N versus S149A/D) (Table 1). Underlying molecular mechanisms for theobservations remain unsolved. In this regard, changes in the size and/or electrostatic
characteristics of a side chain in the linker region and MHR could modify differently thealready optimized ﬂuctuation proﬁles of HIV-1NL4-3 wild-type CA. Such differences inthe ﬂuctuation proﬁles with distinct mutation types may be a factor to cause distinct
effects on Gag-Gag interactions and thus viral replication phenotypes. Results for the
MHR mutants may imply that greater deviations of the CA ﬂuctuation proﬁles from thatof the wild type can cause greater effects on the viral replication phenotypes. In
addition, changes in other structural factors for molecular interactions, such as those in
the secondary structure on the interaction sites and the 3D distributions of the

FIG 10 Legend (Continued)
RMSF in the Gag-CA immature and mature proteins of NL4-3 (A) and those in the CA mature proteins of NL4-3 andmutants (B) are shown. Numbers on the horizontal axes indicate amino acid positions in the HIV-1NL4-3 Gag-CA
protein. Arrows indicate the positions of mutated residues. #, positions of CypA binding loop; orange asterisks, sites
upstream of WM184/185 residues in H9.
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interaction-prone patches, may be involved in shaping the interaction efﬁciencies.
Further study is necessary to address each of these issues.
Our in silico analyses provide structural insights into the experimental observations
on the linker and MHR mutants of HIV-1 Gag-CA. They revealed that the linker and MHRmutations could induce changes in ﬂuctuations at the regions for Gag assembly anddisassembly, most of which were located away from the mutation sites (Fig. 8 and 10).
Since structural ﬂuctuations play key roles in molecular interactions (50–53), physicalchanges induced by the linker mutations would affect interaction efﬁciencies between
CA contact sites and/or between CA and the trans-acting molecule(s) for Gag assemblyand disassembly. This, in turn, could lead to the observed defects in Gag assembly, virus
production process, and viral infectivity (Table 1 and Fig. 3 to 6). In this regard,
structural inﬂuences caused by mutations were much greater for MHR, covering many
important interaction sites, than those for the linker region (Fig. 7 to 10). The results are
consistent with experimental data in that Gag assembly was more severely impaired by
MHR mutations than by linker mutations. Thus, the present experimental and in silico
ﬁndings strongly suggest that the HIV-1 Gag-CA linker region and MHR are bothcis-acting protein elements governing structural properties of various Gag-CA sites for
Gag assembly and disassembly. Taken together, our results have demonstrated that
Gag interdomain linker has a role for HIV-1 replication in the late phase in addition tothat in the early phase by inﬂuencing Gag-Gag interactions.
Our data thus illustrate a hitherto-unknown linker function, an allosteric regulationof CA structure by a disordered protein element, for HIV-1 replication. Although nearly
half of human proteins contain intrinsically disordered peptide segments (56), theirfunctions largely remain unknown. It is possible that the segments function to tune theprotein structure and activity. In fact, the unstructured segment has been reported
recently to be important in the regulation of structural dynamics and activity of human
UDP-␣-D-glucose-6-dehydrogenase (52). In the case of proteins of highly mutable
pathogens, such tuning mechanisms may be necessary to maintain their replication
abilities under constantly changing protein backbone sequences. In this regard, Shannon entropy analysis has shown that the linker region of HIV-1 subtype B allows
practically no amino acid variations except for at position 148 (Fig. 2). The results
strongly suggest the presence of potent selective constraints that restrict structuralchanges in the linker region during subtype B circulation and evolution in the ﬁeld.
Such constraints may be generated by strict requirement for the linker size andchemical property to deliver proper structural ﬂexibility to CA during Gag assembly.
Increasing evidence suggests that Gag assembly is a highly ordered CA-mediatedprocess involving CTD-CTD interactions, interhexameric CTD-CTD interactions, intrahexameric CTD-CTD interactions, and interhexameric NTD-NTD interactions (21, 49).
Thus, it is reasonable to postulate that the maintenance of appropriate physicochemical
properties of the linker region is critical to successfully accomplish the assembly inorder. Consistently, experimental data showed that a single mutation in the linker
region often leads to reduction in particle production along with Gag assembly andinfectivity defects (Table 1 and Fig. 3 to 6). Interestingly, however, position 148, in thecenter of the linker region, was found to accept amino acid changes (Fig. 2). This ﬁnding
may imply that the interdomain linker of HIV-1 CA has a certain genetic space formaintaining the tuning function.
In conclusion, we demonstrated that the Gag-CA interdomain linker of HIV-1 plays
a role in virus particle production via effects on Gag-Gag interactions/assembly. Since
immature capsid assemblies would be a potential target for antiretroviral therapy (24,57), it is necessary to comprehensively understand this process through clariﬁcation of
the critical viral and cellular factors which are involved in virus particle production, as
well as of the structural characteristics of the whole Gag precursor protein.
MATERIALS AND METHODS
Cells. Monolayer cell lines HEK293T (ATCC CRL-1573), HeLa (ATCC CCL-2), and HeLa-derived reporter
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Journal of Virology

inactivated fetal bovine serum as previously described (59). Lymphocyte line H9 (ATCC HTB-176) was
cultured and maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum aspreviously described (59).
Plasmid DNAs. Proviral full-length clone pNL4-3 has been previously described (60). Its site-speciﬁc
Gag-CA mutant clones (Table 1) were generated by PCR-based standard cloning procedures. Parentalclones in this study, designated pNL4-3ΔPro/ΔEnv (Fig. 3 and 4) and pNL4-3Gag-EGFPΔEnv (Fig. 5), wereconstructed from pNL4-3. Brieﬂy, two mutations that abolish functional expression of viral protease(NL-Hc) (61) and Env (NL-Kp) (62) were introduced into pNL4-3 to generate pNL4-3ΔPro/ΔEnv, and the
Env mutation was inserted into a protease-deﬁcient pNL4-3Gag-EGFP (63) to construct pNL4-3GagEGFPΔEnv. The test clones designated HaloTag-fused Gag and NanoLuc-fused Gag (Fig. 6) have beenpreviously described (43). Finally, to perform the experiments whose results are shown in Fig. 3 to 6,
Gag-CA mutant clones derived from the four parental test clones (pNL4-3ΔPro/ΔEnv, pNL4-3GagEGFPΔEnv, HaloTag-fused Gag, and NanoLuc-fused Gag) were constructed by standard recombinant
DNA and PCR-based cloning techniques.
Assays for virus replication. Input virus samples for infections (Table 1) were prepared from
HEK293T cells transfected with various full-length clones by the calcium phosphate precipitation method
as previously described (60, 64). Virus amounts were determined by virion-associated reverse transcriptase (RT) assays as previously described (64, 65). To monitor multicycle virus growth property, 1 ⫻ 105 RT
units of virus samples were inoculated into 1 ⫻ 105 of H9 cells, and culture supernatants were collected
every 3 days for RT assays. To evaluate viral single-cycle infectivity, 1 ⫻ 104 RT units of virus samples were
inoculated into 4 ⫻ 103 TZM-bl cells, and on day 1 postinfection, cell lysates were prepared for luciferase
assays (Promega) as previously described (59). To determine single-cycle virion productivity, 1 ⫻ 106 H9cells were transfected with 2 ␮g of proviral clones by the Amaxa Cell Line Nucleofector kit V (Lonza) using
Nucleofector II (Lonza) with program X-005. To prevent reinfection of progeny virions, CXCR4 antagonist
AMD3100 (1 ␮M) was added to culture medium. Amounts of Gag-p24 in culture supernatants at 48 h
posttransfection were measured by the HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA)
kit (ZeptoMetrix Corporation) according to the manufacturer’s protocol.
Analysis for Gag velocity sedimentation. HeLa cells were transfected with pNL4-3ΔPro/ΔEnv or its
derivative Gag-CA mutant clones by the polyethylenimine method (22), and at 24 h posttransfection,
cells were harvested for analysis for Gag velocity sedimentation as previously described (22). Preparedcell lysates were then centrifuged through a sucrose gradient (22) using a P40ST rotor (Hitachi, Tokyo,
Japan) at 36,000 rpm (162,700⫻ g) for 160 min at 4°C. After centrifugation, gradients were fractionatedfrom top to bottom. Sedimentation coefﬁcients were calculated with a computer program, himac ASSIST(Hitachi), based on their actual Brix values. To detect Gag precursor protein, Western blot analysis using
mouse anti-HIV-1 Gag p24 monoclonal antibody (NIH AIDS Reagent Program; catalog no. 6457) was
performed as previously described (59). Gag bands were quantiﬁed by the Quantity One imaging
program (Bio-Rad).
Membrane ﬂotation assays. HeLa cells were transfected with various pNL4-3ΔPro/ΔEnv clones as
described above. On day 1 posttransfection, cells were harvested and lysates were prepared for theﬂotation assay by centrifugation through a sucrose gradient as previously described (22). Brieﬂy,transfected cells were lysed with hypotonic lysis buffer using a Dounce homogenizer, and nuclei were
removed by centrifugation. Postnuclear supernatants were mixed with the solution with a high sucrose
concentration (ﬁnal sucrose concentration was 75%). Samples were then placed at the bottom of an
ultracentrifugation tube and overlaid with 65% to 10% sucrose solution. After centrifugation using a
P40ST rotor (Hitachi) at 28,000 rpm (98,400 ⫻ g) for 20 h at 4°C, fractions (from top to bottom) were
examined for Gag precursor by Western blotting as previously described (22).
Confocal microscopy analysis. HeLa cells were transfected with pNL4-3Gag-EGFPΔEnv or derivative
Gag-CA mutant clones by Lipofectamine 2000 (Thermo Fisher Scientiﬁc), and at 10 h posttransfection,
cells were ﬁxed with paraformaldehyde for confocal microscopy as previously described (63, 66). Nuclei
were stained with 4=,6-diamidino-2-phenylindole (DAPI; Dojindo), and confocal images were obtainedwith an LSM 700 confocal microscope (Carl Zeiss). More than 100 EGFP-positive cells were examined foreach sample to determine the subcellular localization pattern of Gag-EGFP.
NanoBRET analysis. HEK293T cells in 96-well plates were transfected with vectors encoding
HaloTag-fused Gag (100 ng) and NanoLuc-fused Gag (1 ng) by the Effectene transfection reagent(Qiagen). At 48 h posttransfection, NanoBRET activity was measured by the NanoBRET Nano-Glo detection system (Promega) in accordance with the manufacturer’s protocol. To remove minor effects of
CA-independent Gag-Gag interactions, NanoBRET data were normalized by subtracting the signal value
for a Gag CA linker/CTD deletion mutant (deletion of 146 to 231 amino acids in CA).
Shannon entropy analysis. Amino acid variation at each position of Gag-CA was analyzed with
Shannon entropy as described previously (34–36). Full-length capsid amino acid sequences of HIV-1
subtype B (n ⫽ 14,120) and group M (n ⫽ 25,222) were obtained from the HIV Sequence Database(http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html). Shannon entropy was calculated on
the basis of Shannon’s equation (67):H(i) ⫽ ⫺冘

p(xi)log2 p(xi)xi(xi ⫽ G, A, I, V, . . .)where H(i), p(xi), and i indicate the amino acid entropy score of individual position, the probability ofoccurrence of a given amino acid at the position, and the number of the position, respectively. An H(i)score of zero indicates absolute conservation, whereas a score of 4.4 bits indicates complete randomness.
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Journal of Virology

Molecular modeling of HIV-1 capsid protein. HIV-1 capsid monomer structures with various linker
and MHR mutations were constructed by the homology modeling method with Molecular Operating
Environment (MOE; Chemical Computing Group Inc., Montreal, QC, Canada) as described previously formodeling of various HIV-1 capsid mutants (64, 68–70). The reported structures of HIV-1 immature capsid(PDB code 4USN) (25) and mature capsid (PDB code 3J34) (55) were used as templates for modeling theimmature and mature CA proteins, respectively, of HIV-1NL4-3 and its derivatives.
Protein patch analysis. Assessment and visualization of hydrophobic patches on the CA models
were carried out using the Protein Patch Analyzer tool in MOE (44–47). This program calculateshydrophobic patches with minimum area of 50 Å2 to show critical regions for protein-protein interactions(71, 72). The hydrophobic potential is calculated using the Wildman and Crippen octanol-water partition
coefﬁcients f ⫽ log P (73).
MD simulation of HIV-1 capsid monomer. HIV-1 capsid models were subjected to MD simulations
essentially as described previously for viral structural proteins and enzymes (36, 74–76). Brieﬂy, thesimulations were done by the pmemd module in the Amber 16 program package (54) with the ff14SB
force ﬁeld (77) and the TIP3P water model for simulations of aqueous solutions (78). A nonbonded cutoffof 10 Å was used. Bond lengths involving hydrogen were constrained with SHAKE, a constraint algorithm
to satisfy a Newtonian motion (79), and the time for all MD simulations was set to 2 fs. After heating
calculations for 20 ps 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 200 ns.
Calculation of RMSF. RMSFs were calculated as described previously (74–76) to quantify structuraldynamics of molecules in the MD simulations. RMSFs of the C␣ atoms were calculated to obtain
information on atomic ﬂuctuations of individual amino acid residues during MD simulations. The 15,000
snapshots in the equilibrium states during 130 and 200 ns of MD simulations were used to calculate
RMSF. The average structures were used as reference structures for RMSF calculation. RMSF, whichquantiﬁes the differences between the average values and those obtained at given times of MD
simulations, was calculated using the ptraj module in Amber, a trajectory analysis tool (54).

ACKNOWLEDGMENTS
This study was supported in part by grants to M.N., K.M., and H.S. from the Japan
Agency for Medical Research and Development (AMED; Research Program on HIV/AIDS;
fund identiﬁer, JP18fk0410004).
The numerical calculations for the MD simulations were carried out on the TSUBAME3.0
supercomputer in the Tokyo Institute of Technology. We are indebted to the NIH AIDS
Research and Reference Reagent Program for Gag-p24 antibody. We appreciate theSupport Center for Advanced Medical Sciences, Tokushima University Graduate Schoolof Biomedical Sciences, for experimental facilities and technical assistance. We thankKazuko Yoshida (Tokushima University, Tokushima, Japan) and Hiromi Nakamura(National Institute of Infectious Diseases, Tokyo, Japan) for assistance with editorial
matters and Shannon entropy analyses, respectively. We also thank Yuko Morikawa(Kitasato University, Tokyo, Japan) for the generous gift of pNL4-3Gag-EGFP.
We declare no competing interests.

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