Download Replication-dependent fitness recovery of Human immunodeficiency

Journal of General Virology (2006), 87, 961–965
Short
Communication
DOI 10.1099/vir.0.81473-0
Replication-dependent fitness recovery of Human
immunodeficiency virus 1 harbouring mutations of
Asn17 of the nucleocapsid protein
József Tözsér,1,23 Sergey Shulenin,134 Matthew R. Young,1
Carlton J. Briggs1 and Stephen Oroszlan1
Correspondence
Stephen Oroszlan
[email protected]
Received 30 August 2005
Accepted 6 December 2005
1
National Cancer Institute, Frederick, MD 21701, USA
2
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine,
Debrecen University, H-4012 Debrecen, Hungary
The genetic stability of attenuated Human immunodeficiency virus 1 (HIV-1) variants harbouring
mutations (Gly or Lys) of Asn17, the protease-cleavage site of the proximal zinc finger of the
nucleocapsid protein, was studied. All possible codons for the Gly mutants were tested as starting
sequences. Long-term replication assays revealed that the mutants were unstable; mutations
of Gly17 to Arg, Ala, Ser and Cys, as well as a Lys17Asn reversion, were observed. Replication
kinetic assays in H9 cells revealed that the replication of Ala, Ser and Arg mutants was improved
substantially compared with the Gly variant; the infectivity of Ala17 and Ser17 viruses was
equal to, and that of Arg17 was almost equal to, the infectivity of the wild-type virus. Kinetic analysis
of the cleavage of oligopeptides representing the corresponding nucleocapsid-cleavage sites
revealed that all mutations improved cleavability, in good agreement with the previously proposed
role of nucleocapsid cleavage in HIV-1 replication.
In addition to the well-established role of the retroviral protease (PR) in the late phase of the virus life cycle, evidence
suggests that it might also be involved in the early phase, as
reviewed recently (Tözsér & Oroszlan, 2003). We have
proposed previously that one possible target of the PR in the
early phase is the mature nucleocapsid (NC) protein, based
on PR-mediated NC processing in the core particles of
Equine infectious anemia virus, a lentivirus similar to Human
immunodeficiency virus 1 (HIV-1) (Roberts & Oroszlan,
1989, 1990; Roberts et al., 1991a, b), and on the presence of a
corresponding cleavage site in the NC protein of HIV-1
(Tözsér et al., 1993, 2004). Our proposal is supported
further by the finding that specific inhibitors of HIV-1 PR
are able to block primary infection (Baboonian et al., 1991;
Venaud et al., 1992; Nagy et al., 1994; Panther et al., 1999;
Goto et al., 2001), although others have not observed this
effect (Jacobsen et al., 1992; Kaplan et al., 1996; Uchida et al.,
1997).
The NC domain (p7) of the HIV-1 Gag polyprotein has
several functions in the life cycle of the virus. It is required
for efficient packaging of viral RNA into the assembling
virion and for its dimerization. The NC protein participates
in cDNA synthesis and virus assembly, and interacts with
3These authors contributed equally to this paper.
4Present address: A&G Pharmaceutical, Suite U, 9130 Red Branch
Road, Columbia, MD 21045, USA.
0008-1473 Printed in Great Britain
viral protein R and reverse transcriptase (reviewed by Rein
et al., 1998; Hsu & Wainberg, 2000; Bampi et al., 2004). The
HIV-1 NC protein is also a target for antiretroviral therapy
(reviewed by Musah, 2004).
To study the proteolytic processing of HIV-1 NC protein
and its role in the early phase of replication, we previously
constructed HIV-1 NC mutants with substitutions at Asn17
of the PR-cleavage site within the NC zinc finger and
described detailed biochemical studies of the proteolytic
processing of wild-type and mutant NC proteins (Tözsér
et al., 2004). Introduction of the Gly17 and Lys17 mutations, which substantially impaired the NC and corresponding oligopeptide-substrate processing, into an infectious
HIV-1 clone resulted in viruses with impaired infectivity,
whilst introduction of the protease-sensitive Ala17 mutation
resulted in a virus capable of replicating like the wild type, as
demonstrated previously for this mutant (Dorfman et al.,
1993). The effect of these and other mutations at the 17th
amino acid position of NC on virus production and
infectivity and on various steps of the early phase of the viral
life cycle have also been studied (R. J. Gorelick, J. A. Thomas,
L. V. Coren, W. J. Bosche, T. D. Gagliardi, S. Shulenin & S.
Oroszlan, unpublished data). Here, we present the results of
studies on the genetic instability of Gly17 and Lys17 mutant
viruses. Mutations observed during long-term replication
of these viruses were tested for their effect on proteolytic
susceptibility, as well as for their effect on replication capability. Taking into consideration the high mutation rate in
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J. Tözsér and others
HIV-1 (Coffin, 1995), we assumed that deliberate mutations producing viruses with impaired infectivity would be
unstable and would lead eventually to better-replicating
variants. Studies of the instability of the selected NC mutant
viruses were interesting from a number of viewpoints. First,
analysis of the different types of NC mutants could reveal
what types of residue were optimal, suboptimal or deleterious at a given position and how the observed ‘goodness’
correlates with proteolytic susceptibility. Such correlation
studies may support the proposed role of PR in NC protein
processing. Furthermore, due to increased interest in the NC
protein as a potential target for chemotherapy (Huang et al.,
1998), mutational analysis may provide insights into
possible changes in the HIV-1 zinc finger that could alter
its structure and lead to drug resistance. To explore the
genetic stability of the wild type as well as the Gly17 and
Lys17 mutants, AD293T cells were transfected with the
appropriate plasmid DNA. After 3 days, virus was collected
and used to infect H9 cells. Infected cells were passaged every
3–4 days (split 1 : 4) and, after five to seven passages, cellfree virus was collected and used to infect fresh H9 cells.
RNA was purified from the viruses at the end of each
transfer. DNA fragments encoding the NC and PR regions
were generated by RT-PCR and sequenced. The time points
when the mutations were first observed are presented in
Table 1. No mutations were observed in the PR-encoding
region of any of the clones. Furthermore, no mutations were
found within 232 days of virus cultivation of the wild-type
sequence (data not shown).
Analysis of the stability of the Asn17Gly mutation in the HIV1 NL4-3 strain revealed that Gly17RAla and Gly17RArg
mutations had occurred. These substitutions (utilizing GGA
as the codon for Gly) were the result of single base changes at
the first (GRA, Arg) or second (GRC, Ala) positions of the
GGA codon, respectively. Using the same (GGA) Gly codon,
two additional mutations are possible, resulting in Val or
Glu substitutions, but these were not observed during virus
cultivation. To increase the chance of obtaining additional
mutations, we introduced the three other codons for Gly to
replace Asn17 in the NC domain of HIV-1 strain NL4-3
(Table 1). In these sets of experiments, we found Gly17RSer,
Gly17RArg and Gly17RCys mutations. All substitutions
that produced these mutations were in the first base position
of the codons and the majority were GRA transitions
(Table 1); no second-base changes were observed in these
experiments. Analysis of the Lys17 mutant suggested that
this mutant was also unstable and reverted quickly back to
wild type (Table 1).
When mutations were observed, the analysis frequently
showed a mixture of the parental and new mutant viruses,
with the mutants becoming dominant in most cases by the
end of the next transfer. Once they appeared, the new mutations were stable and no further changes were observed
in experiments lasting an average of 150–160 days. These
observations, in addition to the instability of the Gly17
and Lys17 mutations, suggested that the new mutant viruses
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had growth advantages when compared with the parental
viruses. Altogether, 90 % of the mutations occurred in the
first position of these codons and 75 % of the mutations
were GRA base transitions. The observed high frequency
of GRA base mutations correlates with published data
describing a prevalence of this type of mutation (so-called
‘hypermutation’) in retroviruses (Huang et al., 1998),
including HIV-1 (Martı́nez et al., 1995; Vartanian et al.,
1991). We also observed a GRT transversion in the first
position of Gly codons, resulting in the appearance of Cys,
suggesting that transversions could occur, although at a very
low frequency compared with GRA transitions. We did not
find GRA transitions in the second positions of the codons,
even though a strong preference for GRA transitions within
the GpA dinucleotide has been reported (Vartanian et al.,
1991). GRA transition in the second position of codons
would have led to Glu or Asp mutations, but these were not
observed. The absence of these mutations suggested that
viruses harbouring these mutations would not be more
infectious than the Gly17 parental virus. Among all possible
changes in the second position of the Asn17 codon, only the
GRC transversion producing the Asn17GlyRAla mutation
was found. The relatively rare appearance of the Ala17 mutation was probably caused by the low frequency of GRC
transversions. In contrast to the Gly17 mutants, mutations
at all three base positions would produce changes in the
Lys17 mutant (Table 1). The observed mutation Lys17RAsn
(reversion to wild type) was produced by an ART transversion in the third position of the AAA codon used for
Lys. The mutant viruses in two out of four cases reverted to a
wild-type amino acid sequence. The first mutation was
found after 65 days of virus cultivation and the second after
135 days (Table 1).
As Ser, Arg and Cys substitutions were observed in Gly17containing viruses after long-term cultivation, we introduced
two of these mutations (Ser17 and Arg17) into pNL4-3 for
further characterization. These two mutants, in addition to
wild-type, Ala17 and parental Gly17 viruses, were used for
a comparative analysis of infectivity. Viruses were again
produced by transfection of AD293T cells and viruscontaining supernatants were used to infect H9 cells. The
infectivity of the Ala17 and Ser17 viruses was similar to the
wild-type levels, with Arg17 being slightly less infectious
than the wild type, but more infectious than the parental
Gly17 virus (Fig. 1). The somewhat lower infectivity of
the Arg mutant is in good agreement with the findings of
mutations observed during long-term replication: only a
small amount of Arg17 virus had evolved from Gly17 by
day 74 of replication, but subsequent analysis at day 105
exclusively showed the sequence corresponding to the Ala17
mutation.
Originally, the attenuating substitutions were designed to
alter the PR-cleavage site within the NC protein (see Tözsér
et al., 2004). To obtain a measure of the proteolytic susceptibility of NC proteins, oligopeptides with the observed
mutations were tested as substrates of HIV-1 PR (Table 2).
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Journal of General Virology 87
Fitness recovery of HIV-1 nucleocapsid mutants
Table 1. Possible mutations of the codons used for Gly and Lys in place of Asn17 of the proximal zinc finger of HIV-1 NC
protein and actual mutations found in the virus
Gly codon
GGA
Base position*
First
Second
GGT
First
Second
GGG
First
Second
GGC
First
Second
Lys (AAA)
First
Second
Third
Possible mutations
Mutations found
Codon
Amino acid
Codon
Amino acid
Time found (days cultivation)D
Frequency
AGA
CGA
TGA
GAA
GCA
GTA
AGT
CGT
TGT
GAT
GCT
GTT
AGG
CGG
TGG
GAG
GCG
GTG
AGC
CGC
TGC
GAC
GCC
GTC
TAA
CAA
GAA
ATA
ACA
AGA
AAT
AAC
AAG
Arg
Arg
(Stop)
Glu
Ala
Val
Ser
Arg
Cys
Asp
Ala
Val
Arg
Arg
Trp
Glu
Ala
Val
Ser
Arg
Cys
Asp
Ala
Val
(Stop)
Gln
Glu
Ile
Thr
Arg
Asn
Asn
Lys
AGA
Arg
42, 55, 107
3/17
GCA
Ala
105, 107
2/7
AGT
Ser
55, 107, 107
3/7
TGT
None
Cys
61
1/7
AGG
Arg
55, 55 (66), 55 (50), 107
4/7
AGC
Ser
55, 55 (75), 107, 107, 107 (50)
5/8
TGC
None
Cys
55, 55
2/8
Asn
65, 135
2/4
None
None
None
AAT
*Mutations in the third position do not lead to replacement of Gly. H9 cells (16106) were incubated for 2 h at 37 uC with 0?5 ml clarified
supernatant from transfected AD293T cells containing an equivalent amount of virus, as determined by using a p24 ELISA kit (DuPont/NEN
Research Products). Cells were washed twice with RPMI 1640 medium and suspended in 2 ml RPMI 1640 medium containing 10 % fetal calf
serum and maintained in 24-well plates. Cultures were fed every 3–4 days by removing 1?5 ml suspended cells and replacing with fresh RPMI
1640 medium. After a few passages (usually five to eight), cells were spun down and the supernatant was used to infect fresh H9 cells. At the time
of transfer, 140 ml virus-containing supernatant was used for RNA isolation (QIAamp Viral RNA kit; Qiagen). cDNA was produced by using 4 ml
of the total (50 ml) viral RNA as a template, together with the 39 primer RT-PCRNC3 (59-CTGTTGGCTCTGGTCTGCTCTC-39 nt 2137–2158)
and SuperScript II RT enzyme (Gibco-BRL). cDNA was amplified by PCR using a GeneAmp PCR Reagent kit with AmpliTaq DNA polymerase
(Perkin Elmer), the 59 primer RT-PCRNC5 (59-CTGAAGCAATGAGCCAAGTAACAA-39; nt 1880–1903) and the 39 primer RT-PCRNC3. After
purification by using a QIAquick PCR Purification kit (Qiagen), the DNA was sequenced by using the dsDNA Cycle Sequencing System (GibcoBRL).
DIf the sequencing produced a mixture of the mutant and the original nucleotide, the percentage of the mutant nucleotide sequence is given in
parentheses.
Our previous study established a strong correlation between
peptide-hydrolysis rates and in vitro processing of recombinant NC proteins (Tözsér et al., 2004). Analyses of the
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cleavage of peptides containing mutations Ser17, Arg17 and
Cys17 showed that the rate of cleavage increased when
compared with cleavage of the Gly17 peptide (Table 2).
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J. Tözsér and others
Fig. 1. Replication of wild-type, Asn17Gly and ‘revertant’
viruses in H9 cells. Samples were isolated by using supernatants from transfected AD293T cells, adjusted to 100 ng
p24 and used to infect 16106 H9 cells. Virus replication was
monitored by reverse transcriptase activity in culture supernatants as described previously (Popovic et al., 1984). m, Wild
type; %, Asn17Gly; n, Asn17Ala; #, Asn17Ser; w, Asn17Arg.
It should be noted that, in all cases, mutations observed in
virus cultivation yielded an improvement in the proteolytic
susceptibility of NC. Interestingly, the Gly17RAla mutation
Table 2. Proteolytic processing of substituted oligopeptides
representing the cleavage site inside the proximal zinc finger
of the HIV-1 NC protein
Assays were performed as described previously (Tözsér et al.,
2004). Briefly, the reaction mixture [0?25 M phosphate buffer
(pH 5?6), 7?5 % glycerol, 1 mM EDTA, 5 mM dithiothreitol, 2 M
NaCl] containing the appropriate oligopeptide in various concentrations and HIV-1 PR was incubated at 37 uC for 1 h and the
reaction was stopped by the addition of guanidine/HCl. An aliquot was injected onto a Nova-Pak C18 RP-HPLC column.
Substrates and the cleavage products were separated by using an
increasing water/acetonitrile gradient in the presence of 0?05 % trifluoroacetic acid. The composition of the cleavage products was
determined by amino acid analysis after perchloric acid oxidation.
Kinetic parameters were determined at less than 20 % substrate
turnover by non-linear regression analysis. Standard errors were
less than 20 %.
Mutation
Peptide
kcat/Km (mM”1 s”1)
Wild type
N17RA
N17RG
N17RK
N17RR
N17RS
N17RC
KIVKCF-Q-NCGK
KIVKCF-Q-ACGK
KIVKCF-Q-GCGK
KIVKCF-Q-KCGK
KIVKCF-Q-RCGK
KIVKCF-Q-SCGK
KIVKCF-Q-CCGK
0?040*
0?200*
0?001*
0?006*
0?019
0?006
0?162
*These values have been published previously (Tözsér et al., 2004).
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resulted in a virus that was similar to wild-type virus with
respect to both cleavage of the NC protein by PR and
infectivity. This variant is known as a pseudorevertant (the
true revertant would be Gly17RAsn). There was also
an apparent correlation between the stability of the Lys17
mutant and its NC protein processing. The NC protein
harbouring the Lys17 mutation was a very poor substrate
of HIV-1 PR and the mutant virus also appeared to be
unstable. For revertants of the Gly17 mutant, the cleavage
rate observed for oligopeptide substrates (as a measure of
NC susceptibility) correlated well with virus infectivity,
except for Ser. Cleavage susceptibility was increased in the
order Gly17>Arg17>wild type (Asn17)>Ala17. However,
whilst the Ser17 mutant replicated as well as the wild type
(Fig. 1) and was genetically stable, the peptide with Ser in
place of Asn was a poor substrate, although better than the
peptide with Gly at the same position. Additional experiments need to be performed to resolve this discrepancy.
Although the altered proteolytic processing of NC at the
studied site is a feasible explanation for the altered infectivity, other explanations may also be valid. Detailed virological studies of these mutants are in progress to identify
unambiguously the replication step affected by these mutations. Asn17 of the HIV-1 NC protein is conserved and is
involved in the interaction of the two zinc-finger domains
through hydrogen bonding in a structure that has been
determined with SL3 RNA (De Guzman et al., 1998), but it is
open towards the solvent in another structure involving SL2
RNA (Amarasinghe et al., 2000). A strong influence of the
mutations on intramolecular and protein–RNA interactions
is unlikely, as the mutants appeared to package wild-type
amounts of viral RNA (R. J. Gorelick, J. A. Thomas, L. V.
Coren, W. J. Bosche, T. D. Gagliardi, S. Shulenin & S.
Oroszlan, unpublished data). Taking into account the
various effects of the NC, even in the early phase of virus
replication (Bampi et al., 2004) where the Gly17 and Lys17
NC mutants appear to be defective, many other so far
unknown protein–protein or protein–nucleic acid interactions mediated by the NC could be influenced by these
mutations.
Acknowledgements
This research was supported in part by the Intramural Research
Program of the NIH, National Cancer Institute, Center for Cancer
Research, in part under contract with ABL and by the Hungarian
Science and Research Fund (OTKA T 43482). We would like to thank
Dr Peter Bagossi for analysis of the NC crystal structures.
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