Requirements for efficient minus strand strong-stop DNA transfer in human immunodeficiency virus 1

Abstract

After HIV-1 enters a human cell, its RNA genome is converted into double stranded DNA during the multistep process of reverse transcription. First (minus) strand DNA synthesis is initiated near the 5' end of the viral RNA, where only a short fragment of the genome is copied. In order to continue DNA synthesis the virus employs a complicated mechanism, which enables transferring of the growing minus strand DNA to a remote position at the genomic 3' end. This is called minus strand DNA transfer. The transfer enables regeneration of long terminal repeat sequences, which are crucial for viral genomic DNA integration into the host chromosome. Numerous factors have been identified that stimulate minus strand DNA transfer. In this review we focus on describing protein-RNA and RNA-RNA interactions, as well as RNA structural features, known to facilitate this step in reverse transcription.

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RNA Biology 8:2, 230-236; March/April 2011; © 2011 Landes Bioscience
230 RNA Biology Volume 8 Issue 2
The human immunodeficiency virus type 1 (HIV-1) belongs to
the retrovirus family, members of which have two copies of a
single stranded RNA genome. The genome encodes only a small
set of genes important for viral survival, relying primarily on
host cell factors to complete the viral life cycle. As with all ret-
roviruses, the replication of HIV-1 requires the RNA genome to
be reverse transcribed into DNA soon after the virus enters the
cell. The DNA copy of the virus is subsequently transported into
nucleus for incorporation into human DNA to become perma-
nently linked with its host. The DNA form of HIV-1 or provirus,
may stay latent for a long period of time, but eventually will be
expressed to form new viral particles, which destroy the cell and
go on to infect new cells.
Reverse transcription has been a main target in treatment of
HIV infections, as it is an imperative step in viral replication.
Extensive research is being conducted to understand the mecha-
nisms of this multistep process. Drugs developed to interfere
*Correspondence to: Robert A. Bambara; Email: robert_bambara@urmc.
rochester.edu
Submitted: 10/01/10; Revised: 01/05/11; Accepted: 01/11/11
DOI: 10.4161/rna.8.2.14802
After HIV-1 enters a human cell, its RNA genome is converted
into double stranded DNA during the multistep process of
reverse transcription. First (minus) strand DNA synthesis is
initiated near the 5' end of the viral RNA, where only a short
fragment of the genome is copied. In order to continue DNA
synthesis the virus employs a complicated mechanism, which
enables transferring of the growing minus strand DNA to a
remote position at the genomic 3' end. This is called minus
strand DNA transfer. The transfer enables regeneration of long
terminal repeat sequences, which are crucial for viral genomic
DNA integration into the host chromosome. Numerous factors
have been identied that stimulate minus strand DNA transfer.
In this review we focus on describing protein-RNA and RNA-
RNA interactions, as well as RNA structural features, known to
facilitate this step in reverse transcription.
Requirements for ecient minus strand
strong-stop DNA transfer in human
immunodeciency virus 1
Dorota Piekna-Przybylska and Robert A. Bambara*
Depart ment of Biochemistr y and Biophysics and the Ce nter for RNA Biology; Un iversity of Roches ter School of Medicine a nd Dentistry; Roc hester, NY USA
Key words: minus strand DNA transfer, reverse transcription, RNA genome circularization, HIV-1, retroviruses
Abbreviations: LTR, long terminal repeat; PBS, primer binding site; (-)ssDNA, minus strong stop DNA; RT, reverse transcriptase;
NC, nucleocapsid protein; DIS, dimerization initiation site; TAR, transacting responsive hairpin
with viral reverse transcription have provided effective treatment,
because they prevent the completion of proviral DNA synthesis
and its integration in the host genome. In this review, we focus on
an early step in HIV-1 reverse transcription, called minus strand
DNA transfer. This is a complicated, but obligatory event in con-
version of the RNA genome into DNA and critical in generation of
the DNA provirus in a form for its integration into host cells DNA.
First (minus) Strand DNA Transfer
The genomic RNA of HIV-1 consists of several major genes that
code for essential proteins and enzymes needed for viral replica-
tion and maturation. The protein coding region is flanked by two
unique sequences, U5, at the 5' end and U3 at the 3' end and two
identical sequences of the repeat (R) elements at both ends. These
regions do not encode proteins, but contain regulatory sequences
important for viral replication. During the process of reverse
transcription, U5 and U3 are duplicated to create U3-R-U5 at
both ends of the proviral DNA, segments known as long terminal
repeats (LTRs). These are important regions used by the virus to
mediate its own integration into the host chromosome. In addi-
tion, the upstream LTR acts as a promoter and enhancer and the
downstream LTR acts as transcription termination and polyad-
enylation site.1 The binding of cellular and viral proteins to these
regions regulates HIV gene expression.2 ,3
Duplication of unique sequences U3 and U5 in the LTRs is a
result of the minus strand DNA transfer during reverse transcrip-
tion (Fig. 1A). Conversion of RNA into DNA is performed by
the viral enzyme reverse transcriptase (RT). The process of first
(minus) strand DNA synthesis is initiated from a host cellular
tRNA3Lys that is captured within the viral particles when they
are formed. The 18 nt at the 3' end of the tRNA are bound to
the complementary sequence of the primer binding site (PBS)
localized 182 nt from the 5' end of the HIV-1 RNA genome, just
upstream of the unique sequence U5. The RT extends the primer-
tRNA copying the U5 and R elements and synthesizing 181 nt
long cDNA known as a minus strong stop DNA ((-)ssDNA).
The synthesis of minus strand DNA can be continued only after
the (-)ssDNA is transferred to the 3' end of viral RNA genome

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different RNA-RNA and RNA-protein interactions have evolved
to ensure that it is rapid and efficient.5,6
Protein Activities Promoting Minus Strand Transfer
Conversion of genomic RNA into the DNA provirus requires two
proteins encoded by the genome, the RT and nucleocapsid pro-
tein (NC). In HIV-1, the polymerization activity of RT is accom-
panied by an R Nase H activity. RNase H is essential for RNA
degradation within the hybrid duplexes of synthesized cDNA and
the RNA that was used as a template during reverse transcription.
The RT is composed of two subunits, p66 and p51 (66 and 51
kDa). Both activities are located in subunit p66, whereas p51 acts
as a structural polypeptide for proper conformation of p66.7, 8 The
p51 is formed by proteolytic cleavage of the C-terminal domain in
p66 subunit.9 The RNase H active site in RT is located in the C
domain and is separated from the polymerization site by 18 bp in
DNA:RNA heteroduplex substrates.10,11 This configuration allows
RT to make cleavages in RNA during cDNA synthesis, which is
known as polymerization-dependent RNase H activity.12 However,
the polymerization rate of RT is greater than the rate of RNA
hydrolysis, thus polymerization-independent cuts, made during
revisits of RT molecules to remaining RNA oligonucleotides, are
necessary for complete removal of the genomic template.12,13 A sin-
gle virion contains about 50 molecules of RT enzyme.14 The excess
RT molecules, beyond what is apparently required for synthesis,
may be important in carrying out RNase H cleavage in order to
clear the minus strand DNA to allow efficient strand transfer,
accurate priming of plus strand synthesis and unimpeded synthesis
of the plus strand.
Secondary structures in the RNA genome, such as hairpins
and G-quartets can pause the RT during DNA synthesis pro-
moting RNase H cleavages in the RNA template.15,1 6 Studies in
vitro have demonstrated that the TAR hairpin at the 5' end of
the RNA genome causes a major pause in synthesis by RT.17 The
associated RNase H activity of RT cleaves RNA approximately
14–20 nucleotides downstream from the pause site within the
polyA hairpin, that serves as an initiation site for the invasion-
driven mechanism of minus strand transfer (Fig. 1B). The RNase
H cuts create a short gap where the homologous sequence of R
from the 3' end can invade and anneal to the cDNA and initi-
ate displacement of adjacent segments of the 5' end RNA. The
strand exchange continues by a branch migration process until
it reaches the 3' terminus of synthesized (-) ssDNA completing
minus strand DNA transfer.18,19
The polymerization activity of RT enzyme is greatly facili-
tated by NC protein. In fact, many steps of the retroviral life
cycle, including the assembly of virus particles, genomic RNA
dimerization and packaging, reverse transcription and integra-
tion into the host genome require NC involvement.20 -24 NC is a
small (55 amino acid) protein with nucleic acid binding activity,
processed from a precursor polypeptide encoded by the gag gene.
A single virion contains 2,000–3,000 molecules of NC, coating
the RNA genome with binding sites of 7–8 nt.25,26 The key func-
tion of NC is chaperone activity, which is refolding of nucleic
acids into the most thermodynamically stable conformations that
and this process is known as a minus strand DNA transfer. Since
both R elements in the viral genome are identical, the (-)ssDNA
will interact with the 3' end of the RNA genome through comple-
mentarity of their sequences. The consequence of minus strand
transfer is that the U3 region can be copied and the first U3-R-U5
sequence of the 3' LTR is created. Subsequently, this region is used
as a template to synthesize a second (plus) strand DNA, which is
next transferred to the beginning of the viral genome (plus strand
transfer) in order to create the 5' LTR (see Fig. 1 in ref. 4).
Since replication at the ends of the genome is not continuous
and requires interaction between distant related sequences of R at
the 3' end and in growing cDNA at the 5' end, a number of fac-
tors are involved to ensure effective reverse transcription, which is
necessary for viral fitness and long term survival. The first strand
transfer is a rate-limiting step in reverse transcription and many
Figure 1. Minus strand DNA transfer in HIV-1. (A) After entering a cell,
HIV-1 undergoes a process of conversion of its RNA genome into double
stranded DNA. The DNA synthesis is initiated by the extension of the 3'
end of tRNA3Ly s (red) used as a primer to start reverse transcription. The
U5 and R regions are copied into the minus strong stop DNA ((-)ssDNA)
which is transferred to the 3' end of the RNA genome during the pro-
cess of minus strand transfer. The nal product of reverse transcription
is the DNA provirus with two LTRs anking the ends of the HIV genome.
(B) In the invasion-driven mechanism of transfer, the pausing of RT (blue
oval) at the base of the TAR (hairpin with green loop) initiates RNase
H cleavages (blue triangle) within the polyA hairpin (orange loop)
creating a gap for the invasion of the 3' end of the viral genome and
interaction with the nascent cDNA. This initiates the strand exchange
and displacement of adjacent segments of the 5' end RNA until the 3'
terminus of the synthesized (-)ssDNA is fully transferred (not shown).

232 RNA Biology Volume 8 Issue 2
The TAR hairpin has the same structure in LDI and BMH.
Interestingly, structure analyses of both RNA donors, D199 and
D520, revealed that each adopts a different conformation. The
longer RNA donor has a structure similar to LDI, whereas the
shorter RNA donor with lower efficiency of minus strand transfer
resembled the 5'-side of the BMH.40 Possibly the conversion of
one structure to the other occurs during (-)ssDNA synthesis and
transfer, and aids the process. Interestingly, the BMH structure
was proposed to favor RNA genome dimerization and pack-
aging into the virion.42-44 With the DIS formed as a hairpin
in BMH conformation, the kissing loop base pairing between
have the maximal number of base pairs.27 The protein has two
zinc fingers for interaction with ssR NA, ssDNA and dsDNA,
and has the ability to destabilize nucleic acid helices and cause
nucleic acid aggregation.2 8,29
Studies in vitro have shown that the presence of NC during
reverse transcription increases the efficiency of the various steps
and reactions. NC transiently eliminates secondary structures
such as hairpins.30-33 This activity of NC greatly reduces RT paus-
ing and increases the efficiency of DNA synthesis. During syn-
thesis of minus strand DNA the highly structured TAR hairpin
at the 5' end of the RNA template is destabilized with the help
of NC. Although, the pausing of RT is reduced at TAR, the effi-
ciency of minus strand transfer is higher in the presence of NC.
Studies by Purohit and coworkers showed that while the pausing
of RT diminishes in the presence of NC protein, some RNase H
cleavages increase due to enhanced annealing of cDNA to the
RNA template.34 Moreover, NC enhances annealing between
nascent DNA and the invading 3' R sequence and subsequently
promotes strand exchange, which progresses continuously until
the minus strand transfer process is completed.18,19,29,35 ,36
Analyses of minus strand transfer in vitro have demon-
strated that NC protein also significantly inhibits a self-priming
effect.37, 3 8 The 3' end of (-)ssDNA corresponds to the sequence
of TAR, and so this region has the potential to fold back and
form a similar hairpin, which can self-prime DNA synthesis and
inhibit minus strand transfer. The primary basis of self-priming
suppression in the presence of NC is promotion of an exchange
of the very 5'-most RNA oligomer left from polymerization-
dependent RNase H with the homologous RNA sequence of
the genomic 3' end, leading to minus strand DNA transfer.39
Local RNA Structure as an Important Factor
in Minus Strand Transfer
The reconstituted systems used to analyze minus strand DNA
transfer in vitro have demonstrated that using different lengths of
the RNA representing the 5' end of HIV-1 (donor RNA) results in
different transfer efficiencies. The cDNA synthesized in vitro on
the RNA template spanning the region from the 5' end up to PBS
(D199) transfers with low efficiency to the second RNA (acceptor
RNA) representing the 3' end of the virus. However, the efficiency
of transfer increases dramatically when donor RNA is extended at
its 3' end (D520) to include naturally occurring sequences present
beyond the PBS.40 The transfer of cDNA synthesized from both
donor RNAs uses the same acceptor invasion-driven mechanism,
but that mechanism is more effective when a longer donor RNA is
used. This implies that folding properties of donor RNAs having
different lengths influences the transfer reaction, indicating that
local structure is an important influence on minus strand transfer.40
Analyses in vitro demonstrated that the 5'-untranslated region
in HIV-1 can adopt two distinct structures (Fig. 2A).41 A long-
distance base-pairing interaction (LDI) between the polyA and
dimerization initiation site (DIS) can be formed into a ther-
modynamically stable structure. However, the sequence can
refold into the branched multiple hairpin (BMH) conformation,
which allows the polyA and DIS motifs to fold into hairpins.
Figure 2. The local structure of the 5' end of the HIV-1 RNA genome
and its proposed interactions with the 3' end. (A) The untranslated
RNA at the 5' end can adopt two alternative structures: a long distance
interaction (LDI) and a branched multiple-hairpin (BMH) structure.
Sequences of loops of the hairpins TAR (green, in HIV-1 positions
1–57), polyA (orange, 66–95), PBS (black, 182–199), DIS (red, 243–277),
SD (blue, 282–300), Ψ (yellow, 306–331) are indicated. (B) Proposed
RNA-RNA and cDNA-RNA interactions during circularization of the RNA
genome in HIV-1. Sequences of loops of the hairpins TAR (green, 1–57
and 9,075–9,135), polyA (orange, 66–95 and 9,141–9,170) and PBS (black,
182–199) are indicated. Sequences of motif 9 nt (light blue, 9,033–9,041),
segment 1 (dark blue, 9,008–9,019), gag (619–696), tRNA (red) and the
cDNA (grey) are also shown. The bulk of the genome is designated by a
dotted line. Potential regions of interaction are shown in proximity.

www.landesbioscience.com RNA Biology 233
indicated in retroviruses 35 years ago.56,57 In subsequent years it was
discovered that many single-stranded RNA viruses adopt a circular
conformation, which is essential for different stages of their viral
life cycles.58 Viral genome circularization is known to stimulate
initiation of translation, as is the case for many cellular mRNAs.58
Juxtaposing of viral ends also facilitates transcription, genome rep-
lication and viral packaging.59-62 In many positive strand viruses,
such as ratoviruses, picornaviruses and pestiviruses, a 5'-3' end con-
tact is mediated through RNA binding protein interactions, which
can involve both viral and cellular factors.60,63-66
Alternatively, viruses can circularize their genomes via long-
distance RNA-RNA interactions. With application of atomic
force microscopy it was shown that the dengue virus genome can
form a circle in the absence of proteins.67 RNA-RNA genomic
cyclizations have been documented in many plant viruses,59 fla-
viviruses,60,68-70 hepatitis C virus,71 FMDV (Foot-and-mouth dis-
ease virus),72 and also in HIV-1.73
Several different types of RNA-RNA interactions were
described that could mediate HIV-1 genome circularization. A
palindromic sequence of 10 nucleotides in stem-loop of the TAR
hairpin in the R element was indicated as a possible place of
interaction involved in genome dimerization, in addition to the
primary site of dimerization at DIS.74 Since the TAR is present
at both ends of the viral RNA, the palindromic sequences could
interact and circularize the HIV-1 genome (Fig. 2B).75 This is a
way that the sequences of R elements involved in minus strand
transfer would self-juxtapose by direct interaction. Stem-loops
and loop-loop kissing structures formed by sequences at both
ends of the RNA genome were demonstrated to participate in
genome circularization in many plant viruses.59 Howe ver, ana l-
yses of mutations in TAR sequences that should have disrupted
the putative binding showed that 3'-5' end interactions in vitro
were not significantly affected.75 Apparently, other factors in
addition to TAR hairpins maintain genome circularization,
whereas putative base pairing between TAR sequences at both
ends of the genome might still help in juxtaposing R elements
for minus strand transfer.
Another type of RNA-RNA interaction involves a region in
gag and the 3' U3/R (Fig. 2B). Here, a structure is formed with
extensive base pairing among nucleotides within the gag ORF
(positions 619–696, clone pNL43) and sequences flanking the
TAR hairpin, 18 nt of U3 (positions 9,054–9,071) and the polyA
hairpin of the 3' R (positions 9,136–9,170).73
Interactions between cis-acting sequences of viral RNA
genomes were initially documented for many flaviviruses.61 ,67-7 0
Here, genome circularization is maintained by short comple-
mentary cyclization sequences (CS) present in the capsid coding
region and at the 3' end of the RNA genome. For dengue virus,
additional sequences described as UARs (upstream AUG regions)
were also found in proximity to the CS, and UARs were shown
to be significant for viral viability.67 A similar 3'-5' RNA-RNA
interaction was reported for yeast LTR Ty1 retrotransposon.76
LTR retrotransposons are thought to be ancestors of retrovi-
ruses.7 7,7 8 The interactions involve base pairing of complementary
14 nucleotide long sequences, called CYC5 in the gag ORF and
CYC3 in U3.76 The reconstituted system in vitro demonstrated
complementary sequences in the loop will lead to dimerization
of viral genomes.45,46 These structural inter-conversions may
coordinate sequential events in virus assembly, reverse tran-
scription and protein translation.
Extensive studies in vitro revealed that the structure of the
sequences involved directly in the minus strand transfer is also
important. The sizes of the R elements in different retroviruses
vary significantly from 16 bases in mouse mammary tumor virus
(MMTV) to 247 bases in human T-cell leukemia virus type 2.4 7,4 8
However, a significant shortening of the R sequences affects the
efficiency of minus strand transfer.49-51 Moreover, early strand
transfers of partially synthesized cDNA are not frequently seen
wit h HI V-1.52 Although the (-)ssDNA synthesized at the 5' end
of the viral genome has to hybridize with the complementary R
region at the 3' end, the length of complementarity is apparently
not the only factor important for efficient minus strand transfer.
The structure of the R elements is also crucial. A stable TAR hair-
pin at the 5' end is important for efficient minus strand transfer,
but disruption of this hairpin at the 3' end of viral genome is ben-
eficial for the transfer in vitro. However, stabilizing the polyA hair-
pin in the 5' and 3' R will inhibit the transfer reaction.53 Studies in
vitro have demonstrated that pre-incubation of NC with the 3' end
RNA template rather than with just the 5' end RNA, significantly
enhances the efficiency of minus strand transfer. This implies that
destabilization of hairpins at the 3' end by NC is beneficial for the
reaction.54 Overall, strong structure in the genomic 3' end appears
to decrease the efficiency of the (-)ssDNA transfer, but a highly
structured 5' end of the genome promotes the transfer.
The stimulatory effect of the folded genomic 5' end on minus
strand transfer is presumably important for the invasion-driven
mechanism of transfer, as described above (Fig. 1B). TAR struc-
ture pauses RT during cDNA synthesis, promoting R Nase H
cleavages in the copied template creating an invasion site for the
3' R RNA.17 Lack of structure in the 3' RNA likely promotes its
ability to invade and base pair with the cDNA.
Proximity of R Elements
in Minus Strand DNA Transfer
Analyses in vitro and in vivo have demonstrated that the length
of homology between the 97 nucleotide R elements in HIV-1
influences the efficiency of minus strand transfer.50,55 However, it
was shown that transfer within a homology of just 20 nucleotides
will not be affected in vitro, as long as the mutated 3' R sequence
still contains an unchanged polyA hairpin, needed for invasion
of the (-)ssDNA.55 The interaction between the invasion site in
the (-)ssDNA and the invading polyA hairpin of the 3' R brings
sequences involved in the completion of transfer at the (-)ssDNA
terminus into proximity.
The 5' and 3' R elements are over 9,000 nucleotides apart
in the linear form of the HIV-1 RNA genome. However, RNA
molecules are capable of forming complex tertiary interactions
between related sequences over very long distances. Circularization
of the viral genome in a way that juxtaposes the R elements should
facilitate the process of minus strand DNA transfer. The possibility
of genome circularization during reverse transcription was already

234 RNA Biology Volume 8 Issue 2
in Drosophila melanogaster.85 Here, the primer tRNA2Lys inter-
acts at the PBS and within the region upstream of the PPT and
U3 located about 450 nucleotides from the 3' end of the Gypsy
genomic RNA. The second interaction involves the nucleotides
of the TψC arm and Variable loop in tRNA2Lys.
In order to maintain the proximity of R elements for effi-
cient minus strand transfer, the scenario of the 5'-3' end interac-
tions in HIV-1 genomic RNA could be very complex. Consider
that RNA genome circularization might be already established
by the time of packaging of the viral genome. The circular
form of HIV-1 RNA could be maintained via interactions
between gag and U3/R sequences at the genomic 3' end. The
5' end would form a BMH conformation with DIS and TAR
hairpins exposed. Within the virion, the dimerization of two
RNA genomic molecules via TAR hairpins might no longer be
essential and could be replaced to allow base pairing between
TAR hairpins at the 5' and 3' ends. Proximity of the genomic
ends due to gag—U3/R contact will facilitate self-juxtaposing
of R elements via stem-loop kissing base pairing of TAR hair-
pins. Soon after reverse transcription begins, the interactions
between U5 and the anticodon stem in tRNA3Lys are unwound
enabling tR NA to bind with motif 9 nt in U3. This interac-
tion could help to maintain the proximity of R elements dur-
ing minus strand transfer, when contacts of the 3' R with gag
sequence need to be removed. As the synthesis of minus strand
DNA proceeds towards the 5' end of the genome, TAR inter-
actions will be disrupted. Furthermore, minus strand transfer
occurs within the 3' R element, and so the interactions with gag
have to be disrupted too. The binding between motif 9 nt and
tRNA3Lys would maintain circularization and bring segment 1
into proximity with the 3' end of the tRNA primer, facilitat-
ing tRNA displacement from the PBS. After the transfer of (-)
ssDNA the growing cDNA will disrupt all binding between U3
sequences and gag and the tRNA. If accurate, this interpreta-
tion explains why there must be a stepwise series of interactions.
Conclusion
The ability of HIV-1 to recombine frequently and error prone
reverse transcription are survival features of the virus that allow
it to evolve rapidly and avoid inactivation by the human immune
system. Drugs currently used to treat HIV infections target the
virus replication process. Although they can effectively inhibit the
viral replication machinery, HIV-1 is so effective at evolving drug
resistance that new treatments need to be developed. Targeting
the steps in viral replication, which are unique to the virus, offers
opportunities for treating viral infections without disruption of
human cellular function. Minus strand DNA transfer might be
a good target for interference of viral replication, as it is an early
and mandatory process preparing viral genetic material for inte-
gration into the host. Moreover, efficient minus strand transfer is
critical for maximum viral replication fitness.
Acknowledgements
Work from our laboratory described herein was supported by the
US National Institutes of Health research grant GM049573.
that contact between CYC5 and CYC3 is important for effi-
cient minus strand transfer and also to enhance initiation of
reverse transcription. Additionally, CYC5-CYC3 interactions
are required for Ty1 transposition.76 In the case of HIV-1, with
application of a reconstituted system, mutation analysis of a 3'
U3/R-gag interaction showed that this structure also enhances
efficiency of minus strand DNA transfer, in experiments using a
DNA primer to start reverse transcription.75
Genome circularization in retroviruses via tertiary interac-
tions between 5' and 3' genome ends may also involve other
nucleic acid molecules, such as the cDNA made de novo and
tRNA3Lys used by HIV-1 as a primer to start reverse transcription.
Specifically, it was proposed that growing cDNA extended from
the tR NA3Lys primer could interact via kissing loop contact of the
TAR hairpin segment in (-)ssDNA and TAR at 3' RNA genome
(Fig. 2B).53 Also, the invasion mechanism of minus strand trans-
fer itself is a mechanism for genome circularization (Fig. 1B).
Here, the interaction between the polyA hairpin at the 3' end
of the RNA and the invasion site in (-)ssDNA could hold both
of the viral genome ends together. In both cases, the interaction
only becomes possible after some (-)ssDNA synthesis.
The primer tRNA3Lys might also serve as a bridging factor inter-
acting with the 3' end of the viral genomic RNA (Fig. 2B). The
interaction would occur between nine nucleotides in U3 (motif
9 nt, positions 9,033–9,041) adjacent to the 3' R element and
nucleotides of the anticodon stem in tRNA3Ly s (positions 38–46).79
Computational analysis revealed that sequences flanking motif 9
nt also have complementarity to tRNA3Ly s, and the whole region
spanning extensive sequences in U3/R might represent an entire
intron-containing tRNA gene incorporated during evolution
of the HIV-1 genome.80 Analyses in vitro and in vivo have dem-
onstrated that motif 9 nt and complementary sequences in U3
(segment 1, positions 9,008–9,019) stimulate minus strand trans-
fer.7 9-81 Interactions between U3 sequences and tRNA3Lys appear
most likely to be established after initiation of reverse transcrip-
tion. The nucleotides of the anticodon stem in tRNA3Lys are first
involved in an interaction with sequences upstream of the PBS in
U5 in order to form the initiation complex for DNA synthesis.82, 83
In the case of segment 1, nucleotides are complementary to the
3' end of tRNA3Lys, where 18 nucleotides of the tRNA serve as a
primer and base pair directly with the PBS. Here too, the interac-
tions might take place only after the start of reverse transcription.
Whereas the binding between tRNA3Lys and motif 9 nt in
U3 could help in maintaining the R elements in proximity, the
significance of interactions with segment 1 might be different.
For example, segment 1 could help in displacement of the tRNA
from the PBS to allow copying of the 3' end of the tRNA into
DNA in preparation for second strand transfer.
Primer tRNA serving as a bridging factor in genome circular-
ization was also indicated for LTR retrotransposons and endog-
enous LTR retroviruses. The tRNAiMet is used as a primer to
start reverse transcription in Ty3 yeast retrotransposons. Whereas
the 3' end of this tRNA is bound at the PBS, the TψC arm and
D arm are involved in interactions close to the 3' end of the Ty3
RNA causing retro-element genome circularization.84 A similar
situation was described for the endogenous retrovirus Gypsy

www.landesbioscience.com RNA Biology 235
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