healthmedicinet

Retroviruses and the retroviral life cycle

Most retroviruses belong to the ortho-retrovirinae sub-family that is divided, according
to genome organization, into two major groups, the simple retroviruses and the complex
ones 1]. All retroviruses contain three principal coding domains with information for virion
proteins: (1) gag that directs the synthesis of internal virion proteins that usually form the matrix
and capsid structures. (2) Pol that encodes for the RT and IN enzymes. (3) Env that encodes for the surface and transmembrane glycoproteins of the viral envelope.
An additional smaller domain that is present in all retroviruses is pro that encodes the viral protease. This pro gene can be part of the gag (as in the case of the alpha-retroviruses), as an independent gene (in the beta-retroviruses),
or part of pol (as in gamma-retroviruses, lentiviruses or in delta-retroviruses) 1]. Simple retroviruses usually carry only this essential genomic information, whereas
the complex retroviruses encode also for additional small regulatory proteins that
are derived from multiply-spliced viral mRNAs.

The most unique process in the life cycle of retroviruses is the complex reverse transcription
step that takes place in the cytoplasm of the virus-infected cells 3], 4], 10], 11]. After penetrating the target cells, the retroviral single-stranded plus-sense RNA,
still contained within the viral core complex proteins, undergoes reverse transcription.
In this critical step, the viral RNA is copied into a double-stranded DNA by a combination
of the enzymatic activities of the core-associated RT enzyme. These activities are
the DNA polymerase, capable of copying both RNA and DNA, and the ribonucleases H (RNase
H) that hydrolyzes the RNA template in RNA–DNA heteroduplexes formed during reverse
transcription. This multi-step process involves also two template switches (or strand
transfers) that result in a duplication of sequences located at the 5? and 3? ends
of the virion RNA, so they are eventually fused in tandem to both ends of the generated
viral DNA, forming the long terminal repeats (LTRs). After completion of DNA synthesis,
the resulting product, still in complex with viral proteins (now called pre-integration
complex—PIC), is translocated into the nucleus and the DNA is integrated into the
host cell DNA by the enzymatic activities of the viral IN 2], 10], 12]. This integrated provirus becomes part of the cellular genome and, after activation,
is transcribed into the viral mRNAs. The unspliced full-length RNA can serve as the
viral progeny RNA genome. In addition, this mRNA and the spliced mRNA species are
used to synthesize the various viral proteins.

After infection with simple retroviruses, the transcription control is mediated primarily
by interactions of cellular factors with the viral LTR, whereas in complex retroviruses,
some viral regulatory proteins can affect transcription as well. Since most cells
targeted by exogenous infective retroviruses can survive the infection, once integrated
into germ cell genomes, the retroviral genomes can be transferred vertically in the
infected animals for millions of years. These sequences of the endogenous retroviruses
(ERVs), after being fixed in the evolutionary lineage, became integral part of most
eukaryotic cells. Consequently, these endogenous viruses vastly outnumber the exogenous
retroviruses 10], 13], 14]. Thus, human endogenous retroviruses (HERVs) constitute nearly 8 % of the human genome
15], 16]. Many of these sequences were fixed in the germ line of old-world monkeys after their
evolutionary separation from new-world monkeys about 35 million years ago.

dUTPases in general

Cellular dUTPases (EC 3.6.1.23) hydrolyze dUTP to dUMP and PPi, thus serving two essential
functions in DNA metabolism (Fig. 1). First, the dUMP product is a primary substrate for thymidylate synthase in the
major dTTP biosynthesis pathway. Second, dUTPases help to maintain low intracellular
dUTP/dTTP ratios. This is necessary to minimize the misincorporation of uracil into
DNA, since most DNA polymerases (except for some archaeal enzymes) cannot distinguish
between thymine and uracil and the uracil/thymine incorporation ratio depends on the
relative level of dUTP and dTTP. The misincorporation into the synthesized DNA of
the non-canonical deoxyribo-nucleotide, dUTP, can eventually result in mutagenesis,
for review, see 7], 9]. An excessive DNA repair of the uracilated DNA is initiated by uracil DNA glucosidases
(UNG) that remove the uracil, forming an apurinic DNA 7]–9]. This DNA is then repaired by a chain of enzymatic activities of apurinic endonuclease,
DNA polymerase beta and DNA ligase (Fig. 1). UNGs have evolved in all organisms as the most common form of DNA-repair enzymes.
Thus far, six groups of the UNG superfamily have been discovered and studied to varied
extents 17]. However, under constant high dUTP/dTTP ratios, uracil residues will be incorporated
again and again instead of thymidines during repair synthesis. This vicious circle
of uracil re-incorporation and repair is likely to lead to an accumulation of many
double strand breaks and strand exchanges that will eventually result in thymidine-less
cell death 18].

Given the critical role of dUTPases in cell metabolism, it is not surprising that
their presence was shown to be essential for the survival of both prokaryotic cells,
such as Escherichia coli19], and eukaryotic cells, such as Saccharomyces cerevisiae20]. The enzyme was also identified in Plasmodium falciparum, Mycobacterium tuberculosis, trypanosome and human cells, as well as in DNA viruses and in several groups of
retroviruses and even some bacteriophages (for an updated review—see 7]). Accordingly, inhibiting cellular dUTPase activities by drugs can impair cell growth.
Therefore, this approach might be also applied for treating infections by specific
pathogens. However, to attain selectivity against the pathogens (without debilitating
the host cells), this strategy is particularly applicable for treating infections
by protozoan organisms, as their dUTPases have evolved differently from bacteria and
eukaryotic cells, thus forming a completely distinct family of proteins 21].

In many cases, from mammal to plant cells, cellular dUTPases were shown to be both
development and cell cycle regulated, with elevated activity in undifferentiated dividing
cells and low levels in terminally-differentiated and/or non-dividing cells 22]–24]. Consequently, the levels of cellular dUTPase activity may parallel the size of the
deoxynucleotide pool, which is high in dividing cells, such as activated lymphoblasts,
and very low in non-dividing cells, such as macrophages 25]. In some eukaryotes, two dUTPase isozymes are generated by mRNA alternative splicing
or by using alternative promoters. Thus, human cells contain nuclear and mitochondrial
isoforms, where the nuclear one is under cell cycle control, while the mitochondrial
isoform is constitutive 26].

Interestingly, recent reports that use different models have done away with the dogma
that DNA uracilation is always deleterious. Thus, deamination of cytosine bases in
DNA to uracil by the activation-induced deaminase is obligatory for the diversity
of immunoglobulin genes 27], 28]. Moreover, rather than being dangerous, HIV DNA uracilation can benefit the early
phase of the viral life cycle by inhibiting auto-integration 29]—see below. Finally, it was recently demonstrated that Drosophila melanogaster tolerates high levels of uracil in DNA during some developmental stages, suggesting
a novel role of uracil-containing DNA in Drosophila30]. Assuming that DNA uracilation is beneficial to some biological processes, and since
cellular dUTPases are involved in this scenario, it may be that dUTPases can also
perform other unrelated cellular functions. Indeed, a line of novel studies has shown
that, apart from their pivotal role in lowering dUTP levels, cellular dUTPases play
other roles in regulating several key cellular processes—by serving as signaling molecules
in both prokaryotic and eukaryotic cells. Thus, dUTPases were shown to be involved
in the transfer of mobile genetic elements that carry and disseminate virulence genes
in prokaryotes and in the regulation of the immune system in autoimmunity and apoptosis.
These unexpected dUTPase-associated “moonlighting” activities (defined as activities
of catalytically-active proteins with divergent dual functions) open new research
opportunities to explore the mechanisms, by which they serve as cellular regulators.
Since these diverse regulatory functions of dUTPases were extensively reviewed recently
31], we will not elaborate here on these exciting and promising novel aspects of dUTPases.
However, it should be noted that even in some DNA viruses, dUTPases affect host-cell
interactions via mechanisms independent of dUTP hydrolysis. Recent studies have shown
that the dUTPases, encoded by the gamma-herpesviruses, Epstein-Bar virus, Kaposi’s
sarcoma-associated herpesvirus and MHV-68, control the immune system. This is done
by various mechanisms, such as, upregulating cytokines, activating NF-kB pathway,
targeting cytokine receptors or down-regulating ligands recognized by NK cells (thus,
controlling NK attacks) and inhibiting type I interferon signaling pathway 32]–34]. dUTPases of other human herpesviruses were found to modulate dendritic cell function
and innate immunity 35]. Moreover, some of the data show that enzymatically-inactive dUTPases can also perform
these regulatory functions, suggesting that their biological roles are not merely
associated with their dUTPase enzymatic activity. Further studies are required to
explain how these functions evolved to be associated with dUTPase enzymes. Likewise,
new interesting evidence, regarding the potential involvements of the dUTPases of
HERVs in human diseases, also alludes to possible cellular regulations that are independent
of catalytic activity of dUTPase 36], 37], see below.

Structure and catalysis of dUTPases

While the gamma-phosphate group of nucleoside triphosphates is relatively reactive
(as in ATP or GTP), the alpha-phosphate position, as well as the phosphodiester bonds
in nucleic acid, are significantly more inert, thus help prevent aberrant modifications
38]. Reactions at these sites require powerful enzyme catalysts, such as nucleases, polymerases
39] and dUTPases 7]. Almost all studied dUTPases, including those of bacteria, human and retroviruses,
are homo-trimeric proteins 7]. The homo-trimers form three identical active sites in a symmetric fashion—see Fig. 2. In contrast, herpesviruses 40], 41] and Caenorhabditis elegans42] dUTPases are distinct as monomers. Here, trimer–mimicking monomers are formed from a genome that encodes all three enzyme monomers,
within the same gene (with linker regions located among the subunits). A third occasion
was reported for protozoan dUTPases that function as dimers, but contain none of the
five conserved sequence motifs typical of the dUTPases of the former two groups. Thus,
they have probably evolved differently from dUTPases in bacteria and other eukaryotes
21], 43].

Fig. 2. The three-dimensional structures of human and bacterial dUTPases. The homo-trimeric
E. coli (2HRM) and Human (3ARN) dUTPases were illustrated using the Jsmol internal viewer
(http://www.PDB.org). Structures are presented in the front C3 axis orientation, with each subunit in
a different color. Ligands (methylene-dUTP in the E. coli enzyme and dUTPase inhibitor in the Human counterpart) are presented in stick configuration.

In general, homo-trimeric or trimer-mimicking dUTPases, including the retroviral ones,
are characterized by a series of five conserved amino acid motifs 7] (see also below). Among mammalian viruses, this common set of motifs was initially
identified by sequence comparisons in herpesviruses, retroviruses and poxviruses.
Thought initially thought to be “pseudo-protease”, comparisons of these motifs with
the E. coli dUTPase sequence and other known dUTPases revealed their identity as unique dUTPase
motifs 5], 44]. The importance of these conserved motifs for the catalytic function was established
by mutagenesis and by X-ray crystallography of a variety of dUTPases. To prevent wasteful
and undesired hydrolysis of energy-rich NTPs or dNTPs, dUTPases must be highly specific
to their related substrate. This is provided by two major mechanisms, reviewed in
7]. In short, the first one is a steric exclusion of purines, thymine and ribose and
the second is hydrogen bonding that is specific to uracil. The steric hindrance is
mediated by residues from motif 3, which form a tight beta-hairpin that binds uracils
and deoxyribose and exclude thymine and purines. Altogether, the specificity of dUTP
binding by dUTPases is provided by the uridine moiety that fits precisely into the
enzyme’s active site 7]. The dUTPase binding pockets, present in each of the three subunits are highly specific
for uracil. Phosphate chain coordination involves magnesium ions and is analogous
to that in DNA polymerases. Due to conformational changes in the enzyme during catalysis,
most crystal structures have not resolved the residues in the C-terminus. All homo-trimeric
dUTPases share similar mechanisms that lead to efficient catalysis. In several crystal
structures, a water molecule is positioned for a nucleophilic attack on the dUTP alpha-phosphate
that leads to phosphate ester hydrolysis. This attack is coordinated by the side chain
of the highly conserved aspartate that is located in motif 3 45], 46]. Interestingly, in herpesvirus dUTPases, though the conserved motifs are preserved,
they are arranged in a different manner. Yet, these motifs still fold similar to usual
trimeric dUTPases 7], 41].

The dUTPase-encoding genes in retroviruses

Despite their variable positions, dUTPase-encoding genes in many dUTPase-encoding
organisms were observed to be adjacent to other genes that are involved in nucleotide
metabolism, such as ribonucleotide reductase, transcription initiation factors, primase,
and DNA synthesis flavoprotein 44]. A similar pattern of gene localization also exists in retroviral dUTPases, where,
in most cases, the dUTPase-encoding genes are located in the same genome segment encoding
for the viral RT, IN and or PR proteins. In addition, in the case of beta-retroviruses
the nucleic acids binding protein (NC) is an integral part of the viral dUTPase (see
Fig. 3).

Fig. 3. The biogenesis of dUTPase-expressing retroviruses. This schematic description of the
various precursor polyproteins, encompassing the dUTPase proteins, reflects also the
position of the dUTPase-encoding genes in the different retroviral groups (described
in detail in the text). These schemes are not drawn to scale.

Unlike most other retroviral enzymes, in the dUTPase-expressing viruses, the encoding
gene is situated in two different genomic locations, suggesting two diverse evolution
pathways (Fig. 3). In the beta-retroviruses, the gag–pol genes have three reading frames (gag, pro and pol) 1] and the dUTPase protein is encoded by both the gag and pro reading frames 47]–51]. Consequently, the dUTPase protein is actually a trans-frame polypeptide. To be more
specific, the dUTPase N-terminal segment (of about 90 residues) is derived from the
C-terminus of the Gag polyprotein. Therefore, this segment is identical to the entire
viral nucleocapsid (NC) protein 52] (also known in these viruses as p14). The C-terminus of the dUTPase is encodes by
the 5? portion of the pro gene, ending adjacent to the PR-encoding segment. In these viruses, the total length
of the dUTPase is about 240 amino acids residues and it is a proteolytic product of
the Gag-PR polyprotein precursor. This fused Gag-PR polyprotein results from ribosomal
frameshifting that occurs during translation (see 53]). In contrast, the gag–pol genes of lentiviruses have only two reading frames. In the dUTPase-expressing lentiviruses,
the encoding gene is located within the pol gene, between the RT’s RNase H (C-terminal) and the IN-encoding parts. Thus, dUTPase
is a proteolytic product of the Gag–Pol polyprotein precursor. Interestingly, the
polypeptide length of the majority of the lentivirus-associated dUTPases is about
half (~130 residues) of that of the beta-retroviral dUTPases. A rare exception to
the pattern in non-primate lentiviruses was reported for scarcely studied endogenous
retroviruses (ERVs) 54]–56]. Here, the dUTPase gene is located also within the pol gene, but rather at its 3? terminus; hence, it is C-terminal to the IN (Fig. 3).

Since all reoviruses have very small genomes, any genetic information included in
these genomes must be vital to the viruses. Therefore, even with little knowledge
about the importance of virus-coded dUTPases, it is highly likely that they are essential
for replication. Despite sharing similar mechanisms of replication, only several groups
of retroviruses encode dUTPases, while others lack this enzyme. The major dUTPase-expressing
retroviruses are the beta-retroviruses and the non-primate lentiviruses, whereas most
other viral groups (including primate retroviruses) lack the enzyme. It might be that
this difference in the requirements for a viral-encoded enzyme depends on the cells
infected by the viruses. Thus, alpha or gamma-retroviruses that lack a viral dUTPase
replicate mainly in dividing cells, which have high endogenous dUTPase levels, while
the dUTPase-expressing retroviruses can infect also non-dividing cells with low dUTPase
activity (vide supra). In several retroviruses, early DNA sequence comparison analyses
have suggested some resemblance of unidentified genes to the viral protease; hence,
they were initially termed protease-like domains or pseudo-proteases 57]. Only later on, the homology to the dUTPase gene was confirmed 5]. The key study by Elder and associates has subsequently confirmed the presence of
a catalytic dUTPase activity in particles of several retroviruses 58].

In the dUTPase-expressing retroviruses, the location of encoding gene can affect the
level of the protein’s expression. The gag portion of the gag–pol polycistronic mRNA is translated approximately 20 times more than the entire polycistron
that has to undergo one or two ?1 nucleotide frameshifting events to complete translation
1], 53]. Thus, in the beta-retroviruses, where the N-terminal part of dUTPase is derived
from Gag, there is a higher level of dUTP expression compared to non-primate lentiviruses.
Indeed, such a high expression enabled one of us to detect the first retroviral dUTPase-related
protein in virions of mouse mammary tumor virus (MMTV), already in 1987 49]. Relatively large amounts of the protein (designated at the time as p30, due to its
~30 kDa size) were isolated from virions and analyzed by protein sequencing. The data
showed that this protein is a trans-frame protein, as its N-terminal sequence was
identical to the viral NC, and its C-terminus was derived from the N-terminal half
of Pro. Only later on, after a study on the presence of dUTPases in retroviral virions
was published 58], this MMTV p30 was expressed as a recombinant protein and shown to possess a dUTPase
catalytic activity 48], 51].

Like most dUTPases, all studied retroviral dUTPases are homo-trimeric in their three-dimensional
structure (Fig. 4). Accordingly, enzymatically active retroviral dUTPases possess the five conserved
domains, typical to all homo-trimeric or trimer-mimicking dUTPases, Fig. 57]. Some families of endogenous retroviruses, notably, HERVs K and ERVs share also dUTPase-related
motifs with the five conserved segments 13], 59], 60]—see below. In contrast, the putative dUTPases of the non-primate lentivirus, bovine
immunodeficiency virus (BIV) was recently shown by us to have a sequence with only
a partial resemblance to these conserved motifs with no detectable enzymatic activity
61]—see below. The phylogenetic tree of most exogenous retroviral dUTPases, discussed
below, is shown in Fig. 6. It is highly likely that after their separation throughout evolution, the beta-retroviral
dUTPase-encoding gene has evolved separately than that of the non-primate lentiviruses.
For more general phylogenetic trees that include also dUTPases from endogenous retroviruses,
see 13], 60].

Fig. 4. The three dimensional structure of three representative retroviral dUTPases. The structures
of the homo-trimeric dUTPases of MPMV (3TRL), FIV (1F7D) and EIAV (1DUN) were illustrated
employing the Jsmol internal viewer (http://www.PDB.org). The structures are presented in the back C3 axis orientation with each subunit
in a different color.

Fig. 5. Multiple sequence alignment of the retroviral dUTPases. The sequences of dUTPases
in the major exogenous retroviruses described in the text were analyzed. The five
highly-conserved domains typical of dUTPases are indicated below the sequences. In
the case of the beta retroviruses, the NC-derived N-terminal sequences were not included
in the alignment. The sequences of the following dUTPases are as follows: EIAV (GI:
157830894); FIV (GI: 1942421); Visna (GI:9626549); BIV (GI: 9626219); CAEV (GI: 266706151);
JDV (GI:733067);MMTV (GI: :9626965);MPMV (GI: 9627210);SRV (GI: 334748);JSRV (GI:
9626914). The figure was prepared using the T-COFFEE alignment tool.

Fig. 6. Phylogenetic tree of dUTPase sequences from exogenous infective retroviruses. This
phylogenetic tree was constructed using amino acid multiple alignments and the neighbor-joining
method with ETE-toolkit (http://etetoolkit.org/treeview/), with the sequences shown in Fig. 5. Scale bar represents the p-distance (the observed number of nucleotide differences per site).

Beta-retroviruses

All beta-retroviral dUTPases are bi-functional due to their chimeric nature. Here,
the viral Gag-derived NC proteins are fused at their C-termini to the Pro-derived
dUTPase conserved domains. The dUTPase proteins from two prototype viruses of this
group, MMTV and Mason Pfizer monkey virus (MPMV), were the mostly studied. The information
pertaining to dUTPases from other viruses in this group, Jaagsiekte sheep retrovirus
(JSRV) and simian retrovirus (SRV), was mainly based on sequence homology to the more
studied dUTPases 47], 62], see Fig. 5. In all beta-retroviruses, the NC is 81–95 residues long and the Pro-derived segment
is 153–154 residues, out of a total of ~240 residues long polypeptide 63]. This means that the NC sequence can encompass more than a third of the whole enzymatically-active
dUTPase protein. Retroviral NC has a variety of activities that are central to viral
replication, as it has a nucleic acid chaperoning activity through its conserved basic
residues and zinc-finger structures, for a review—see 52]. This chaperone function, in conjunction with the protein’s aggregating function,
is up-modulated by successive NC processing events, resulting in the condensation
of the viral NC. Reverse transcription also depends on NC processing. Inducing NC
dissociation from double-stranded DNA leads to the formation of the PIC that is capable
of host chromosomal integration. In addition, NC interacts with cellular proteins,
some of which are involved in viral budding, and also with several viral proteins.
This collection of activities is likely to substantially affect the mature beta-retroviral
dUTPases (that were shown always to retain the NC). Indeed, three retroviral proteins,
IN, capsid and NC, were found to be capable of physical interaction with MPMV dUTPase
64]. This protein is present in stable form that resists proteolysis by retroviral and
cellular proteases in virions as well as in virus-infected cells. MPMV dUTPase retains
both nucleic acid binding and dUTP hydrolyzing catalytic activity. Sequence comparison
of beta-retroviral dUTPases with other dUTPases reveals that the beta-retroviral enzymes
have evolved to support the proper function of the NC protein fused to their dUTPase
domain. This evolution affected the whole sequence, except for the five conserved
motifs (see Fig. 5), as shown by the relatively low sequence similarity (30 %) between beta-retroviral
and other dUTPases 63]. The modifications, however, do not provoke changes in the protein’s overall fold.
Yet, due to the basic nature of the NC segment, the beta-retroviral dUTPases have
a much higher, basic pI, compared with the more acidic isoelectric points observed
in most dUTPases 7], 63]. The catalytic rate constant of the recombinant protein is, however, about tenfold
lower than in other dUTPases (including those from lentiviruses) 47]. In the case of retroviral dUTPases, this feature may compensate for the higher levels
of beta-retroviral dUTPase expression, relative to lentiviral dUTPases (see above).
In all, it may be that the coupling, within a single protein subunit, of the nucleic
acids binding function with dUTPase activity, could facilitate the attachment of the
dUTPase to sites, where DNA synthesis by RT takes place, thus hydrolyzing in situ
the incoming dUTP.

Enzyme kinetics of recombinant MPMV dUTPase and a truncated protein segment (without
the NC domain) suggested that the NC domain has no adverse effects on enzymatic activity
and that oligonucleotide binding to the NC domain may modulate enzymatic activity
47]. These results failed to provide an explanation for the ~tenfold lower catalytic
rate constant. A shorter linker region located between conserved motifs 4 and 5 was
observed in all beta-retroviral dUTPases relative to other dUTPases (including a four-residue
deletion relative to the non-primate lentiviral dUTPases)—see Fig. 5. This may suggest that the missing connecting residues can cause a steric constraint
that lowers the k
_cat
value. High-resolution X-ray structures, combined with modeling, indicate that the
fusion with NC domains alters the conformation of the flexible C-terminus by disturbing
the orientation of a critical beta-strand. Accordingly, this segment is capable of
double backing upon the active site of its own monomer and is stabilized by non-covalent
interactions formed with the NC terminal segment. In this case, the homo-trimeric
dUTPase fold is modulated in a specific manner that allows the accommodation of the
additional NC segment. Such a co-folding of the dUTPase terminal segments, which was
not observed in other dUTPases, results from the presence of the fused NC domain 63]. Elaborate studies on MPMV dUTPases were conducted to show the mechanism of the alpha
attack-mediated dUTP hydrolysis that is carried out by the enzyme 64]. Here, a combination of diverse structural methods, as well as the knowledge of other
dUTPases, unveiled molecular details of the catalytic nucleophilic attack and identified
novel enzyme-product intermediates.

Although the dUTPase of MMTV was identified and characterized before the MPMV counterpart
was investigated 48], 49], 51], substantially fewer studies were performed on the MMTV enzyme. A comparative study
of the dUTPases of MMTV, herpes simplex and E. coli showed that the two viral enzymes are less specific to dUTP than the bacterial one
65]. The MMTV enzyme has a reduced discrimination against dTTP and UTP, while it is still
selective against dCTP.

Despite the relatively large body of research conducted on beta-retroviral dUTPases,
we could not find any studies on the biological importance of the enzyme to the life
cycle and infectivity of the viruses. However, this missing information can be complemented
by the studies described below on involvement of dUTPases in the infectivity of the
non-primate lentiviruses.

Lentiviruses

Non-primate lentiviruses

Among all exogenous lentiviruses, dUTPase-encoding genes were observed in only the
non-primate lentiviruses. However, there are traits of these genes in some ERVs, including
in many HERVs (see below). Among the non-primate lentiviruses, dUTPase-encoding genes
are present in feline immunodeficiency virus (FIV) 66], puma lentivirus 67], equine infectious anemia virus (EIAV) 68], 69], caprine arthritis-encephalitis virus (CAEV) 70] and visna virus of sheep 71]. Additionally, in the bovine lentiviruses, BIV that is associated with a debilitating
cattle disease 72], and Jembrana disease virus (JDV), a homologous dUTPase-encoding gene is present
73], 74], see above (Fig. 5). In a rare case of the infectious small-ruminant genotype E lentivirus (isolated
from goats and sheep), almost the entire dUTPase genome is deleted 75]. As mentioned above, the dUTPase encoding gene in this retroviral group is part of
the pol gene and is situated between the RT and IN encoding segments (Fig. 3). Another feature that sets the non-primate lentiviral dUTPases apart from the beta-retroviral
counterparts (and from other known dUTPases as well) is the relatively shorter polypeptide
subunit, of about 130 residues (which is roughly half of the beta-retroviral dUTPases).
Despite this major difference, similar to other studied dUTPases, the three-dimensional
structure of EIAV dUTPase exhibits a homo-trimeric arrangement, where each subunit
folds into a twisted antiparallel beta-barrel with the N and C-terminal portions interacting
with the adjacent subunits 76]. A generally similar structure was reported also for FIV dUTPase 77], see also Fig. 4.

The majority of the biochemical studies on the dUTPases of the non-primate lentiviruses
were conducted on recombinant EIAV dUTPase. This enzyme was shown to be highly specific
to dUTP and sensitive to inhibition by dUDP, with little inhibition by other nucleotides
or the reaction products, dUMP and PPi 78]. In this study, mutational analyses were also performed by targeting a conserved
domain present at the C-terminus of all dUTPases. This domain shares high homology
with the phosphate binding loops (P-loops) of several ATP and GTP-binding phosphatases.
The P-loop-like motif of dUTPases is glycine rich, but lacks the invariant lysine
found in authentic P-loops. Deletion of this motif led to a loss of the enzymatic
activity. In addition, a series of point mutations in EIAV dUTPase that inactivate
these P-loops also abolished the dUTPase activity; thus establishing the importance
of these loops for catalysis. Another study compared EIAV dUTPase with the E. coli counterpart 79]. The results showed that the viral enzyme was as potent as the bacterial one in hydrolyzing
dUTP, albeit less specific. The inhibition of the EIAV enzyme by dTTP, dUMP and a
synthetic analog is stronger by one order of magnitude than that of the bacterial
counterpart. Transient kinetics of EIAV dUTPase showed that the rate constants for
the association and dissociation of substrate and inhibitors were consistent with
a one-step substrate binding mechanism 80]. After the flexible C-terminal part of the protein was removed by a limited proteolysis,
the dUTPase activity was totally quenched, although substrate binding was hardly affected.
This suggests that this terminus is indispensable for catalysis but not a for substrate
binding.

Most studies on the effects of dUTPases on the biology of non-primate lentiviruses
concluded that this enzyme is critical for replication only in non-dividing cells
(such as primary macrophages). In contrast, dUTPase-defective viruses can grow quite
well in dividing cells, where the dUTPase activity is supplied by the infected cell.
This result is consistent with the data presented above, showing that cellular dUTPases
are cell cycle regulated, with an elevated activity in dividing cells and low levels
in terminally differentiated non-dividing cells. This finding regarding the non-primate
lentiviruses was reported for the dUTPase of CAEV and visna 70], EIAV 68], 81], 82] and FIV 66]. The effects of viral dUTPase in virus-infected animals were also evident. Thus,
in FIV-infected cats, virus burden was reduced due to dUTPase impairment, particularly
in tissues, such as spleen and salivary gland 83]. The viral RNA load in plasma of Shetland ponies, infected with a dUTPase-defective
EIAV, was 10 to 100-fold lower than in animals infected with the wild-type virus 68]. In the case of CAEV, the dUTPase is necessary for the development of bilateral arthritis
lesions in the carpus of infected goats 84]. However, this is not always the case. Thus, visna virus dUTPase was found to be
dispensable for neuro-pathogenicity 71]. Likewise, the dUTPase gene of FIV is not essential for neuro-pathogenesis in cats
85].

As expected, the importance of the dUTPase to the retroviruses is linked to the misincorporation
of dUTP instead of dTTP. Several studies have linked the lack of dUTPase activity
with an increased incidence of mutations in the viral DNA, especially G to A substitutions
when the viruses replicate in terminally differentiated non-dividing cells. This result
was found in CAEV 84], EIAV 81] and FIV 83]. These findings indicate that uracil accumulation in the viral DNA can be detrimental
to the viral life cycle, although the precise mechanism it still not fully understood.
HIV-1 RT was shown to introduce G to A mutations in a simple in vitro DNA synthesis,
using highly-biased dNTP concentrations 86]. This can explain why many of the spontaneous mutations found in HIV-1 are G to A.
As mentioned above, the levels of cellular dUTPase activity may parallel the size
of the deoxynucleotide pool, which is high in dividing cells, such as activated lymphoblasts,
and very low in non-dividing cells, such as macrophages 25]. Therefore, it is possible that in these dNTP-deficient cells, there are dU misincorporations
across the template G, due to low dCTP concentrations, high dUTP (due to the lack
of dUTPase) and the relative stability of dU-G mispairs, These conditions will eventually
result in selective G to A transitions 84]. Apparently, despite the numerous studies conducted on dUTP misincorporation, this
hypothesis still calls for further experimental support.

The distinct case of BIV The putative dUTPase genes of BIV and JDV are distinct, though both follow the pattern
of non-primate lentiviruses 87], 88]. As in all non-primate lentiviruses, these genes are located between the RT and IN-encoding
genes. However, in both cases the encoded polypeptide is substantially shorter than
the ~130-residues protein of other lentiviruses, as it is only about 74 residues long
61], 73], 74]—see Fig. 5. This truncated polypeptide lacks extensive parts of the five conserved motifs, characteristic
of the homo-trimeric dUTPases, or the whole motifs 61]. As far as we know, no other dUTPase-related protein, including all viral, prokaryotic
or eukaryotic enzymes is so small. Still, it is highly likely that this dUTPase-related
peptide has an important biological role, since it is conserved in both BIV and JDV
87], 88]. Our recent study showed that recombinant wild-type BIV dUTPase and infectious wild-type
BIV virions were both dUTPase-defective, as no detectable enzymatic activity could
be shown 61]. To assess the importance of the dUTPase gene to BIV replication, we generated virions
of wild-type BIV or BIV with mutations in this gene. The two mutant dUTPases were
the double mutant, D48E/N57S (located in the putative dUTPase active site and its
vicinity) and a 36 residues deletion. Both mutant viruses were defective, as no progeny
viruses were generated. Surprisingly, the cells infected with the mutant virions carry
in their genomic DNA levels of integrated BIV DNA that were as high as in wild-type
BIV-infected cells. This result shows that the dUTPase-mutated BIV strains could infect
cells, as viral cDNA was synthesized and integrated. Yet, no new virions were generated
from the infected cells 61]. Interestingly, all experiments were conducted in dividing cells, where, as mentioned
above, the endogenous cellular dUTPase levels are supposed to be high. Therefore,
according to this prediction, there should be only a minor effect of mutating the
dUTPase-encoding gene, even in the case of an enzymatically-active dUTPase activity
(let alone in the present case, where no activity was noticeable).

To explain these puzzling results, we speculated that either the integrated cDNA of
the BIV mutants is defective (due to potential multiple mutations introduced during
reverse-transcription) or that dUTPase mutations led to blocks in viral replication
at steps post integration. These suppositions may implicate the involvement of BIV’s
dUTPase in processes other than dUTP hydrolysis, thus highlighting its importance
to BIV replication, despite the lack of any detectable catalytic activity. At this
stage, several unexplored alternatives are open. For example, it might be that the
viral protein interacts with cellular proteins (like UNG-see above) or it participates
in late (post integration) stages of the retroviral replication cycle. Likewise, given
the evidence on novel “moonlighting” activities of dUTPases, see above, such activities
may be associated with the dUTPase of BIV. The information listed below on the possible
involvement of HERV-K dUTPases with human diseases can also support this possibility.
In any case, further studies are being performed now by us to answer the raised questions.

Human and primate lentiviruses

Primate exogenous lentiviruses (such as HIV-1, HIV-2 and simian immunodeficiency virus-SIV)
evolved differently than other lentiviruses and are devoid of a dUTPase-encoding gene.
Yet, one study proposed a weak albeit significant sequence similarity between HIV-1
gp120 envelop protein and human dUTPase 89]. This information may suggest that an ancestral dUTPase gene has evolved into the
present CD4 receptor interacting region of gp120. Since these primate viruses can
replicate also in non-dividing cells, where the dUTP/dTTP ratio is high (due to low
cellular dUTPases, see above), it is likely that they can utilize other equivalent
means to counteract the emergence of uracilated viral DNA genomes. Indeed, as an alternative,
they can recruit one of the cellular UNG enzymes (UNG2) that are involved in the base-excision
repair pathway (see above and Fig. 1). In HIV-1, there are conflicting reports about the identity of the viral protein
that is responsible for this recruitment. Some studies implicated the viral IN 90]–92], whereas others the accessory protein, Vpr 8], 93], 94]. Interestingly, the HIV-1 associated UNG2 could be replaced by packaging into the
virions a heterologous dUTPase from CAEV. This finding suggests that UNG2 can counteract
the dUTP misincorporation that results from the lack of the dUTPase 95]. A recent study established the essential steps through which UNG2 initiates the
degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration
96].

Remarkably, there is another entirely independent mechanism to form uracil-containing
DNA in many lentiviruses that eventually leads also to G to A mutations. This process
is mediated by incorporating the cellular restriction proteins, APOBEC cytosine deaminases
into Vif-deficient virions. This eventually leads to the impairment of virus replication
due to C to U deamination of the synthesized viral cDNA. The APOBEC cytosine deaminase
activity is largely specific to single-stranded DNA substrates and requires a minimum
of five contiguous deoxy-nucleotides (three on the 5? side of the target cytosine
and one base on the 3? side of the target cytosine). In wild-type viruses, the viral
Vif protein recruits cellular CBF-? to form an E3 ubiquitin ligase complex that usually
leads the APOBEC degradation. Due to its cardinal importance to HIV infectivity, this
innate cellular anti-viral activity was heavily investigated, as part of the intensive
investigations of cellular factors that restrict HIV-1 (for comprehensive reviews—see
97]–100]). Therefore, we will not elaborate here on this process.

Taken together, as in the case of the other retroviruses, it is believed that uracilation
of the viral cDNA is detrimental to the retroviral life cycle. This conclusion was
also supported by in vitro evidence showing that the incorporation of uracils into
minus-strand DNA during HIV-1 reverse transcription affects the specificity of plus
strand synthesis initiation 101]. An interesting revision to the belief that uracilation has negative consequences
was proposed by showing that HIV-1 could tolerate, or even benefits, from non-mutagenic
uracil incorporation during reverse transcription 29]. Here, uracilation of the viral cDNA obstructs the strand transfer of the DNA ends
that is catalyzed by the viral IN, thereby inhibiting the suicidal auto-integration
side pathway and facilitating the correct chromosomal integration of viral cDNA and,
consequently, the viral replication.

Endogenous human retroviruses

About 8 % of the human genome comprise HERVs that represent fossilized sequences of
ancient exogenous retroviruses 15], see also above. These elements, distributed in about 400,000 loci and transmitted
vertically in a Mendelian manner, are classified into 30–40 families. Each family
can encompass up to thousands of loci 102]. HERVs were suggested to be associated with a variety of human diseases, including,
autoimmune diseases, neurological disorders and multiple malignancies, as well as
involvement in placentation 16], 103]. Several HERV families were reported to harbor dUTPase domains. A sequence survey
of various HERV families for the presence of dUTPase has found that ancestors of all
HERV-K families but one encode dUTPases 60]. This phylogenetic analysis shows a monophyletic origin for the different HERV-K
dUTPases. Sequences of the consensus dUTPase domains suggest that the various exogenous
ancestors of HERV-K once encoded active enzymes. Interestingly, a recombinantly-expressed
dUTPase was catalytically-active when a consensus sequence was constructed from independent
genomic clones of HERV-K. This presumably-ancestral wild type HERV-K dUTPases was
meticulously studied employing biochemical, mutagenic and structural approaches 59]. Despite this study, there are no available convincing reports that show the intracellular
expression of HERV-encoded and catalytically active dUTPases. Even so, it was speculated,
with no experimental proof, that HIV can lack a dUTPase-encoding gene, because the
host human cells, infected by this exogenous virus, already express an endogenous
dUTPase that is encoded by a HERV 104].

Possible links of HERVs dUTPases to human diseases

As mentioned, the high presence of HERVs in numerous sites over the human genes has
suggested their potential linkages to human diseases. Interestingly, stimulating new
evidence suggest an unexplored specific linkage between the HERV-K encoded dUTPase
and human psoriasis. Psoriasis is a chronic inflammatory immune disease of the skin
that is characterized by an elaborate interplay between multiple-risk genes and their
interactions with environmental factors. The psoriasis susceptibility locus 1 (PSORS1)
mutation resided within a region close to human leukocyte antigen-C, designated risk
haplotype (RH) 1/2, which is located within marker M6S168. This target region harbors
fragments of a HERV-K. Two single nucleotide polymorphisms with alleles differing
between high and low-risk haplotypes are located within the HERV-K dUTPase 105]. One of these haplotypes encodes a non-conserved Glu to Arg mutation. The HERV-K
dUTPase is expressed in peripheral blood and in normal as well as in lesional psoriatic
skin, thus suggesting that it can be a candidate gene for the PSORS1 mutation. To
investigate the direct role of the HERV-K dUTPase in psoriasis, purified recombinant
dUTPase versions with the wild-type sequence, and with mutations, reflecting the genotype
characteristics of high and low-risk haplotypes, were evaluated to see whether they
could modulate innate and/or adaptive immune responses 36]. The outcomes show that both wild-type type and mutant HERV-K dUTPase proteins induced
NF-kB activation through Toll-like receptor 2 that is independent of the enzymatic
activity. In both cases, the treatment of human primary cells with the recombinant
dUTPase proteins triggered a secretion of TH1 and TH17 cytokines that are involved
in forming psoriatic plaques, including IL-23, IL-12p40, IL-17, tumor necrosis factor-alpha,
IL-8, and CCL20. This result was observed in dendritic/Langerhans-like cells and,
to a lesser extent, in keratinocytes. An independent study described a variant discovery
and case–control association of HERV-K dUTPase variants in 708 psoriasis cases and
349 healthy controls. Five common HERV-K dUTPase variants exhibited a high association
with psoriasis, with the strongest association with a missense single-nucleotide polymorphism
that leads to a K158R mutation. Haplotype analysis revealed that HERV-K haplotypes
with the non-risk alleles significantly reduced the risk of psoriasis 37]. Moreover, functional testing showed higher antibody responses against recombinant
HERV-K dUTPase in psoriasis patients compared with controls, as well as higher T cell
responses against a single HERV-K dUTPase peptide.

The described studies support a novel and independent role for the HERV-K dUTPase
in the susceptibility to psoriasis. Nevertheless, the mechanisms underlying the linkage
between the HERV-K dUTPase and psoriasis are still elusive, especially since it does
not require an enzymatically-active protein. In this respect, there is a partial similarity
to our recent study on effects of the enzymatically-inactive BIV dUTPase 61], see also above. It is likely that, as described earlier for several outstanding
cases 31], the HERV-K protein serves as a signaling molecule that is involved in affecting
basic cellular functions. Apparently, the most exciting question is how the retroviral
enzyme evolved to function in these “moonlighting” activities. It is expected that
further detailed phylogenetically studies on retroviral dUTPases (similar to those
already performed 13], 60]), in combination with intensive cell biology, will uncover new answers for this fundamental
question.