Review
Comparative analysis of the molecular
mechanisms of recombination in
hepatitis C virus
Andrea Galli and Jens Bukh
Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital,
and Department of International Health, Immunology, and Microbiology, Faculty of Health and Medical Sciences, University of
Copenhagen, Copenhagen, Denmark
Genetic recombination is an important evolutionary
mechanism for RNA viruses. The significance of this
phenomenon for hepatitis C virus (HCV) has recently
become evident, with the identification of circulating
recombinant forms in HCV-infected individuals and by
novel data from studies permitted by advances in HCV
cell culture systems and genotyping protocols. HCV is
readily able to produce viable recombinants, using replicative and non-replicative molecular mechanisms.
However, our knowledge of the required molecular
mechanisms remains limited. Understanding how HCV
recombines might be instrumental for a better monitoring of global epidemiology, to clarify the virus evolution,
and evaluate the impact of recombinant forms on the
efficacy of oncoming combination drug therapies. For
the latter, frequency and location of recombination
events could affect the efficacy of multidrug regimens.
This review will focus on current data available on HCV
recombination, also in relation to more detailed data
from other RNA viruses.
Genetic recombination in RNA viruses
HCV is an important human pathogen, causing an estimated 180 million chronic infections and annually 3–4
million new infections worldwide [1]. Infected individuals
are at increased risk of developing chronic liver diseases,
including cirrhosis and hepatocellular carcinoma [2]. HCV
is a single-stranded, positive-sense RNA virus, with a
genome of around 9600 nucleotides that contains one long
open reading frame (ORF) flanked by two distinct untranslated regions. The ORF encodes a single polyprotein that is
cleaved by viral and cellular proteases into structural
(core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A
and B, NS5A and B) proteins. Due to its extensive genetic
heterogeneity, HCV has been classified into seven major
genotypes (see Glossary) with genomes differing between
Corresponding author: Bukh, J. ([email protected]).
Keywords: hepatitis; HCV; RNA virus; copy–choice recombination;
breakage–rejoining recombination.
0966-842X/$ – see front matter
ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.02.005
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Trends in Microbiology, June 2014, Vol. 22, No. 6
25% and 30% at the nucleotide and amino acid level.
Genotypes are further divided into subtypes that typically
diverge by more than 15%.
HCV is classified as a member of the family Flaviviridae, genus Hepacivirus [3]. The genera Pestivirus (e.g.,
bovine viral diarrhea virus, BVDV; classical swine fever
virus, CSFV), Flavivirus (e.g., dengue virus, yellow fever
Glossary
Co-infection: simultaneous infection of the host or target cell by two or more
strains of the same virus. More broadly, it can indicate infection of the host by
different viruses, such as HCV and hepatitis B virus in hepatocytes.
Copy–choice recombination: a recombination model according to which
recombinant molecules are generated by the strand switching activity of the
viral polymerases. During synthesis of novel genomic strands, the nascent
molecule can be transferred to a different template thus producing a chimeric
genome.
Genotype: a group of phylogenetically closely related viruses, defined by its
genetic distance from other genotypes. HCV genotypes diverge around 30%
from each other at the nucleotide level. In HIV classification, the term ‘group’ is
used instead of genotype for the same level of correlation [91,92].
Inter-genotype recombination: recombination occurring between viral strains
belonging to different genotypes.
Intra-genotype/inter-subtype recombination: recombination occurring between viral strains belonging to the same genotype but different subtypes.
Intra-subtype recombination: recombination occurring between viral strains
belonging to the same subtype. In HCV classification, such strains would
typically differ around 5% at the nucleotide level [57].
Random breakage–rejoining recombination: a recombination model according
to which recombinant genomes can be generated by random breakage and
ligation of different genomic molecules. Breaks generated by mechanical
forces or enzymatic activity produce a population of genomic fragments of
different origin with compatible ends. Subsequently, cellular ligases or selfligation can randomly rejoin different fragments thus producing chimeric
genomes.
Replicon system: a cell system in which a sub-genomic or, less commonly, fulllength viral RNA is transfected into permissive cells and leads to viral RNA
replication and production of viral proteins. Most sub-genomic replicons also
carry a selectable marker gene to facilitate isolation of replicating cells. Despite
several HCV full-length replicons being fully replication competent, consistent
particle release could not be achieved with these systems [57].
Sequence homology: a measure of the level of evolutionary correlation
between DNA sequences. Highly homologous sequences share a common
ancestor. It is commonly, although incorrectly, used as a synonym for
sequence similarity [93].
Subtype: a group of phylogenetically highly related viruses, clustering within a
known genotype, defined by its genetic distance from other subtypes. In
general, HCV subtypes diverge at least 15% from each other at the nucleotide
level [91].
Super-infection: separate subsequent infections of the host or target cell by
two or more different strains of the same virus.
Super-infection exclusion: cellular mechanism(s) that prevent(s) re-infection of
an already infected target cell by the same virus.
Review
virus, and West Nile virus), and Pegivirus (e.g., GB virus C,
GBV-C, also called hepatitis G virus, HGV) belong to the
same family [4].
A common feature of RNA viruses is the high mutation
rate, due primarily to the lack of proofreading activity of
the viral polymerases and the high viral turnover. This has
been traditionally considered the main method of RNA
virus evolution; in recent years, however, it has become
increasingly clear that RNA recombination plays an important role in the generation of genetic diversity in RNA
virus populations [5]. Among the Flaviviridae, natural
recombinant forms have been identified for HCV [6–19],
GBV-C [20], dengue viruses [21], and BVDV [22–24]. In
addition, recombination has been demonstrated, among
others, in Picornaviridae (Poliovirus) [25,26], Tombusviridae (e.g., tomato bushy stunt virus, TBSV; cucumber
necrosis virus, CNV) [27,28], Bromoviridae (brome mosaic
virus, BMV) [29], Coronaviridae (mouse hepatitis virus,
MHV; infectious bronchitis virus, IBV) [30,31], and Retroviridae (e.g., human immunodeficiency virus 1, HIV-1;
murine leukemia virus, MLV) [32–34].
Through genetic recombination, genomic fragments
belonging to distinct viral strains are combined in a single
genome [35]. More rarely, genetic material from the host
cell can be incorporated in a viral genome [24]. Coupled
with the high mutation rate of viral polymerases, the
substantial exchange of genetic information allowed by
recombination represents a powerful tool in the evolution
of RNA viruses [5]. Recombination can allow non-replicative genomes to gain viability [36–38], increase viral
pathogenicity [24,39,40], eliminate deleterious insertions
to increase fitness [36,41], and contribute to the development of single- and multi-drug resistance [42]. Several
viruses, including human pathogens, have been shown to
be the result of recombination events that occurred in the
past between genetically distinct viruses, emphasizing
the significant role of recombination in the evolution
of RNA viruses [43–47]. Furthermore, recombination
events play essential roles in the life cycle of retroviruses
such as MLV and HIV-1 [48], allowing the complete
replication of the viral genome, and of pestiviruses such
as BVDV, generating the cytopathogenic biotype of this
virus [24].
The development of JFH1-based recombinants of HCV
capable of replicating in cell culture made it possible to
study the mechanisms of viral recombination in the context
of fully replicating viral genomes [36,49–51]. In addition,
the very recent availability of full-length single-strain
replicating genomes of different genotypes represents
the ideal tool to further analyze details of HCV recombination [52–54]. With the oncoming availability of several
new classes of antivirals against HCV and the recent
demonstration of the recombination capacity of this virus,
it becomes imperative to better understand the mechanisms of HCV genetic recombination. This would allow
evaluating the possible impact of recombinants on combination therapies and the effects of recombination on HCV
epidemiology. In this article, we review the current knowledge on recombination in HCV, with reference to related
RNA viruses for which more detailed information are
available.
Trends in Microbiology June 2014, Vol. 22, No. 6
Types of recombination
In this review, we will classify recombination based on the
structure of the crossover junction: homologous recombination occurs at the same site in both parental templates and
produces molecules with the same genetic structure as the
parental strains, whereas non-homologous recombination
occurs between different sites of the involved molecules,
producing genomes with duplications, deletions, or insertions. Consequently, homologous recombination occurs between related viruses that share similar genetic structure,
whereas non-homologous recombinants can also be generated from molecules of very different origin (Box 1). Because
the underlying recombination mechanisms giving rise to the
observed recombinants are often unknown and different
mechanisms could theoretically result in recombinants with
comparable structures, we will consider the mechanisms of
recombination separately.
Two mechanisms of recombination are generally accepted for RNA viruses, the replicative, copy–choice model and
the non-replicative, breakage–rejoining model (Figure 1)
[35,55,56]. In the copy–choice model, the viral polymerase
changes template during synthesis of the nascent strand,
producing a chimeric genome that contains fragments of
Box 1. Definitions of recombination
Early classification of viral recombination was proposed in analogy
to DNA recombination and defined homologous and non-homologous recombination based solely on the level of sequence
homology between the parental strains [94].This definition was
later refined to take into account the site of crossing over that
determines the structure of the resulting recombinant [35]. Recombination occurring between similar or closely related viral genomes
was classified as homologous if it occurred at the same site on both
molecules (type I) or aberrant homologous if it occurred at different
sites on the two parental strains (type II). Non-homologous
recombination, by contrast, occurred between highly dissimilar
molecules, such as different viruses or viral and cellular RNAs (type
III). Although better suited to describe viral recombination, this
definition still suffered from its origins in the field of DNA
recombination. The level of similarity between the parental
templates rather than the structure of the resulting recombinant
was still the main distinction between homologous and nonhomologous recombination.
An alternative definition that focused on the underlying mechanism of recombination rather than the level of sequence homology
between strains was subsequently proposed [87]. In this new
classification, recombination could be driven by sequence similarity
(class 1, similarity-essential recombination), factors other than
sequence similarity (class 2, similarity-nonessential recombination),
or a combination of the two (class 3, similarity-assisted recombination). Importantly, this definition established the notion that the
types of recombinant genomes produced are independent of the
recombination mechanism at play. In fact, all three classes of
recombination could in principle lead to precise (homologous) or
imprecise (aberrant or heterologous) recombinants.
Subsequent studies generally classified recombination as homologous or heterologous based on the crossover site of the generated
recombinants, independently of the level of homology between
parental strains [5]. Homologous recombinants have the same
structure as the parental viruses, whereas non-homologous recombinants have aberrant structure including deletions, insertions, or
duplications. Recombination mechanisms are considered separately. This is currently the most commonly used definition of
recombination; it has the advantage of being straightforward and
allows descriptive analysis of recombinant forms without prior
knowledge of the underlying recombination mechanism
[5,32,36,37,89].
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Trends in Microbiology June 2014, Vol. 22, No. 6
Replicave recombinaon
(A)
Non-replicave recombinaon
(D)
Overlap region
li-
3′ 5′
Polymerase
(B)
3′ 5′
(E)
Ligaon
3′ 5′
(C)
(F)
Duplicaon
TRENDS in Microbiology
Figure 1. Mechanisms of replicative and non-replicative RNA recombination. In replicative recombination (A–C), the nascent nucleotide strain can dissociate from the
template during replication of the viral genome (A), leading the polymerase complex to detach from the template and re-associate to a different one (B); synthesis of the
nascent molecule then resumes on the new template, resulting in a chimeric genome (C). In non-replicative recombination (D–F), genomic fragments generated by
enzymatic activity or mechanical damage (D) can be randomly rejoined by self-ligation or cellular ligases (E), resulting in chimeric genomes that can contain duplications,
insertions, or deletions (F).
both parental templates. The general model for copy–
choice recombination postulates that the nascent strand
can dissociate from the RNA template (donor) and interact
with a different template (acceptor) or with a different
region of the same template. This association results in
the transfer of the replication complex to the acceptor
molecule, where RNA synthesis resumes. Template
switching can produce homologous as well as non-homologous recombinants, as will be discussed in more detail in
the following sections.
In the non-replicative mechanism, genetic fragments of
different origins can be joined together generating hybrid
genomes. It is generally accepted that breakage of RNA
molecules due to mechanical forces, endoribonuclease activity, or cryptic ribozymes can produce fragments with
termini compatible with self-ligation or ligase-mediated
rejoining. Breakage and rejoining can occur between fragments of the same virus, among different viruses, and
between viral and cellular molecules, resulting in a broad
range of recombinant structures. Although this mechanism can theoretically result in both homologous and
non-homologous recombinants, it is most likely to produce
non-homologous recombination. Additionally, given the
random nature of the breakage and rejoining mechanisms,
recombinants are more likely to acquire deletions or insertions.
Recombination in HCV
Several requirements have to be met in order for viral
recombination to occur in vivo between viral strains, and
most of these are fulfilled for HCV (Box 2). Different
genotypes, subtypes, and isolates of HCV co-circulate in
several regions of the world [57], and multiple infections in
individuals have been reported [58–60]. However, there is
compelling evidence that super-infection exclusion is present in HCV-infected cells [61,62]. Data indicate that host
356
ver cells can be simultaneously co-infected by multiple
strains of HCV but the frequency of productive superinfection is very low [61].
HCV genomes are known to replicate within discrete
areas of the cytoplasm defined as replication complexes,
thus colocalization of different genomes is possible for this
virus. In fact, experimental results suggest that once cells
are co-infected, the different viral genomes can undergo
recombination efficiently [63]. Moreover, studies using
cells infected with distinct poliovirus strains showed that
the majority of replication complexes contained both viral
genomes, indicating that genomes originating from distinct viruses colocalize in the same subcellular regions
[64]. Our understanding of the topology and molecular
details of HCV replication remains, however, incomplete
and the existence of factors influencing the localization of
viral genomes cannot be ruled out. Nonetheless, circulating recombinant forms have been isolated from HCVinfected individuals and in experimental settings, indicating that this virus can undergo recombination productively [6–19].
Circulating HCV recombinant forms
As mentioned, multiple genotypes, as well as subtypes and
isolates, co-circulate in several areas of the world, fulfilling
a prerequisite of recombination. To date, at least nine
inter-subtype and eight inter-genotype recombinants have
been identified in the HCV-infected population in different
geographic regions (Figure 2) [6–19]. Many such recombinant forms displayed crossovers in the NS2/NS3 junction
region, suggesting that exchange between structural and
nonstructural proteins is most likely to produce viable
recombinants. Experimental generation of inter-genotype
recombinants also indicated that chimeras carrying a genotype-specific core-to-NS2 region in the genotype 2a background of the JFH1 strain lead to viable recombinants.
Review
Trends in Microbiology June 2014, Vol. 22, No. 6
Box 2. Recombination checkpoints
RNA viruses capable of recombination will always produce recombinant genomes during infection; however, only recombinants originating from distinct viral strains can be easily detected. Several
requirements have to be met for recombination to occur between
different virus strains in vivo.
(i) Co-circulation. Different strains need to co-circulate in the same
geographic area. This is the primary prerequisite for recombination because recombinants between very similar genomes are
extremely difficult to identify. Viruses for which recombinant
forms have been identified in vivo display extensive co-circulation of different viral forms. This is, for example, true for HIV-1
[69], HCV [57], dengue virus [21], and BVDV [95].
(ii) Co-infection. The hosts have to be co-infected by distinct viral
strains. This means on the one hand that the different cocirculating viruses must have reasonably high prevalence in the
infected population, and on the other hand that they need to be
circulating in the same risk population. This is normally true for
most viruses, but cases have been described where specific risk
groups are associated with infection by a subset of circulating
viral strains, with limited exchange with the general infected
population [96].
(iii) Cellular co-infection. Target cells have to be co-infected by
different virus strains. Super-infection inhibition can reduce the
occurrence of multiply infected cells in several viruses [61,97].
However, low blood concentration of circulating virus could also
Nonetheless, recombinant forms with breakpoints in other
genetic regions have been isolated, indicating that several
areas of the HCV genome can undergo recombination.
At least one recombinant form, named RF1_2k/1b, has
epidemiological relevance [15]. This recombinant was originally isolated in patients from St. Petersburg, but has
reduce the number of co-infected cells, resulting in low apparent
recombination frequency.
(iv) Subcellular colocalization. Viral genomes subsequently need to
localize in the same region of the cytoplasm for their interaction to
occur. RNA viruses, including HCV, that replicate their genomes
within special membranous enclosures naturally concentrate
genomic RNAs within discrete areas. If distinct genomes can
distribute freely among replication complexes, their accumulation
within this compartment would support recombination. However, if
genomes of different origin were segregated in distinct replication
complexes, their apparent recombination frequency would be
lower than expected. For most RNA viruses, the distribution of
genomes within the cytoplasm, as well as the mechanisms involved
in their regulation, remain unknown. However, it has been shown
that HIV-1 genomes engineered to use separate nuclear export
pathways recombine with lower efficiency [98], whereas MLV
displays lower-than-expected recombination rates, probably due to
differential packaging of distinct genomic strains [99].
(v) Survival. Finally, generated recombinant forms have to be viable
to spread in the viral population. The different fragments of
recombinant genomes must be able to function together to
overcome structural and functional constraints that could impair
their viability. Consequently, closely related strains should have
higher chances to produce viable recombinants than distant
strains, as has been shown for HIV-1 [71].
been subsequently isolated from several unlinked individuals and in different countries, confirming its genetic
stability and infectious capacity. Moreover, there is evidence suggesting a possible selective advantage of the RF1
recombinant form under antiviral treatment [65]. Other
recombinant forms have been isolated in single instances
E1
E2
p7
NS2
NS3
NS4B
5
6a
NS5A
NS5B
UTR
C
NS4A
UTR
HCV genomic structure
Inter-genotypic recombinants
RF1_2k/1b [15]
2i/6p [18]
RF3_2b/1b [14]
2/5 [16]
2b/6w [6]
2b/1b [10]
2b/1a [8]
2b/1b [7]
Intra-genotypic recombinants
RF2_1b/1a [12]
1a/1c [13]
1a/1c [19]
1b/1a [17]
4d/4a [11]
6a/6o [9]
6e/6h [9]
6e/6o [9]
6n/6o [9]
Key:
1a
1b
1c
2
2b
2i
2k
4a
4d
6e
6h
6n
6o
6p
6w
TRENDS in Microbiology
Figure 2. Genomic structures of all hepatitis C virus (HCV) recombinants characterized so far in vivo. Recombination points are illustrated in reference to the HCV genomic
structure depicted at the top. Breakpoint locations and genotypes involved in each recombinant form were obtained from their respective publication, indicated in square
brackets next to each recombinant name. The location of recombination points is approximate and meant for illustrative purposes only. Color coding of genotypes and
subtypes is shown at the bottom. The full-length sequence of recombinant 4d/4a has not been reported.
357
Review
and their epidemiological relevance is still unclear. The
availability of novel classes of antivirals against HCV,
including directly acting antivirals that target specific viral
components, might further the emergence of novel recombinant forms with enhanced resistance profiles.
The relative paucity of identified recombinant forms
fostered the idea that HCV recombines at low frequency.
However, the inherent difficulties in identifying recombination between closely related strains and the limitations of
the genotyping strategies used to monitor HCV infections
could have resulted in an underestimation of this phenomenon in HCV [66,67]. Genotype determination is routinely
performed using a single region of the HCV genome, thus
failing to identify any recombinants that do not have breakpoints within the analyzed region. Even when genotyping is
performed using multiple or extended regions of the genome, the phylogenetic methods used cannot detect
recombination between highly similar genomes, so that
intra-subtype recombinants, among others, are difficult to
identify. In addition, recombination between identical or
nearly identical genomes, such as quasispecies, remains
undetected, contributing to the underestimation of HCV
recombination capacity. Interestingly, a retrospective study
evaluating the presence of recombinant forms in a cohort of
more than 100 HCV-infected patients, found evidence for
recombination in 18% of the subjects [68]. This figure is
comparable to what has been estimated for HIV-1 [69],
which is commonly considered a virus with a high recombination frequency [70], supporting the notion that biases in
the detection strategies of HCV recombination might have
reduced the number of identified recombinants. Importantly, all recombinants isolated in vivo must survive strong
selection pressure imposed by the host environment and
compete against wild type parental viruses. As such, functional constraints could exist that prevent the survival of the
majority of recombinant forms, as demonstrated in HIV-1
[71]. As a result, the limited number of recombinant forms
identified to date could be caused by genetic bottlenecking
acting on the recombinant population, rather than limited
occurrence of recombination.
Experimental systems permitting studies of HCV
recombination
Despite the identification of several inter- and intra-genotype HCV recombinants in infected patients, very few
studies have investigated the mechanism of HCV RNA
recombination in experimental settings. Multiple intragenotype recombinants were detected in chimpanzees experimentally infected with HCV genotype 1a and 1b
strains within 30 days after infection [63]. This first report
further demonstrated the ability of HCV to productively
recombine in vivo and produced a rough estimate of HCV
recombination frequency of around 310–5 crossovers/nucleotide, under the assumption of a constant recombination
frequency across the genome. Interestingly, the identified
recombinants showed crossovers in the E1/E2 genes, in
domains of high sequence homology that were flanked by
more variable regions. These genes encode the envelope
glycoproteins, which are important targets of the humoral
immune system. Recombination within E1/E2 could therefore aid the generation of escape genomes able to evade the
358
Trends in Microbiology June 2014, Vol. 22, No. 6
immune response. Most HCV recombinants identified so
far in infected humans have breakpoints within the NS2/
NS3 junction, whereas only one genotype 1 recombinant
showed crossover in the E1/E2 genes (Figure 2). Recombination in the NS2/NS3 area effectively uncouples structural and nonstructural genes, arguably increasing the
likelihood of producing viable recombinant genomes. By
mixing functional nonstructural genes with different capsid/envelope gene sets, HCV could for example produce
viable viruses with diverse antigenic epitopes. Thus, the
results from the experimental infection in chimpanzees
provided additional evidence that HCV can recombine in
genome regions outside NS2/NS3.
Successively, a study measured recombination frequencies between pairs of HCV replicons of genotype 1b expressing neomycin-resistance genes. Replicons in each pair
carried point mutations that either restored neomycin
sensitivity or abrogated replication; recombination events
occurring between different mutants would produce replicating neomycin-resistance replicons that could be easily
detected in culture [72]. The data presented confirmed the
ability of HCV to produce intra-genotype recombinants at
low frequency, with a normalized recombination frequency
of 410–8 crossovers/nucleotide. The recombination frequency estimate was much lower than what was previously
published; however, given the fundamental differences in
the systems used, direct comparisons must be made with
caution. Nevertheless, both the in vivo and in vitro studies
agree as to the capability of HCV to generate recombinants
in experimental conditions, albeit at lower frequencies
compared to other RNA viruses. In addition, both studies
isolated only homologous recombinants, which had most
likely been generated through the replicative copy–choice
recombination mechanism.
In the development of recombinant HCV expressing
marker genes within the NS5A region, heterologous recombinant viruses were observed carrying short deletions
in domain II of NS5A [41]. The acquired deletions most
likely compensated for the additional domain inserted in
the NS5A gene because the novel recombinants were
stable over several passages in cell culture and maintained the expression of the marker gene. The observed
heterologous recombination events were probably the result of intra-strain copy–choice recombination, again suggesting efficient strand-switching capability of the HCV
polymerase.
In addition, HCV recombinant genomes containing deletions of the essential stem-loop I region in the 50 untranslated region were shown to regain viability by inserting in
its place RNA fragments with similar structures of viral or
cellular origin [38]. In one case, additional nucleotides had
been removed from the parental strain at the site of
insertion. The presence of additional deletions and the
insertion of exogenous sequences suggested the occurrence
of the non-replicative breakage–rejoining recombination.
More recently, the generation of recombinant forms of
HCV was investigated more systematically by using efficient cellular systems that recapitulate the entire viral life
cycle [36]. Genomes derived from genotype 2a strains J6
and JFH1, and carrying different inactivating mutations/
alterations, were transfected in pairs into Huh7.5 cells and
Review
monitored over time. HCV could efficiently produce functional recombinants with variable crossover junctions
along the genome. More importantly, HCV recombination
was demonstrated for the first time to occur in the absence
of a functional viral polymerase, with an average recombination frequency of 410–9 crossovers/nucleotide. The
majority of chimeric genomes were heterologous non-homologous recombinants, containing extensive duplications
of the HCV genome; however, two homologous recombinants were also identified. Additionally, the generated
heterologous recombinants could undergo further recombination events that reduced the extent of the duplications
and arguably produced more fit genomes.
The estimated non-replicative recombination frequency
of HCV is markedly lower compared to other RNA viruses
such as poliovirus (710–6 crossovers/nucleotide [25]),
BMV (310–7 crossovers/nucleotide [73]), and HIV
(1.510–4 crossovers/nucleotide [74]). However, these viruses were assayed under conditions favoring replicative
recombination. The stochastic nature of the non-replicative, breakage–rejoining mechanism most likely provides
fewer chances to produce functional genomes than the
copy–choice mechanism, thus partly explaining the lower
recombination frequency observed in this study. Nonetheless, the frequency of replicative recombination of HCV
measured using the replicon system (410–8 crossovers/
nucleotide [72]), is still at least tenfold lower than other
viruses, suggesting that HCV has a reduced recombination
capacity.
In the in vitro culture study, viable recombinants between a defective 2a genome and non-replicating genomes of
genotypes 1a, 1b, 3a, and 4a could be isolated only in two
instances, resulting in an estimated inter-genotype recombination frequency around tenfold lower than the intragenotype frequency [36]. This is consistent with the difficulties encountered in the experimental generation of functional recombinant HCV strains [51,75,76], and is probably due
to functional rather than mechanistic constrains that limit
the viability of inter-genotypic recombinants. Indeed, all
studies conducted on HCV recombination so far have analyzed recombination in the presence of strong selective
pressure, limiting the analysis to only functional recombinant forms.
Taken together, data available on HCV recombinants
indicate that both replicative and non-replicative recombination mechanisms are at work in the production of HCV
chimeric forms. The molecular details of the two mechanisms and their correlation with other virus models will be
discussed further in the following sections.
Replicative recombination in HCV
It is generally accepted that the prevalent recombination
mechanism in RNA viruses is the replicative copy–choice
model, resulting mostly in homologous recombination. In
the case of HCV, the observed homologous recombinants
could have arisen directly through replicative homologous
recombination or as the result of successive non-homologous replicative recombination events. In the latter scenario, first-generation non-homologous recombinants that
could have arisen through either replicative or non-replicative recombination would evolve into homologous recom-
Trends in Microbiology June 2014, Vol. 22, No. 6
binants over one or more subsequent recombination
events. Such additional recombination events could be
observed in cell culture and were probably replicative in
nature, given their propensity to eliminate a replicated
region and the fact that a functional NS5B polymerase was
available to the virus. In at least one case [36], it appeared
that secondary recombination events resulted in a recombinant genome with homologous structure. Indeed, copy–
choice recombination has been shown to be capable of
precisely removing genomic duplications with high efficiency in retroviruses and in a pestivirus genome [77–79].
Details on the molecular aspects of HCV copy–choice
recombination are lacking; however, several studies have
clarified aspects of replicative recombination in the related BVDV and in other RNA viruses. Retroviruses possess a
very different replication cycle compared to other RNA
viruses, and have been consistently considered separately
when discussing viral recombination. Despite the differences in the life cycle, however, there are several similarities in the molecular aspects of recombination between
retrovirus and other RNA viruses. In vivo studies of tombusvirus, bromovirus, BVDV, and poliovirus showed a
correlation between sequence homology and overall
frequency of recombination [25,80–82]. Similarly, sequence similarity is well known to influence the frequency
of retroviral strand switching and recombination
[71,78,83,84]. In addition, the degree of sequence homology between recombining regions of BVDV genomes can
affect the choice of crossover sites, determining homologous or non-homologous recombination [77,81].
It is generally accepted that recombination during replication is due to strand switching of the viral polymerase
between different genomes or different regions of the same
molecule. In vitro studies demonstrated the strand-switching capacity of several viral polymerases [85]. Studies
conducted on tombusvirus and bromovirus indicated that
structural features capable of pausing the viral polymerase, such as breaks or hairpins, promote strand switching
[80,82]. Moreover, low nucleoside triphosphate (NTP) concentrations as well as AU-rich sequence stretches can
increase strand switching of different viral polymerases
[28,85]. Retroviruses recombine mostly through a replicative homologous mechanism, due to the processivity
requirements of the viral reverse transcriptase (RT). A
key step in retroviral strand switching is the RNaseHdriven degradation of the RNA template during DNA
synthesis, which leaves single-stranded nascent DNA
available to interact with the acceptor template. The dynamic copy–choice model for retroviral recombination predicts that factors that decrease RT function, such as
reduced dNTP pool, mutations, or secondary structures,
allow more efficient degradation of the upstream RNA
template resulting in higher rates of template switching
(reviewed in [32,86]). Conversely, reduced activity of the
RNaseH domain results in lower frequency of template
switching.
Given the parallels between retroviral and RNA virus
recombination aspects, we propose a unified model that
recapitulates recombination in positive-sense RNA viruses
(Figure 3). For recombination to occur, the nascent molecule needs to be free from the donor template and interact
359
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Trends in Microbiology June 2014, Vol. 22, No. 6
Infecon
Release
Lumen
Cytoplasm
Non-replicave recombinaon
Uncoang
Crypc ribozyme
(A)
Endoribonuclease
(B)
Breakage
3′P 5′OH
Translaon
Ligaon
(C)
Maturao
Maturaon
(D)
Replicaon
Replicave recombinaon
RCs
AU-rich Box
Golgi
Golgi
E
(E)
RNA Polymerase
As
Assembly
(F)
ER
ER
(G)
Nucleus
N
l
TRENDS in Microbiology
Figure 3. Proposed models for replicative and non-replicative recombination in positive-sense RNA viruses. A model based on experimental evidence obtained from HCV and
other RNA viruses is exemplified in the case of dual infection by two strains of HCV. After infection by different viral strains and uncoating of the viruses, both RNA genomes are
localized in the cytoplasm. In non-replicative recombination (A–D), different mechanisms such as cellular endoribonucleases, cryptic ribozymes, or mechanical damage can
produce a population of randomly cut genomic RNA (A), carrying 30 -P and 50 -OH termini (B). Different fragments can be randomly joined through self-ligation or with the aid of
cellular ligases (C), resulting in a recombinant genomic RNA (D). Parental and functional recombinant genomes are subsequently translated and targeted to the replication
complex for replication. In replicative recombination (E–G), during synthesis of the novel RNA strain, the nascent strain can become unpaired from the donor template and
initiate a strand-switching event (E). Factors that destabilize pairing (e.g., AU sequences) or reduce polymerase processivity (e.g., low NTP concentration, secondary structures,
and breaks) will favor strand switching. Depending on the sequence similarity between donor and acceptor template, the single-stranded nascent strain can: anneal extensively
with the acceptor template; partially anneal with the acceptor upstream of the crossover junction and induce a downstream strand switch through base pairing and/or secondary
structure interaction; or not anneal and promote strand switching through secondary structure interaction (F). Once the polymerase has switched to the acceptor template, RNA
synthesis is resumed and a recombinant genomic RNA is produced (G). Genomes are further packaged and assembled into mature virions ready to be released through the
secretory pathway. Abbreviations: ER, endoplasmic reticulum; HCV, hepatitis C virus; NTP, nucleoside triphosphate; RCs, replication complexes. Dark blue and green
nucleotides represent distinct HCV strains; light blue and green nucleotides represent newly synthesized HCV genomes.
360
Review
with the acceptor molecule. This is achieved through degradation of the template in retrovirus, and by strand
dissociation in other RNA viruses. Factors that favor dissociation, such as AU-rich regions, would promote strand
switching, whereas factors reducing dissociation, such as
defective RNaseH, would result in lower switching frequencies. Similarly, factors that slow down the polymerase, such as limited NTP pools, breaks, or secondary
structures, allow more time for the nascent strand to
detach from the donor in the polymerase proximity, thus
promoting strand switching.
The fate of the nascent strand would then depend upon
the level of sequence homology with the acceptor template
[35,87]. In the case of high sequence homology, the two
strands could dimerize extensively and induce a precise
strand switch resulting in homologous recombination. In
the case of lower sequence homology, the nascent and
acceptor strands could partially dimerize upstream of
the crossover junction and then promote downstream
strand switching via local partial annealing and/or secondary structure interaction. In this case, sequence or structural differences between donor and acceptor template
could result in mis-annealing of the nascent strand producing insertions, deletions, or duplications. Finally, if
sequence homology between donor and acceptor molecule
were too low to allow even limited dimerization, strand
switching would rely only on secondary-structure interaction. Viable recombinants would then have to survive
natural selection to spread to other cells and become
predominant in the viral population.
This model is in agreement with evidence obtained in
several RNA viruses, including retroviruses [25,32,85].
However, additional evidence is required to confirm its
general validity for the HCV system in particular.
Non-replicative recombination in HCV
There is now evidence that HCV can recombine through a
non-replicative model, as demonstrated by the generation of viable chimeric viruses in the absence of a functional viral polymerase [36]. Despite the strong selective
pressure imposed on the generated recombinants, breakpoints could be identified at variable locations along the
genome. These findings suggest that random breakage of
the parental genomes, rather than cryptic ribozyme activity as previously proposed for other RNA viruses [88],
is largely responsible for the generation of HCV genetic
fragments. However, whether they are produced by physical breakage or by endoribonuclease activity remains to
be determined.
Non-replicative recombination compatible with a breakage–rejoining mechanism has been demonstrated in the
related pestivirus BVDV [37,89] and in poliovirus [90]. In
addition, both systems showed enhanced re-ligation of
fragments carrying 30 -phosphate and 50 -hydroxyl ends,
equivalent to those generated by endoribonucleolytic cleavage or mechanical breakage. These findings support a
breakage–rejoining model in which fragments generated
by endoribonucleases or physical damage subsequently are
rejoined by cellular ligases or through self-ligation. Furthermore, a linear correlation between RNA concentration
and frequency of recombination was observed in pestivirus
Trends in Microbiology June 2014, Vol. 22, No. 6
[89] and HCV [36], as would be predicted by a random
breakage–joining mechanism.
Most of the recombinants identified in non-replicative
recombination studies on BVDV [37] and HCV [36] were
non-homologous, in accordance with the random breakage–rejoining model. The higher number of possible nonhomologous combinations of genomic fragments compared
to homologous junctions entails that the chances of generating a viable chimera are higher in the first case.
However, non-replicative recombination in poliovirus produced mostly homologous recombinants [90]. This could be
a feature of the non-replicative recombination mechanism
of poliovirus or, alternatively, the result of rapid evolution
of non-homologous recombinants leading to the excision of
the duplicated regions. In fact, the HCV study showed that
non-homologous recombinants in the polymerase region,
the same gene analyzed in the poliovirus study, lost their
duplication very rapidly [36], suggesting that insertions in
the polymerase gene are particularly deleterious to the
virus. Finally, the limited sequence overlap (178 nucleotides) between the parental strains of poliovirus could
have restricted the number of possible viable non-homologous recombinants, biasing the assay towards the generation of homologous recombinants.
Based on the data produced so far, a putative model for
non-replicative breakage–rejoining recombination in HCV
and other RNA viruses can be envisioned (Figure 3).
According to this model, viral genomes that are present
in the cytoplasm of infected cells can undergo random
cleavage by endoribonucleases, cryptic ribozymes, or mechanical forces, producing genomic fragments with 30 -P
and 50 -OH ends. Subsequently, cellular ligases or selfligation randomly join different fragments, generating a
large population of non-homologous recombinants with
aberrant genomic structures. A limited number of genomes
will be able to produce functional proteins upon translation
and replicate in the cell. In addition, viable recombinants
could undergo further replicative recombination events,
which could result in partial or complete loss of duplicated
regions. Eventually, the fittest recombinant form will
spread to other cells and become predominant in the viral
population.
Concluding remarks
All HCV recombinants identified so far, either in infected
humans or in experimental studies, represent genomes
that have survived several rounds of selection. Consequently, their number and composition could have been
altered compared to the original pool of generated recombinants. Thus, to fully understand the mechanisms of HCV
genetic recombination it is imperative to analyze the population of recombinant forms before any selection has
taken place. This would not only allow precise quantification and characterization of the recombinant genomes, but
also contribute to the understanding of the molecular
mechanisms driving the evolution of HCV.
Experimental evidence demonstrated that both replicative and non-replicative recombination occur in HCV, however their relative contribution to the composition of the
viral population is unclear. Non-replicative recombination
seems to be the prevalent mechanism in the absence of a
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Review
Box 3. Outstanding questions
What is the relative contribution of replicative and non-replicative
recombination to the generation of novel HCV recombinants? The
two mechanisms have different effects on the sequence space
available to the novel recombinants, and understanding their
respective activities would help understanding modes and rates
of HCV evolution. Assays that can separate non-replicative and
replicative recombination steps are required; the capability of
isolating functional replication complexes [100], and detecting
recombination between non-functional genomes [36] are important steps towards this goal.
What is the recombination potential of HCV in the absence of
selection pressure? To elucidate the relative contribution of native
recombination potential and selective pressure to the overall
recombination frequency, it is essential to analyze these two
mechanisms independently. Single-cycle cell systems, or other
assays that can monitor and quantify recombination before
selection takes place, are a possible and valid approach to this goal.
What are the molecular or cellular features affecting the frequency
of HCV recombination? Once quantitative assays are available to
measure recombination rates (see previous bullet), experimental
alterations of the host cell environment and/or of the viral
genomic structure, could clarify the importance and effect of
different factors to the overall recombination potential of HCV.
functional polymerase, but whether this mechanism is still
relevant in the recombination of functional genomes
remains to be determined. Current data suggest that
homologous replicative recombination is the major force
driving recombination of viable viruses. However, based on
recent experimental evidence it cannot be excluded that
the observed homologous recombinants are the final
results of several rounds of recombination beginning with
the generation of non-homologous recombinants. Additional studies are necessary to clarify this and other aspects of
HCV recombination and effects on viral fitness (Box 3).
Many similarities in different aspects of recombination
can be found among RNA viruses of distinct groups, including HCV, pestiviruses, retroviruses, and bromoviruses, suggesting the existence of common molecular
models of recombination. Intriguingly, and maybe predictably, the two mechanisms seem to cooperate in the generation and evolution of recombinant forms in several virus
groups. Nonetheless, our knowledge of the HCV recombination mechanisms is still extremely limited, and additional experimental evidence is essential in order to verify
the strength of the models proposed here for recombination
of HCV. The recent availability of full-length HCV infectious culture systems of different genotypes and subtypes
will represent an invaluable tool to dissect the molecular
aspects of HCV recombination under conditions that more
closely recapitulate in vivo infection.
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