Opinion
Cellular targets for improved
manufacturing of virus-based
biopharmaceuticals in animal cells
Ana F. Rodrigues1,2, Manuel J.T. Carrondo1,3, Paula M. Alves1,2, and
Ana S. Coroadinha1,2
1
iBET – Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal
2
Instituto de Tecnologia Quı́mica e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
3
FCT-UNL, P-2829-516 Caparica, Portugal
The past decade witnessed the entry into the market of
new virus-based biopharmaceuticals produced in animal
cells such as oncolytic vectors, virus-like particle vac-
cines, and gene transfer vectors. Therefore, increased
attention and investment to optimize cell culture pro-
cesses towards enhanced manufacturing of these bio-
products is anticipated. Herein, we review key findings
on virus–host interactions that have been explored in cell
culture optimization. Approaches supporting improved
productivity or quality of vector preparations are dis-
cussed, mainly focusing on medium design and genetic
manipulation. This review provides an integrated outline
for current and future efforts in exploring cellular targets
for the optimization of cell culture manufacturing of
virus-based biopharmaceuticals.
Virus-based biopharmaceuticals produced in animal
cells
Virus-based biopharmaceuticals (VBBs) comprise several
bioproducts used for vaccination and gene transfer (Box 1).
Vaccines have traditionally received more attention, but
the interest in viral vectors has also been growing, lever-
aged by the recent marketing authorization for the first
gene therapy medicine granted by the European Commis-
sion [1]. Therefore, active investment in the field can be
anticipated, including optimizing VBB manufacturing.
Herein, we revisit key findings on the interactions of
animal viruses and their hosts, and discuss the main cell
culture approaches exploring these interactions to improve
the production of VBBs.
Many VBBs are produced in bacteria, yeast, or plant
cells [2]. However, animal cells gained relevance for VBB
manufacturing because they provide important traits dif-
ficult to achieve with alternative hosts. For instance, the
inability of non-animal cells to support complex post-
translational modifications (PTMs) often impairs the
Corresponding author: Coroadinha, A.S. (avalente@itqb.unl.pt).
Keywords: biochemical pathways; virus–host interactions; viral vectors; vaccines;
medium design; genetic manipulation.
0167-7799/
ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.09.010
602 Trends in Biotechnology, December 2014, Vol. 32, No. 12
capacity of the viral particles to transduce the target
cells, particularly relevant for gene therapy, oncolytic
virotherapy, and vaccine vectors. Even when transduc-
tion is not an issue, animal cells can offer a superior
production platform: virus-like particles (VLPs) of hepa-
titis B virus produced in mammalian cells, although
similar in composition, glycosylation, and lipid content,
were larger in size and presented increased content of
monomers exhibiting higher immunogenicity than the
VLPs produced in yeast [2]. The advantages are not
restricted to complex PTMs. For instance, HIV-based
vectors have only been successfully produced in human
and (non-human) primate cells. Alternative hosts, includ-
ing insect cells, produce high titers of HIV–VLPs, but
have failed to produce infectious virions due to abnormal
activity of the lentiviral protease [3]. In general, animal
cells, and in some cases only those resembling the
natural host, allow for robust production of animal virus-
es and their recombinant derivatives. Here, we discuss
how the understanding of virus–host interactions can
unveil cellular targets to optimize animal cell culture
manufacturing of VBBs.
Virus–host interactions that affect cellular processes
Lipid metabolism
Viruses use lipid scaffolds as physical support for the
concentration of viral and cellular factors/proteins during
replication and assembly [4]. This reliance often imposes
changes to the host lipid metabolism. Increased biosynthe-
sis of fatty acids and phospholipids has been shown to occur
after infection of several viruses [5–7], while the inhibition
of these pathways frequently impairs viral replication or
leads to defective particle formation both for enveloped [8]
and non-enveloped viruses [9]. Increased biosynthesis can
occur through: (i) higher expression and/or activation of the
respective enzymes [8]; (ii) recruitment of biosynthesis-
related transcription factors [10]; or (iii) by reducing lipid
oxidation [11]. There are also viruses inducing downregu-
lated biosynthesis, counterbalanced with the upregulation
of lipid uptake – mainly cholesterol – as in the cases of
hepatitis C virus (HCV) [6,12] and murine leukemia virus
(MLV) [13].
Opinion Trends in Biotechnology December 2014, Vol. 32, No. 12

Box 1. The toolbox of virus-based biopharmaceuticals

VBBs can be defined as any virus-derived component or virus-
based particle with therapeutic or prophylactic application
(Figure I). This definition includes viral vectors for gene therapy
and oncolytic virotherapy, VLPs, vaccine vectors, inactivated (or
attenuated) viruses, and also free viral proteins. The latter are,
however, outside of the scope of this review. Viral vectors for gene
therapy are recombinant viral particles designed to transfer genetic
material into the patient cells to treat or prevent a disease.
Oncolytic virotherapy appears as a subfield of cancer gene therapy,
typically using replicative competent lytic viruses to infect (and
lyse) cancer cells. Viral particles for vaccination can be conceptually
divided into three groups: attenuated (or inactivated) viruses (wild
type or recombinant versions), VLPs, and vaccine vectors. VLPs are
multiprotein structures assembled into a virus-resembling archi-
tecture mimicking the structural conformation of native viruses,
thereby used for antigen display, but lacking viral genome. Vaccine
vectors (or vectored vaccines) can be conceived as frontier particles
between VLPs and viral vectors, typically coupling antigen display
with nucleic acid delivery to raise both antibody and cytotoxic T cell
(CTL) responses.

Virus-based biopharmaceucals (VBBs)

Inacvated/aenuated
Viral vectors Virus-like parcles
viruses

Replicaon-defecve Replicaon-competent Vaccine vectors for angen Virus-like parcles Inacvated or aenuated
vectors vectors delivery (and display) for angen display viruses

Gene therapy Oncolyc virotherapy Vaccines Vaccines Vaccines

Main applicaons
TRENDS in Biotechnology

Figure I. Categorization of virus-based biopharmaceuticals.

Energy metabolism biosynthesis [20] to arresting the synthesis and hijacking

Three main mechanisms – not necessarily independent –
by which viruses usurp the host energy machinery have
been described: (i) adenosine-50 -monophosphate-activated
protein kinase (AMPK) interaction [14]; (ii) Warburg ef-
fect-like metabolic reprogramming [15]; and (iii) mobiliza-
tion of lipid storages [16]. AMPK activation occurs in
response to ATP depletion and stimulates catabolic routes,
such as lipid oxidation and glucose uptake, and has been
described following the infection of a few viruses
[17,18]. Yet, not all viruses lead to AMPK activation and
the interactions of viruses with AMPK may depend on the
type of infection: acute, predominantly associated with
activation, or chronic/latent, related to AMPK inhibition
[14]. Metabolic reprogramming is usually based on higher
expression or activity of the glycolytic enzymes, namely
phosphofructokinase, and used to rapidly increase the ATP
content [7,19,20]. Mobilization of the triacylglycerol
storages and enhanced lipid b-oxidation has also been
described [21].
Nucleic acid metabolism and pentose phosphate
pathway
Virus replication strongly affects the nucleic acid metabo-
lism of the cell host, ranging from increasing total RNA
(or DNA) synthesis and substantial de novo nucleotide
the available nucleic acid content [22]. Increased biosyn-
thesis seems to be predominant in latent/chronic viruses,
while arresting is associated with lytic and acute infec-
tions. The changes in nucleic acid metabolism have been
related to increased enzyme activity and/or expression of
nucleotide biosynthesis preceding routes such as the pen-
tose phosphate pathway (PPP) [23]. Alterations in the pool
of PPP metabolites have also been described, including
elevated levels of ribose-5-phosphate and ribulose-5-phos-
phate [24].
Oxidative stress metabolism
Viral infections can raise the oxidative burden, mediated
by reactive oxygen and nitrogen species [25]. This usually
occurs via the inhibition of antioxidant enzymes and the
activation of pathways generating oxidants. Some
aspects on how the oxidative burden relates to the viral
pathogenesis have also been highlighted, including in-
creased viral genome heterogeneity and escape from
intracellular antiviral defenses. Several studies suggest
that the oxidative environment is a nursing milieu for
viral replication. This conclusion may, however, not be
straightforward, because some viruses that generated
high oxidative load were also shown to upregulate the
expression of antioxidant enzymes during the infection
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Opinion
[26]. Overall, the role of oxidative stress in viral replica-
tion is likely to depend on the life cycle stage and on the
virus itself, deserving further investigation.
Protein processing and post-translational modifications
Viral replication affects the protein processing of the host,
typically by subverting the biosynthetic pathways [27]. Ad-
ditionally, many viral proteins – including most of viral
attachment proteins mediating cell entry – are glycosy-
lated, making glycosylation a particularly relevant PTM in
viral replication. Several viruses have been shown to take
advantage of the host glycosylation machinery to modify
viral proteins and, for most viruses, glycosylation plays a
role in biogenesis, stability, antigenicity, infectivity, and
immune evasion [28]. More than using the available
resources, viruses can hijack and modulate the glycosyla-
tion machinery. For example, infection with human cyto-
megalovirus (HCMV) and varicella zoster virus (VZV)
activated the expression of several fucosyltransferases
(FUT3, FUT5, and FUT6) that were either dormant or
expressed at low levels in uninfected cells [29]. It was also
found that HCMV infection, but not VZV infection, induced
the transcription of FUT1, FUT7, and FUT9, showing that
the takeover of the glycosylation machinery is virus-de-
pendent.
Polyamine metabolism
The role of polyamines in viral replication was most prom-
inently studied in the late 1970s and early 1980s. Inhibi-
tion of herpes simplex virus (HSV) and Semliki Forest
virus production was observed in BHK21 cells when trea-
ted with inhibitors of polyamine synthesis, rescued with
polyamine supplementation, and alleviated by other di-
amine and polyamine analogs [30]. The activity of viral
RNA polymerase was decreased in polyamine-depleted
cells but rapidly increased after addition of spermidine
to the culture medium [30]. Increased activity (>10-fold) of
mammalian type C retroviral DNA polymerases has also
been reported [31]. By contrast, high concentration of
polyamines inhibited the initiation process of simian
vacuolating virus 40 (SV40) DNA replication, but low
levels stimulated replication [32]. In fact, large quantities
of polyamines may lead to oxidation, as oxidized polya-
mines are known to exhibit potent antiviral activity
[33]. Thus, polyamine concentration seems to be a critical
factor aiding viral replication.
Control of cell cycle and apoptosis
Many viruses modulate the cell cycle to increase their
replication efficiency [34]. Some induce a G1 to S phase
transition to replicate the genome with the cellular DNA
synthesis; others induce a G2/M arrest to provide an opti-
mized cellular environment for maximal replication. The
control occurs at different checkpoints including the cyclin
protein family and the nucleolus [34]. Cell cycle arrest is
one of the main causes, although not the only one, leading
to virus-induced apoptosis. As obligatory intracellular
parasites, viruses have also acquired sophisticated anti-
apoptosis artillery. Antiapoptotic viral strategies target
interferon and inflammatory pathways such as in the case
of vaccinia virus, influenza A, or HCV. Others inhibit
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Trends in Biotechnology December 2014, Vol. 32, No. 12
proapoptotic proteins including p53 as in SV40 and HCMV
infection. Others directly target apoptosis by coding for
caspase inhibitors, such as the p35 of baculovirus and
serpins of poxviruses, or for BCL2 homologs, such as
E1B19K of adenovirus or ORF16 of HSV. The fighting of
viruses against apoptosis has been extensively reviewed
[35].
Leveraging virus–host interactions to improve the
manufacturing of VBBs
In this section, the main strategies to improve cell culture
production of VBBs are summarized, focusing on medium
design and genetic modification (Table 1). We further
discuss the major advantages and disadvantages of each
approach.
Medium design
Lipid metabolism is a relatively easy pathway to manipu-
late by medium design as, in the presence of an external
source, most cultured cells readily take them up from the
culture medium. Lipid supplementation has been used to
improve the production of retrovirus-based [36,37] and
lentivirus-based [36,38] vectors, as well as to increase their
stability [39]. It was additionally used to improve the
production of HIV–VLPs by 2.4-fold [40]. Limitations in
cholesterol biosynthesis were shown to be a major cause for
serum heterotrophy in cells producing retroviral vectors
requiring external cholesterol supplementation to support
the removal of serum from the culture medium [36,41].
Energy metabolism has been manipulated mainly at the
level of sugar source and central carbon metabolism inter-
mediates. Yet, the effects are seldom restricted to one
pathway. For instance, in the production of retroviral
vectors, the use of glucose alternatives, such as fructose
and galactose, improved viral titers in several producer
cells [42–45]. Lactate accumulation was substantially de-
creased, but changes in the lipid composition of the cellular
and viral membranes were also described and shown to
confer higher stability to the vectors [43,44]. Increased
glycogen biosynthesis was also reported and related to a
high-productivity metabolic status [42]. The energy metab-
olism of SF9 insect cells producing baculovirus was also
manipulated by medium design: supplementation with
pyruvate or a-ketoglutarate at the time of infection in-
creased the productivity 6- to 7-fold at high cell density
[46].
Nucleic acid metabolism was also targeted by medium
design. Indeed, increased de novo nucleotide biosynthesis
during viral replication is usually associated with higher
consumption of nucleic acid precursor amino acids (gluta-
mine, glycine, and aspartate) and nucleoside backbones
[7,20], suggesting their supplementation as a strategy to
improve viral production. Adding nucleosides to the culture
medium increased the production of retroviral vectors by
1.9-fold [13]. Changes in nucleic acid metabolism during
viral replication have also been associated with increased
activity and/or expression of the PPP enzymes. Thus, these
appear as potential candidates to be targeted by genetic
manipulation although no attempts have been reported yet.
Polyamines can bind to nucleic acids participating in
their stabilization, modulate transcription events, play a
Opinion Trends in Biotechnology December 2014, Vol. 32, No. 12

Table 1. Cellular targets for improved manufacturing of VBBs in animal cells

Metabolic pathway Hallmarks of viral replication Approachesa to improve the manufacturing of VBBs
Lipid metabolism  Increased biosynthesis of fatty acids and  Lipid supplementation improved the production of
phospholipids [5–7] retroviral [36,37] and lentiviral vectors [36,38]
 Increased expression or activity of lipid biosynthetic  Lipid supplementation increased the stability of
enzymes [8] retroviral and lentiviral vectors [39]
 Recruitment of lipid biosynthesis-related  Lipid supplementation improved the production of
transcription factors [10] HIV–VLPs by 2.4-fold [40]
 Reduced lipid oxidation [19]  Expression of lipid metabolism genes to fully
 Upregulation of lipid uptaking machinery [6,12,13] reconstitute HCV life cycle in non-hepatic cells [53]
Energy metabolism  AMPK interaction [14] both at the level of activation  Use of glucose alternatives improved the production
and inhibition of retroviral vectors up to 8-fold [42–45]
 Metabolic reprogramming and enhanced glycolysis  Supplementation with pyruvate or a-ketoglutarate
[15,19] increased baculovirus yields 6- to 7-fold [46]
 Mobilization of lipid storage [21]
Nucleic acid metabolism  Increased nucleotide biosynthesis [20]  Supplementation with nucleosides increased the
and pentose phosphate  Increased consumption of nucleic acid precursor production of retroviral vectors by 1.9-fold [13]
pathway (PPP) amino acids and nucleoside backbones [7,20]
 Increased activity and/or expression of PPP enzymes
[23]
 Changes in the steady-state levels of PPP
intermediates [24]
Oxidative stress metabolism  Modulation of reactive oxygen and nitrogen species  Supplementation with antioxidants and reduced
intracellular load [25] glutathione increased the production of retroviral
vectors by 2.1-fold [13]
 Supplementation with trichostatin A resulted in a 4-
fold increase in adenoviral vector titers [49]
Protein processing and  Subversion of host protein synthesis machinery [27]  Overexpression of vesicle trafficking protein munc18b
post-translational  Modulation of host glycosylation machinery and increased lentiviral vector titers by 2-fold [54]
modifications differential activation of glycosyltransferases [29]  Engineering of the glycosylation patterns increased
retroviral vector stability by 3.5-fold [55]
Polyamine metabolism  Inhibition of polyamine biosynthesis limits viral  Supplementation with polyamines increased the
replication [30] production of retroviral vectors by 2-fold [13]
 Potential roles include viral genome stabilization and
transcription, increasing viral enzyme activity, and
modulation of viral membrane rigidity [31,47]
Control of cell cycle  Control of cell cycle checkpoints to increase viral  Cell cycle synchronization mediated by chemical
and apoptosis replication efficiency [34] blockage combined with temperature shift increased
 Fighting of proapoptotic machinery, upregulating the production of adenoviral vectors up to 7.3-fold [52]
antiapoptotic defenses, or coding for antiapoptotic  Downregulation of proapoptotic intracellular innate
gene homologs [35] defenses increased influenza virus yields by 9-fold [57],
lentiviral and adenoviral vectors by 5- to 10-fold, and up
to 100-fold for Sindbis virus [58]
a
Medium design strategies are in red font and genetic manipulation in blue font.

role in membrane rigidity, and present antioxidant prop-
erties [47]. These properties may have a beneficial effect on
viral replication, particularly by stabilizing viral genome
leading to improved encapsidation and, for enveloped vi-
ruses, by changing the rigidity of the viral membrane and
preventing its lipid peroxidation. A 2-fold increase in ret-
roviral vector production was obtained by medium supple-
mentation with polyamines [13].
Targeting oxidative stress by medium design appears
relatively straightforward as numerous supplements modu-
lating the oxidative environment are available. Yet, defining
a priori whether the choice should fall on oxidizing or antiox-
idant agents is far from trivial. For instance, in cell culture,
oxidizing agents promoted HIV replication while antioxi-
dants had the opposite effect [48]. However, improved pro-
ductivity of retroviral vectors was achieved by adding
antioxidants and reduced glutathione to the medium [13]. An-
other approach used trichostatin A to sustain the expression
of coxsackie adenovirus receptor (CAR) at high levels during
the production of adenoviral vectors, resulting in a 4-fold
improvement of specific productivity [49]. Although the
underlying mechanisms were not discussed, trichostatin A
changes the expression of several oxidative stress and inflam-
mation-related genes [50] – including inflammatory cyto-
kines known to decrease CAR expression – potentially
favoring the synthesis of virus-related proteins.
The control of cell cycle has also been achieved by
environmental manipulation, mainly chemical blockage,
and several methods exist to induce arrest at different time
points [51]. Chemically induced cell cycle synchronization,
combined with temperature shift, was used to increase the
productivity of adenoviral vectors [52].
Genetic manipulation
Lipid metabolism is particularly complex and extensive
rendering its genetic manipulation an underexplored
territory. It has only been reported for the reconstitution
of the HCV life cycle in HEK 293 cells [53]. Although the
titers were modest, this was an important landmark in
the field of cell substrates for the production of HCV, a
difficult virus to replicate in cell culture particularly in
non-hepatic cells.
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Protein processing and secretion pathways have been
manipulated through the overexpression of vesicle traf-
ficking protein munc18b, increasing the production of len-
tiviral vectors by 2-fold [54]. Another interesting approach
targeting PTMs, although not for improved productivity,
describes a strategy to generate more stable retroviral
vectors which, being produced by murine-derived cell lines,
were rapidly inactivated in human serum [55]. By target-
ing the PTM machinery and expressing chimeric glycosyl-
transferases to reduce a-gal-epitope synthesis, vector
stability was increased up to 3.5-fold. Protein processing
and PTMs have been primarily targeted through gene
manipulation, in the context of virus production, but rele-
vant changes based on medium design have been reported
for recombinant protein [56]. This suggests an opportunity
to optimize the production of VBBs also based on medium
design.
Improving virus production by manipulating the anti-
apoptotic machinery has been mainly conducted by genetic
engineering. A 9-fold improvement in influenza A produc-
tion was reported by stable downregulation of interferon 7
(IRF7) [57]. The concept behind this strategy – blocking
proapoptotic intracellular innate defenses – has been al-
ready described and shown to enhance the production of
lentiviral and adenoviral vectors by 5- to 10-fold and up to
100-fold for Sindbis virus [58].
Medium design or genetic modification?
Medium optimization is simpler from an experimental
viewpoint, reasonably cheap at the laboratory scale, and
relatively fast. However, since the use of supplements is
limited to the active pathways (although some supple-
ments can modulate gene expression), productivity
improvements are usually modest. Medium optimization
is also more likely to exhibit beneficial effects with stable
cell lines, continuously secreting the product into the
culture medium. In production systems based on lytic
cycles and associated with growth arrest, cell physiology
is drastically skewed, potentially diluting the supplemen-
tation effects. Furthermore, medium supplements usually
exhibit effects that are not long-lasting, thus necessitating
feeding schemes. Some supplements may become signifi-
cantly costly when moving from laboratory to production-
scale, and a few are perishable (e.g., antioxidants), reduc-
ing the reproducibility of potential improvements, or be-
coming a burden for the culture when provided in higher
concentrations than the cells are able to take up (e.g.,
lipids, which are easily peroxidizable).
Genetic manipulation, in turn, involves labor-intensive
steps, from the introduction of the target genes to the
isolation and characterization of candidate clones. Also,
the effects of point manipulations (single-gene engineer-
ing) are often diluted out without obvious phenotypic
differences, with substantial improvements more likely
to be obtained with changes in several genes [59]. More-
over, the outcome depends on the final expression levels –
which are difficult to control – and on a favorable combi-
nation between the external manipulations and the exis-
tent genetic background. Thus, successful genetic
manipulation usually requires extensive characterization
of the host cell by large-scale ‘omics’ methodologies, ideally
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Trends in Biotechnology December 2014, Vol. 32, No. 12
comparing the production with the non-production scenar-
io, followed by a careful biological analysis to identify the
potential targets [13]. This process can be as time and cost
demanding as the manipulation itself and, although the
pathways herein discussed should provide useful guidance,
others will certainly be of relevance, depending on the
virus and on the cell host. Despite the investment, the
reward in genetic manipulation is often notable. For re-
combinant protein production, average productivity in-
crease by gene engineering in animal cells ranges from
2- to 4-fold for single-gene and up to 30-fold for double-gene
manipulation (reviewed in [60]). No media optimization
based on two metabolites or macromolecules has reached
equivalent improvements. In the case of VBBs, the highest
improvements reported – up to 100-fold – were also
achieved with genetic manipulation (Table 1).
Nevertheless, separating medium from genetic manip-
ulation is, from a biological viewpoint, an abstraction. For a
majority of the pathways, gene engineering will be lever-
aged by proper medium feedings and, in a few cases, it will
be actually useless without them. It is, thus, the combina-
tion of both that will make the most of the final yield and
quality of the viral preparations.
Concluding remarks and future perspectives
The increasing demand for VBBs is raising the interest in
exploring cellular targets to improve their production.
Compared with recombinant protein, the optimization of
virus producer cell factories is still underexplored. But that
landscape might change: the growing market for VBBs
produced in animal cells is pushing the manufacturing
industry to rely on robust and high-yielding hosts to reduce
the production costs, to improve the efficacy in clinical
protocols, and to accelerate the clinical-to-market transi-
tion. To this end, medium design and genetic manipulation
offer a myriad of opportunities worthy to explore and the
pathways herein reviewed highlight potential targets in
the trail of improved manufacturing of VBBs.
Acknowledgments
The authors would like to thank Dr Eric J. Kremer, Institute of Molecular
Genetics of Montpellier, France, for critical revision of this manuscript.
The authors acknowledge Fundação Para a Ciência e a Tecnologia (FCT),
Portugal, for financial support: PTDC/EBB-BIO/118621/2010 and PTDC/
EBB-BIO/118615/2010.
References
1 Moran, N. (2012) First gene therapy nears landmark European market
authorization. Nat. Biotechnol. 30, 807–809
2 Kushnir, N. et al. (2012) Virus-like particles as a highly efficient vaccine
platform: diversity of targets and production systems and advances in
clinical development. Vaccine 31, 58–83
3 Adamson, C.S. et al. (2003) Control of human immunodeficiency virus
type-1 protease activity in insect cells expressing Gag-Pol rescues
assembly of immature but not mature virus-like particles. Virology
308, 157–165
4 Heaton, N.S. and Randall, G. (2011) Multifaceted roles for lipids in
viral infection. Trends Microbiol. 19, 368–375
5 Heaton, N.S. et al. (2010) Dengue virus nonstructural protein
3 redistributes fatty acid synthase to sites of viral replication and
increases cellular fatty acid synthesis. Proc. Natl. Acad. Sci. U.S.A.
107, 17345–17350
6 Lambert, J.E. et al. (2013) Elevated lipogenesis and diminished
cholesterol synthesis in patients with hepatitis C viral infection
compared to healthy humans. Hepatology 57, 1697–1704
Opinion
7 Munger, J. et al. (2008) Systems-level metabolic flux profiling identifies
fatty acid synthesis as a target for antiviral therapy. Nat. Biotechnol.
26, 1179–1186
8 Spencer, C.M. et al. (2011) Human cytomegalovirus induces the activity
and expression of acetyl-coenzyme A carboxylase, a fatty acid
biosynthetic enzyme whose inhibition attenuates viral replication. J.
Virol. 85, 5814–5824
9 Gaunt, E.R. et al. (2013) Inhibition of rotavirus replication by
downregulation of fatty acid synthesis. J. Gen. Virol. 94, 1310–1317
10 Waris, G. et al. (2007) Hepatitis C virus induces proteolytic cleavage of
sterol regulatory element binding proteins and stimulates their
phosphorylation via oxidative stress. J. Virol. 81, 8122–8130
11 Seo, J.Y. and Cresswell, P. (2013) Viperin regulates cellular lipid
metabolism during human cytomegalovirus infection. PLoS Pathog.
9, e1003497
12 Blackham, S. et al. (2010) Gene expression profiling indicates the roles
of host oxidative stress, apoptosis, lipid metabolism, and intracellular
transport genes in the replication of hepatitis C virus. J. Virol. 84,
5404–5414
13 Rodrigues, A.F. et al. (2013) Metabolic pathways recruited in the
production of a recombinant enveloped virus: mining targets for
process and cell engineering. Metab. Eng. 20C, 131–145
14 Mankouri, J. and Harris, M. (2011) Viruses and the fuel sensor: the
emerging link between AMPK and virus replication. Rev. Med. Virol.
21, 205–212
15 Noch, E. and Khalili, K. (2012) Oncogenic viruses and tumor glucose
metabolism: like kids in a candy store. Mol. Cancer Ther. 11, 14–23
16 Herker, E. and Ott, M. (2012) Emerging role of lipid droplets in host/
pathogen interactions. J. Biol. Chem. 287, 2280–2287
17 McArdle, J. et al. (2012) HCMV targets the metabolic stress response
through activation of AMPK whose activity is important for viral
replication. PLoS Pathog. 8, e1002502
18 Kumar, S.H. and Rangarajan, A. (2009) Simian virus 40 small T
antigen activates AMPK and triggers autophagy to protect cancer
cells from nutrient deprivation. J. Virol. 83, 8565–8574
19 Abrantes, J.L. et al. (2012) Herpes simplex type 1 activates glycolysis
through engagement of the enzyme 6-phosphofructo-1-kinase (PFK-1).
Biochim. Biophys. Acta 1822, 1198–1206
20 Munger, J. et al. (2006) Dynamics of the cellular metabolome during
human cytomegalovirus infection. PLoS Pathog. 2, e132
21 Heaton, N.S. and Randall, G. (2010) Dengue virus-induced autophagy
regulates lipid metabolism. Cell Host Microbe 8, 422–432
22 Wagner, E.K. and Roizman, B. (1969) Ribonucleic acid synthesis in
cells infected with herpes simplex virus. I. Patterns of ribonucleic acid
synthesis in productively infected cells. J. Virol. 4, 36–46
23 Diamond, D.L. et al. (2010) Temporal proteome and lipidome profiles
reveal hepatitis C virus-associated reprogramming of hepatocellular
metabolism and bioenergetics. PLoS Pathog. 6, e1000719
24 Delgado, T. et al. (2012) Global metabolic profiling of infection by an
oncogenic virus: KSHV induces and requires lipogenesis for survival of
latent infection. PLoS Pathog. 8, e1002866
25 Akaike, T. (2001) Role of free radicals in viral pathogenesis and
mutation. Rev. Med. Virol. 11, 87–101
26 Tilton, C. et al. (2011) Human cytomegalovirus induces multiple means
to combat reactive oxygen species. J. Virol. 85, 12585–12593
27 Walsh, D. and Mohr, I. (2011) Viral subversion of the host protein
synthesis machinery. Nat. Rev. Microbiol. 9, 860–875
28 Vigerust, D.J. and Shepherd, V.L. (2007) Virus glycosylation: role in
virulence and immune interactions. Trends Microbiol. 15, 211–218
29 Nystrom, K. et al. (2007) Virus-induced transcriptional activation of
host FUT genes associated with neo-expression of Ley in
cytomegalovirus-infected and sialyl-Lex in varicella-zoster virus-
infected diploid human cells. Glycobiology 17, 355–366
30 Raina, A. et al. (1981) Roles of polyamines in the replication of animal
viruses. Med. Biol. 59, 428–432
31 Marcus, S.L. et al. (1981) Polyamines stimulate DNA-directed DNA
synthesis catalyzed by mammalian type C retroviral DNA
polymerases. J. Biol. Chem. 256, 3460–3464
32 Kim, D.G. et al. (2008) The possible roles for polyamines in the
initiation process of SV40 DNA replication in vitro. Oncol. Rep. 19,
535–539
33 Bachrach, U. (2007) Antiviral activity of oxidized polyamines. Amino
Acids 33, 267–272
Trends in Biotechnology December 2014, Vol. 32, No. 12
34 Emmett, S.R. et al. (2005) The cell cycle and virus infection. Methods
Mol. Biol. 296, 197–218
35 Roulston, A. et al. (1999) Viruses and apoptosis. Annu. Rev. Microbiol.
53, 577–628
36 Chen, Y. et al. (2009) Cholesterol supplementation during production
increases the infectivity of retroviral and lentiviral vectors
pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-
G). Biochem. Eng. J. 44, 199–207
37 Rodrigues, A.F. et al. (2009) Retroviral vector production under serum
deprivation: the role of lipids. Biotechnol. Bioeng. 104, 1171–1181
38 Mitta, B. et al. (2005) Detailed design and comparative analysis of
protocols for optimized production of high-performance HIV-1-derived
lentiviral particles. Metab. Eng. 7, 426–436
39 Carmo, M. et al. (2009) Stabilization of gammaretroviral and lentiviral
vectors: from production to gene transfer. J. Gene Med. 11, 670–678
40 Cervera, L. et al. (2013) Generation of HIV-1 Gag VLPs by transient
transfection of HEK 293 suspension cell cultures using an optimized
animal-derived component free medium. J. Biotechnol. 166, 152–165
41 Rodrigues, A.F. et al. (2012) Adaptation of retrovirus producer cells to
serum deprivation: implications in lipid biosynthesis and vector
production. Biotechnol. Bioeng. 109, 1269–1279
42 Coroadinha, A.S. et al. (2006) Retrovirus producer cell line metabolism:
implications on viral productivity. Appl. Microbiol. Biotechnol. 72,
1125–1135
43 Coroadinha, A.S. et al. (2006) Effect of medium sugar source on the
production of retroviral vectors for gene therapy. Biotechnol. Bioeng.
94, 24–36
44 Coroadinha, A.S. et al. (2006) Effect of osmotic pressure on the
production of retroviral vectors: enhancement in vector stability.
Biotechnol. Bioeng. 94, 322–329
45 Merten, O.W. (2004) State-of-the-art of the production of retroviral
vectors. J. Gene Med. 6 (Suppl. 1), S105–S124
46 Carinhas, N. et al. (2010) Improving baculovirus production at high cell
density through manipulation of energy metabolism. Metab. Eng. 12,
39–52
47 Wallace, H.M. et al. (2003) A perspective of polyamine metabolism.
Biochem. J. 376, 1–14
48 Staal, F.J. et al. (1990) Intracellular thiols regulate activation of
nuclear factor kB and transcription of human immunodeficiency
virus. Proc. Natl. Acad. Sci. U.S.A. 87, 9943–9947
49 Liu, X. et al. (2009) The improvement of adenovirus vector production
by increased expression of coxsackie adenovirus receptor. Biotechnol.
Lett. 31, 939–944
50 Adcock, I.M. (2007) HDAC inhibitors as anti-inflammatory agents. Br.
J. Pharmacol. 150, 829–831
51 Banfalvi, G. (2011) Overview of cell synchronization. Methods Mol.
Biol. 761, 1–23
52 Ferreira, T.B. et al. (2009) 293 cell cycle synchronisation adenovirus
vector production. Biotechnol. Prog. 25, 235–243
53 Da Costa, D. et al. (2012) Reconstitution of the entire hepatitis C virus
life cycle in nonhepatic cells. J. Virol. 86, 11919–11925
54 Peng, R.W. et al. (2010) The vesicle-trafficking protein munc18b
increases the secretory capacity of mammalian cells. Metab. Eng.
12, 18–25
55 Hansen, W. et al. (2005) Generation of serum-stabilized retroviruses:
reduction of a1,3gal-epitope synthesis in a murine NIH3T3-derived
packaging cell line by expression of chimeric glycosyltransferases.
Metab. Eng. 7, 221–228
56 Wong, N.S. et al. (2010) An investigation of intracellular glycosylation
activities in CHO cells: effects of nucleotide sugar precursor feeding.
Biotechnol. Bioeng. 107, 321–336
57 Hamamoto, I. et al. (2013) High yield production of influenza virus in
Madin Darby canine kidney (MDCK) cells with stable knockdown of
IRF7. PLoS ONE 8, e59892
58 de Vries, W. et al. (2008) Increased virus replication in mammalian
cells by blocking intracellular innate defense responses. Gene Ther. 15,
545–552
59 Seth, G. et al. (2007) In pursuit of a super producer-alternative paths to
high producing recombinant mammalian cells. Curr. Opin. Biotechnol.
18, 557–564
60 Lim, Y. et al. (2010) Engineering mammalian cells in bioprocessing –
current achievements and future perspectives. Biotechnol. Appl.
Biochem. 55, 175–189
607