UL37 is a herpesviral tegument protein that remains associated with the viral capsid during trafficking within the host cell during viral entry and egress. Previous studies have shown UL37 is required for efficient viral replication. The authors aim to characterize the structure of UL37 from Marek's Disease Virus (MDV) since its avian host has a higher body temperature, hypothesizing MDV UL37 will be more thermally stable. They cloned and expressed MDV UL37 constructs in E. coli and purified one construct. Future work includes further purifying MDV UL37 and crystallizing it to determine its structure, which could aid in designing antivirals targeting UL37.

1. Characterization of herpesviral trafficking protein
UL37, a potential therapeutic target
Eleni Papadopoulos, Andrea Koenigsberg, Ekaterina Heldwein
Department of Microbiology and Cell Biology, Sackler School of Graduate Biomedical Sciences
Tufts University School of Medicine, Boston MA 02111
Acknowledgements
Abstract
Herpes Simplex Virus (HSV) infects most of the world
population for life and causes diseases ranging from cold sores,
genital lesions, chickenpox, to blindness, encephalitis, and cancers.
UL37 protein is a herpesviral tegument protein, that remains capsid
associated during trafficking within the host cell during viral entry and
egress. Previous studies have demonstrated that UL37 is required for
efficient viral replication. HSV-1 UL37-null viruses take three times as
long to reach the nucleus post entry [1], are unable to reach the site of
secondary envelopment during egress causing the capsid to
accumulate in the cytoplasm [2], and have fewer and shorter
movements within the host cell [3]. However, the specific role of UL37
in capsid trafficking remains unclear. Our lab determined the crystal
structure of the N-terminus of UL37 from Pseudorabies virus (PRV), a
related veterinarian alphaherpesvirus [4]. Efforts to purify and
crystallize the C-terminus and full length PRV UL37 protein have
proven to be difficult due to instability of the constructs. We
hypothesize that the avian homolog of UL37 from Marek’s Disease
Virus (MDV) will be more thermally stable since birds have a higher
internal body temperature than mammals. MDV UL37FL will be
expressed and purified from bacteria and then be screened in 800
different crystallization conditions. Any hit conditions will be optimized
and resulting crystals will be tested for x-ray diffraction. Ultimately, the
crystal structure of MDV UL37 can be used to model the structures of
its human herpesvirus homologs, such as HSV-1 and VZV. The
structure and functional information gained will aid in the design of
new antivirals targeting and disabling the protein UL37.
Conclusions
capsid
dsDNA
inner tegument
outer tegument
lipid envelope
glycoproteins
UL37
UL37 Outer Tegument Host trafficking proteinInner Tegument
1 52
Viral
genome
replication/
packaging
NUCLEUS
3 4
Methods
•Expression of HisSUMO-MDV37FL-Strep and TwinStrep-MDV37FL-
6xHis in Escherichia coli grown in either Luria broth (LB) induced with
IPTG or autoinduction in Terrific broth (TB).
•Purification included Ni-NTA and Strep affinity columns followed by Size
Exclusion Chromatography
Strep
Column
Nickel
Column
Size
Exclusion
E. coli
4L
pJP4
Twin
Strep
6xHis
MDV37FL
6xHisTwin Strep MDV37FL
122kDa
Results
IPTG AI
kDa
250
130
10
25
15
35
70
100
50
Figure 2. (A) Cloned construct of TwinStrep-MDV37FL-6xHis (B) expressed in LB media induced with IPTG or
autoinduced in TB media. The cells were lysed by freezing with liquid N2 and thawed at 37oC five times. This construct
of MDV UL37 does not express when autoinduced and is completely insoluble when induced with IPTG.
pJP4
His-
SUMO
Strep
MDV37FL
MDV37FL StrepHis-SUMO
132 kDa
kDa
250
130
10
25
15
35
70
100
50
LB LB TBTB
1x 1x 2x 2x
Figure 3. (A) Cloned construct of His-SUMO-MDV37FL-Strep (B) expressed in LB media induced with IPTG or
autoinduced in TB media. The cells were lysed by freezing with liquid N2 and thawed at 37oC five times. This
construct of MDV UL37 does not express when autoinduced and is soluble when induced with IPTG.
250
100
70
55
35
25
15
10
130
kDa Elution (desthiobiotin)
250
kDa
130
100
70
55
35
25
15
MDV37FL StrepHis-SUMO
Figure 4. (A) His-SUMO-MDV37FL-Strep was expressed in 4L of LB media induced with IPTG and purified using a
Strep Affinity column. (B) Flow through and Wash 1 was put over the strep column again and all of the elution
fractions from both strep purifications were pooled.
A B
250
130
100
70
50
35
25
15
10
kDa
Future Work
kDa
130
100
70
50
250
35
25
15
10
kDa
250
130
100
70
55
35
25
15
10
HS-MDV37FL-St
HS-MDV37N
A B C
Figure 5. (A) Concentrated MDV UL37 before (B) Nickel affinity column (C) UL37 detected on Nickel resin even
after 500 mM imidazole wash.
UL37 full length of the construct HisSUMO-
MDVUL37FL-Strep expresses in LB media under
IPTG induction. UL37 efficiently binds to both Strep
and Nickel Columns.
•Elute UL37 from the Nickel resin by cleaving the His-
SUMO tag on the column, and collecting the cleaved
protein in the flow through. Cleaved UL37 will be pooled
and concentrated for Size Exclusion Chromatography
and eventually set-up for crystallization.
•Expression and purification of MDV UL37C
•Expression of other homologs such as Human Simplex
Virus 1 (HSV), Varicella Zoster Virus (VZV), Equine
Herpesvirus (EHV), Bovine Herpesvirus (BHV).
•Heldwein Lab
•Building Diversity in Biomedical
Sciences program (R25 HL007785)
•The Leadership Alliance
References
A B
B
1. Krautwald M, Fuchs W, Klupp BG, Mettenleiter TC. 2009. Translocation of incoming pseudorabies virus
capsids to the cell nucleus is delayed in the absence of tegument protein pUL37. J. Virol. 83:3389–
3396.
2. Desai P, Sexton GL, McCaffery JM, Person S. A null mutation in the gene encoding the herpes simplex
virus type 1 UL37 polypeptide abrogates virus maturation. J. Virol. 2001;75(21):10259-10271.
3. Luxton GW, Lee JI, Haverlock-Moyns S, Schober JM, Smith GA. The pseudorabies virus VP1/2
tegument protein is required for intracellular capsid transport. J.Virol. 2006;80(1):201–209.
4. Pitts JD, Klabis J, Richards AL, Smith GA, Heldwein EE. Crystal structure of the herpesvirus inner
tegument protein UL37 supports its essential role in control of viral trafficking. J Virol. 2014 Mar 5;
JVI.00163–14. PMID: 24599989
A
Figure 1: Overview of herpesviral replication cycle. (1) Outer tegument shed following
entry (2) Capsid with bound inner tegument traffics to nucleus (3) Viral genome is replicated and
packaged into newly assembled capsids within the host nucleus (4) Post nuclear egress newly
replicated capsids traffic from nucleus to Trans Golgi Network derived vesicles (5) Capsids bud
through secretory pathway to final release into extracellular environment.