The Laboratory of Michael McVoy
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Michael A. McVoy
Department of Pediatrics
Medical College of Virginia campus of
Virginia Commonwealth University
P.O. Box 980163 MCV Station
Richmond VA 23298-0163
office phone:
804-828-1739
lab phone: 804-828-2291
division secretary: 804-828-0132
fax: 804-828-6455
e-mail: mmcvoy@vcu.edu
Federal
Express Address:
Michael McVoy
Sanger Hall Room 12-026
1101 East Marshall Street
Richmond VA 23298
Jianben Wang, M.D.
Research Associate (1996)
M.D.
Harbin Medical Institute, Harbin P.R. China
M.S. Shanxi Medical Institute,
Taiyuan P.R. China
Anne Sauer
Ph.D. student (2003), Molecular Biology and
Genetics
B.S. University of Alabama (Physical
Education, Health, and Recreation)
B.S. Virginia Commonwealth University
(Biology)
EL-Sayed E.
Habib, Ph.D.
Visiting Scientist (2009)
Associate Professor, Department of
Microbiology, Mansoura University, Egypt
Ian McVoy
Grade 7, Binford Middle School,
Richmond Virginia
click here to view: Ian1 , Ian2
Megan Reeves-Crumpler
Ph.D. Student, Department of Microbiology & Immunology (2004-2008)
B.S. University of Florida (2002)
Currently:
Post-doctoral fellow, Virginia
Commonwealth University School of Medicine
Ben
Meza
M.S. Student, Department
of
Physiology
(2007-2008)
Certificate in Biochemistry, Virginia
Commonwealth University (2007)
B.A. Davidson
College (2006)
Currently: M1 student, University of Pittsburgh School of
Medicine
Juan
Lacayo, Ph.D.
Ph.D.
student, Department of Microbiology & Immunology (1999-2003)
B.S.
Virginia Commonwealth University
Currently: Post-doctoral Fellow, NIH
Erin Douglass
Laboratory Specialist (2003-2004)
B.S. Duke University (2003)
M.D. Medical College of Virginia class of 2008
Cristine Howard
Laboratory Specialist
(2001-2003)
B.S.
College of William and Mary
M.S.
College of William and Mary
M.D.
Medical College of Virginia class of 2007
Melissa
Mondello
Laboratory Specialist (2002-2003)
B.S. University of Richmond
M.D. Medical College of Virginia class
of 2007
Will
Bierach
M.S.
Student (2001-2002)
B.S. Campbell University
Carlos Berbes
Ph.D. student, Department of Microbiology & Immunology (1998-2001)
B.S. Virginia Commonwealth University
M.S. Medical College of Virginia Campus of Virginia Commonwealth
University
Frederic Schynts, Ph.D.
Visiting Ph.D. student from the University of
Liege, Belgium (2001 – 2002)
Ph.D. University of Liege, Belgium.
Currently: Head of Molecular analytics
GSKbiologicals, Rixensart Belgium
Stephanie Siegmund
Field Experience Student (2005 - 2006)
Governor's School for Government and
International Studies
Jessica Abbate
Field Experience Student (1998 - 1999)
Governor's School for Government and
International Studies
B.S. The University of Virginia (2003)
Dipti Ramnarain
Field Experience Student (1998 - 1999)
Governor's School for Government and
International Studies
B.S. The College of William and Mary
(2003)
Yulin Liu
Visiting Scientist (2000 - 2001)
M.D. Xinjiang Medical University,
Urumuqi P.R. China
B.S. Xinjiang Medical University,
Urumuqi P.R. China
Jae Kyun Hur
Visiting Scientist (1996 - 1997)
M.D. Catholic Medical College, Seoul Korea
Ph.D. Catholic Medical College, Seoul Korea
Anupam Bapu Jena
(1995 - 1996)
Governor's School for Government
and International Studies
B.S. Massachusetts Institute of Technology
McVoy, M. and S.P. Adler.
1991. Analysis of human cytomegalovirus DNA replicative intermediates:
DNA forms not predicted by the rolling circle model. In M.P
Landini (ed.), Progress in cytomegalovirus research. Elsevier Science
Publishers, Amsterdam
Abstract
Brown, J. C. , M. A.
McVoy and F. L. Homa. 2002. Packaging DNA into
Herpesvirus Capsids. In A. Holzenburg and E. Bogner
(ed.), Structure-Function Relationships of Human Pathogenic
Viruses. Kluwer Academic/Plenum Publishers, London. Abstract
Schynts, F., F. Meurens, B. Muylkens, A. L. Epstein, M. McVoy, and E. Thiry. 2002. Réplication, clivage-encapsidation et recombinaison de I’ADN des herpèsvirus. Virologie 6:353-52. Abstract
Schleiss, M. R. and M. A. McVoy.
2004. Overview of congenitally and perinatally acquired
cytomegalovirus infections: recent advances in antiviral therapy.
Expert Review of Anti-infective Therapy, 2:89-103.
Selected Journal Publications
McVoy, M. and S.P. Adler. 1994. Human cytomegalovirus DNA replicates after early circularization by concatemer formation and inversion occurs within the concatemer. Journal of Virology, 68:1040-1051
McVoy, M.A., Nixon, D.E., and
S.P. Adler. 1997. Circularization and cleavage of guinea
pig cytomegalovirus genomes.
Journal of Virology, 71:4209-4217
McVoy, M.A., Nixon, D.E.,
Adler, S.P., and E.S. Mocarski. 1997. Sequences within the
herpesvirus-conserved pac1 and pac2 motifs are required
for cleavage and packaging of the murine cytomegalovirus genome.
Journal of Virology, 72:48-56
McVoy, M.A. and E.S. Mocarski. 1999. Tetracycline regulation of
reporter gene expression within the human cytomegalovirus genome.
Virology 258:295-303.
McVoy, Michael A., Daniel E. Nixon, Jay K. Hur, and Stuart P. Adler. 2000. The ends on herpesvirus DNA replicative concatemers contain pac2 cis cleavage/packaging elements and their formation is controlled by terminal cis sequences. Journal of Virology, 74:1587-1592.
McVoy, M.A. and D. Ramnarain. 2000. The machinery to support genome segment inversion exists in a herpesvirus which does not naturally contain invertible elements. Journal of Virology, 74:4882-7.
Abbate, J., J. C. Lacayo, M. Prichard, G. Pari, and M. A. McVoy. 2001. A bifunctional protein conferring enhanced green fluorescence and puromycin resistance. BioTechniques, 31: 340-347.
Nixon, D. E. and M. A. McVoy. 2002. Terminally repeated sequences on a herpesvirus genome are deleted following circularization but are reconstituted by duplication during cleavage and packaging of concatemeric DNA. Journal of Virology, 76:2009-2013.
DeWire, S., M. A.
McVoy and
B. Damania. 2002. Kinetics of expression of rhesus monkey
rhadinovirus
(RRV) and characterization of a polycistronic transcript encoding RRV
Orf50/Rta,
RRV R8, and R8.1 genes. Journal of Virology, 76:9819-9831.
Mark R. Schleiss, David I.
Bernstein, Michael A. McVoy, Greg Stroup, Fernando Bravo, Blaine
Creasy, Alistair McGregor, Kristin Henninger, and Sabine Hallenberger.
2005. The Nonnucleoside Antiviral, BAY 38-4766, Protects Against
Cytomegalovirus Disease and Mortality in Immunocompromised Guinea
Pigs. Antiviral Research, 65:35-43.
Michael A. McVoy and Daniel E. Nixon.
2005. The Impact of BDCRB
(2-Bromo-5,6-Dichloro-1-_-D-Ribofuranosyl Benzimidazole Riboside) and
Inhibitors of DNA, RNA, and Protein Synthesis on Human Cytomegalovirus
genome maturation. Journal of Virology, 79:11115-11127.
Book Chapters
Analysis of human cytomegalovirus (HCMV) DNA replicative intermediates: DNA forms not predicted by the rolling circle model
Michael A. McVoy and Stuart P. Adler. In M. P. Landini (ed.), Progress in Cytomegalovirus Research. Elsevier Science Publishers, Amsterdam. 1991.
INTRODUCTION The mechanism of HCMV DNA replication has not been studied in detail but has generally been assumed to be similar to that of Herpes Simplex Virus (HSV). HSV DNA is replicated via a large primary replicative intermediate (PRI) that lacks terminal restriction fragments. The structure of this PRI has not been conclusively established; however, it is likely to be one of two possibilities: i) a head to tail concatemer generated by a rolling circle mechanism or ii) interlocked unit length circles generated by theta replication1. To determine if HCMV DNA is replicated through a similar PRI, we characterized the replicative intermediates following field inversion gel electrophoresis (FIGE).
MATERIALS AND METHODS Infection and Preparation of DNA Human MRC-5 fibroblasts were infected with strain AD169 at an moi of 0.5-1.0. Cells were washed with PBS, scraped, pelleted, re-suspended in 30ul melted 1% seaplaque agarose (FMC) in TE and cast into plugs. Plugs were incubated 48h at 50 C in 0.5ml TE 1% sarkosyl 1ug/ml proteinase K and dialyzed 3x2h with TE and stored at 4 C. Field Inversion Gel Electrophoresis FIGE was performed at 10 C using 0.5X TBE in a horizontal agarose gel electrophoresis box. DNA samples were melted at 65 C and loaded into wells using a wide bore pipet tip. Forty milliliter (8x11cm) gels were run at 80V for 20h with pulses starting at 5s, increasing to 45s and a forward to backward ratio of 3:1. One hundred milliliter (11x14cm) gels were run at 120V for 36h with pulses starting at 5s and increasing to 60s. Southern Hybridization FIGE separated DNA was transferred to Nytran (Schleicher and Schuell) by capillary blotting, UV cross-linked (0.12 J/cm2) and hybridized according to the manufacturer's instructions. Probes were random hexamer labeled (Boehringer Mannheim kit) with 32P dCTP (106 cpm/hybridization). Terminal fragment analysis DNA from cells 5 days post-infection (PI) were separated by FIGE in duplicate on a 40ml 1% seaplaque gel. One lane was cut from the gel and dialyzed 3x30min with 1X EcoRI buffer, then digested for 16h at 37 C with 2500U EcoRI. The digested gel slice was cast laterally into a 100ml 1% agarose gel and separated in a second dimension by FIGE at 120V for 20 h. Pulses increased from 0.1 to 2.0s with forward to backward ratio of 2:1. The gel was aligned with the undigested duplicate lane and overlaid with 1% agarose. The composite gel was blotted and sequentially hybridized with probes (fig.1) specific for the long arm terminal fragment W (pON227), the short arm terminal fragments N and L (Towne XbaI I in pACYC184), or a long unique region control (Towne XbaI E in pACYC184). -irradiation DNA plugs from cells 5 days PI were separated by FIGE on a 40ml gel and the positions of DNA forms were determined by ethidium bromide staining of lanes cut from each edge of the gel. DNA forms were cut into blocks, placed in microfuge tubes containing 1 ml TE, and -irradiated using a CES-I- RAD 1000 137Cs/134Cs source. Irradiated blocks were cast into a 100ml gel and separated by FIGE. Gels were blotted and probed with the Towne XbaI E fragment.
RESULTS DNA prepared five days PI contains four HCMV DNA forms: a high molecular weight (HMW) form that does not migrate from the loading well, an approximately 1000 kb form, a 500-600 kb form, and a unit-length form of 230 kb (fig.2 lane 5). DNA from uninfected cells did not hybridize with the probe. Time course DNA was prepared from cultures one hour PI and each day thereafter. DNA was separated by FIGE, Southern blotted and probed with the XbaI E fragment (fig.2). Viral DNA is first detected in the HMW form 48h PI. Unit length DNA is first detected on day 4 and the 500-600 kb and 1000 kb forms are first detected on day 5. Pulse labeling experiments using 32P phosphate confirmed that the HMW form is first synthesized at about 36-48h PI and is subsequently processed to the smaller forms (data not shown). Terminal Fragment Analysis Probing for the short arm EcoRI terminal fragments N and L with the XbaI I probe (fig.1) reveals that only unit length DNA has short arm termini (N and L are not detected in HMW, 1000 kb, and 500-600 kb DNA but are detected in unit length DNA, not shown). Reprobing for the long arm EcoRI terminal fragment W with the pON227 probe reveals that only unit length DNA has long arm termini (W is not detected in HMW, 1000 kb or 500-600 kb DNA but is detected in unit length DNA, not shown). Reprobing with a long unique region fragment (XbaI E) demonstrates that all four forms have the four EcoRI fragments that overlap with XbaI E (not shown).
-Irradiation Analysis Low dose -irradiation was used to distinguish circular from linear DNA forms (unit sized circles are converted to unit length linear molecules by a single double-stranded break; linear molecules are broken into random-length fragments2). -irradiation of 230 kb and 1000 kb DNA produced a shearing pattern consistent with linear DNA - even smearing with no discrete bands produced (not shown). -irradiation of 500-600 kb DNA produced a band of 230 kb, suggesting that 500-600 kb DNA is composed, at least in part, of unit length circles (not shown). No discrete bands of any size are produced by -irradiation of HMW DNA, indicating that it is not composed of interlocked circles (not shown).
DISCUSSION Our results indicate that the HMW DNA is the primary replicative intermediate of HCMV: it is synthesized first, is large, and lacks termini. It is not composed of interlocked circles and may be a long linear concatemer. The 500-600 kb and 1000 kb DNA forms lack termini and are therefor not products of incomplete cleavage (dimers and tetramers) of a concatemer. Such molecules should have terminal fragments albeit at reduced molar ratios. The 1000 kb form is apparently linear. Its role in the replicative process, if any, is unknown. The 500-600 kb DNA is composed in part of unit length circles and is present in low but significant amounts. Two models satisfy the current data on Herpesvirus DNA replication: (1) the rolling circle model, which is generally favored, suggests that the PRI is a long linear concatemer of head to tail linked genomes; (2) the theta replication model suggests that the PRI is composed of interlocked unit length circles1. Our observation that the HMW DNA is not composed of interlocked circles clearly rules out the theta replication model for HCMV; however, the presence of significant amounts of unit length circular DNA is not consistent with the rolling circle model. The presence of these circles suggests that replication may proceed in a process analogous to that of E. coli phage Lambda3 in which the linear genome circularizes and replicates first by theta replication producing unit length circles. Late in infection replication switches to rolling circle, using the unit length circles produced by early theta replication as templates.
Packaging DNA into Herpesvirus Capsids
Jay C. Brown, Michael A. McVoy and Fred L. Homa. In A. Holzenburg and E. Bogner (ed.), Structure-Function Relationships of Human Pathogenic Viruses. Kluwer Academic/Plenum Publishers, London. 2002.
Injection of DNA into a pre-formed capsid is a central event in
herpesvirus replication.
Similar packaging of DNA into a pre-formed shell is also observed
during
replication of dsDNA bacteriophage such as T4 and l; adenoviruses may
encapsidate
DNA in the same way. Herpesvirus DNA packaging takes place in the
infected cell nucleus where capsid assembly and DNA replication also
occur.
The substrates for packaging are capsids plus the multi-genome,
concatemeric DNA that is the product of virus DNA replication.
During the encapsidation process, double strand cuts are made at
specific sites (pac sites) in
the DNA concatemer so that one complete genome is packaged into each
capsid.
Genetic studies with herpes simplex
virus 1 (HSV-1) have resulted in identification of seven virus genes
whose protein products are specifically involved in DNA
encapsidation. None is required, for example, for capsid
formation or DNA replication. The seven proteins are expected to
be involved in processes such as introduction of specific cuts in the
DNA concatemer, formation of a portal through which virus DNA can enter
the capsid, provision of the energy required for DNA translocation into
the capsid and sealing the capsid once it is filled.
Investigators are studying herpesvirus DNA packaging with the idea that
it constitutes an attractive target for novel therapeutic agents
directed against herpesvirus replication. Encapsidation is
particularly appealing as a target
because it is required for herpesvirus growth, and because most, if not
all, of the proteins involved are virus-encoded.
Recent
studies of herpesvirus DNA packaging have defined the basic nature of
the process and provided information about the components involved in
individual steps. Here we summarize recent progress with emphasis
on areas such as pac site recognition and the function of the
processing/packaging proteins where there has been the most
interest. Discussion is focused on HSV-1 with other
herpesviruses, particularly cytomegalovirus, mentioned when relevant
studies have been done. The mechanism of DNA encapsidation as it
occurs in dsDNA bacteriophage is described briefly because the same
basic mechanism is expected to apply in herpesviruses and because, as
studies with phage are generally more advanced, they have suggested
productive lines of research with herpesviruses. We conclude with
a brief description of small molecule packaging inhibitors that have
the potential to be developed as anti-herpes therapeutics.
Réplication,
clivage-encapsidation et recombinaison de I’ADN des herpèsvirus
Schynts, F., F. Meurens,
B. Muylkens, A. L. Epstein, M. McVoy, and E. Thiry. Virologie, in
press.
*Herpesvirus genomes consist of a large, linear double stranded
DNA. The DNA replication process of herpesviruses takes place in
the nucleus where the genome circularizes and is replicated by a
mechanism resulting in formation of concatemeric molecules
in which the genomic termini are fused in a head-to-tail
arrangement. This process requires the expression of virally
encoded proteins. Concatemers
are then cleaved at specific sites by viral proteins, specified by the
presence of cis-acting sequences, to form unit-length molecules that
are packaged
into newly formed capsids. This paper summarizes the current
knowledge
of both replication and cleavage/packaging of herpesvirus DNA as well
as
antiviral drugs which target these viral processes. Finally,
herpesvirus
DNA recombination is described because it is intimately linked to the
DNA
replication process.
*note: This paper is in
French. The abstract above has been translated to English.
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