Abstracts from 11th Symposium Antiviral Drug Resistance

Rita

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XMRV abstracts from the
11th Symposium on Antiviral Drug Resistance
Hershey, PA, Nov. 7-10- 2010.



http://antiviralresistance.org/ProgramBook_SADR10.pdf



Oral Presentations



http://antiviralresistance.org/abstract25_2010.pdf

INEFFICIENT REPLICATION AND SPREAD OF XENOTROPIC MURINE LEUKEMIA
VIRUS-RELATED VIRUS IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

Chawaree Chaipan1, Kari A. Dilley2, Tobias Paprotka1, Krista A.
Delviks-Frankenberry1, Narasimhan J. Venkatachari1, Wei-Shau Hu2, and
Vinay K. Pathak1*

1Viral Mutation Section and 2 Viral Recombination Section, HIV Drug
Resistance Program, National Cancer Institute at Frederick, Frederick,
MD 21702, USA

Xenotropic murine leukemia virus-related virus (XMRV) is a
gammaretrovirus recently isolated from human prostate cancer and
peripheral blood mononuclear cells (PBMCs) of patients with chronic
fatigue syndrome (CFS). Previously, we showed that host restriction
factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in
human PBMCs, potently inhibit XMRV replication. The recovery of
infectious XMRV from human PBMCs in CFS patients suggested that XMRV
can replicate in these cells despite the expression of APOBEC3
proteins. To determine the extent of XMRV replication and spread, we
infected phytohemaglutinin-activated human PBMCs and A3G/A3F-positive
and negative cell lines (CEM and CEMSS, respectively) with different
amounts of XMRV and monitored virus production using quantitative
realtime PCR. We found that XMRV efficiently replicated in CEM-SS
cells but not in CEM cells and activated PBMCs. However, infectious
XMRV could be recovered from the infected PBMCs by co-cultivation with
canine indicator cells, and we observed low-level hypermutation of
XMRV genomes in PBMCs. Overall, these results indicate that A3G/A3F
expression in human PBMCs constitutes a potent block to replication.



http://antiviralresistance.org/abstract43_2010.pdf

LOOKING FOR XMRV IN PROSTATE CANCER

Amanda L. Aloia1, Karen S. Sfanos2, Jessica L. Hicks2,6, William B.
Isaacs3-6, Qizhi Zheng2,6, Kenneth J. Pienta7, Frank Maldarelli1,
Angelo M. De Marzo2-6, and Alan Rein1

1HIV Drug Resistance Program, National Cancer Institute, Frederick, MD
21702; Department of 2Pathology, 3Urology, and 4Oncology, 5The Brady
Urological Research Institute and the 6Sidney Kimmel Comprehensive
Cancer Center at Johns Hopkins, Baltimore, MD 21231; 7The University
of Michigan Comprehensive Cancer Center, Ann Arbor, MI 48109

Several recent papers have reported the presence of a gammaretrovirus,
termed XMRV (xenotropic murine leukemia virus-related virus) in
prostate cancers (PCa). If confirmed, this could have enormous
implications for the detection, prevention, and treatment of PCa.
However, other papers report failure to detect XMRV in PCa. We tested
nearly 800 PCa samples, using a combination of real-time PCR and
immunohistochemistry (IHC). The PCR reactions were simultaneously
monitored for amplification of a single-copy human gene, in order to
confirm the quality of the sample DNA and its suitability for PCR.
Controls demonstrated that the PCR assay could detect the XMRV in a
single infected cell, even in the presence of a 10,000-fold excess of
uninfected human cells. The IHC used two rabbit polyclonal antisera,
each prepared against a purified MLV protein. Both antisera always
stained XMRV-infected or -transfected cells, but never stained control
cells. No evidence for XMRV in PCa was obtained in these experiments.
It is possible that XMRV is not actually circulating in the human
population; even if it is, the data do not seem to support a causal
role for this virus in PCa.



http://antiviralresistance.org/abstract44_2010.pdf

PROBING THE RELATIONSHIP OF XMRV AND MLV

John M. Coffin1, Oya Cingz1, Ann Wiegand2, Jon Spindler2, and Mary Kearney2

1Department of Molecular Biology and Microbiology and Program in
Genetics, Tufts University, Boston MA 02111; 2HIV Drug Resistance
Program, National Cancer Institute, Frederick MD 21702

Xenotropic MLV-related virus (XMRV) was first described in a few cases
of prostate cancer about 5 years ago. XMRV is closely relate to, but
distinct in sequence from, known endogenous xenotropic MLV (Xmv)
proviruses. It has since been associated with larger numbers of
prostate cancers, as well as a significant fraction of patients with
chronic fatigue syndrome (CFS). In these cases, it has been possible
to isolate infectious virus and study its properties. Some more recent
studies have identified other types of endogenous MLV-related
sequences (polytropic-Pmv- and modified polytropic, Mpmv) in a cohort
of CFS cases, but not in random blood donors. In the latter case,
sequences have been identified only by PCR amplification of a fragment
of the genome. Neither complete genome sequences nor infectious virus
has been obtained. In the meantime, a number of other investigators
have reported nearly complete failure to replicate these results by
finding virus or antiviral antibodies in other cohorts of CFS and
prostate cancer. The very close similarity of XMRV and the large
numbers (around 60 in inbred mice) of endogenous MLVs raise
unclarified issues regarding the provenance of virus and virus-like
sequences identified in clinical samples. Our groups have been
investigating the relationship between endogenous MLVs and XMRV
with the goal of understanding the origin of the virus and the
potential for confusion due to inadvertent contamination of assays
with mouse DNA. Using a highly specific PCR assay for XMRV, based on
characteristic conserved indels in the LTR and gag, we have been
looking for closely related sequences in wild and inbred mouse DNA, so
far without success. In another study, the DRP group has developed
highly specific and sensitive PCR assays (X- single copy assay (SCA)
and X- single genome sequencing
(SGS)) to quantitate and distinguish XMRV and endogenous MLV
sequences. In reconstruction experiments, these assays could detect
single copies of XMRV RNA in plasma and very low numbers of infected
cells in whole blood. They are also very sensitive detectors of mouse
DNA contamination, allowing detection and genetic analysis of the
proviral DNA im less than one-tenth of a cell.

To distinguish mouse DAN contamination, we have developed a PCR assay
based on the LTR sequences of the intracisternal A particle (IAP)
retroelement, of which there are more than a thousand copies in the
mouse genome, but none in human DNA. This assay provides a clear and
unambiguous means of detecting much less than 1 cells worth of mouse
DNA.


Poster Presentations



http://antiviralresistance.org/abstract_poster5_2010.pdf

POSTER 5

BIOCHEMICAL, STRUCTURAL, AND INHIBITION STUDIES OF XMRV REVERSE TRANSCRIPTASE

Tanya Ndognwe1, Karen Kirby1, Bruno Marchand1, Adeyemi Adedeji1,
Eleftherios Michailidis1, Yee-Tsuei Ong1, Atsuko Hachiya1, Emily
Ryan1, Shun-Lu Liu1, Angela Whatley1, Donald H. Burke1, Sanath Kumar1,
Marc Johnson1, Ei-Ichi Kodama2, Krista A. Delviks-Frankenberry3, Vinay
K. Pathak3, Hiroaki Mitsuya4, Michael A. Parniak5, Kamal Singh1, and
Stefan G. Sarafianos1

1Department of Molecular Microbiology & Immunology, University of
Missouri School of Medicine, Columbia, MO; 2Division of Emerging
Infectious Diseases, Tohoku University School of Medicine, Sendai,
Japan; 3HIV Drug Resistance Program, National Cancer Institute,
Frederick, MD; 4Department of Internal Medicine, Kumamoto University
School of Medicine, Kumamoto, Japan & Experimental Retrovirology
Section, HIV/AIDS Malignancy Branch, NIH, Bethesda, MD; 5Department of
Molecular Genetics & Biochemistry, University of Pittsburgh School of
Medicine, Pittsburgh, PA

The Xenotropic Murine Leukemia Virus-Related Virus (XMRV) was
originally identified in biological samples from familial prostate
cancer patients and was more recently reported in patients with
chronic fatigue syndrome (CFS). Although other studies have failed to
detect XMRV in CFS or prostate cancer patients, given the potential
importance of this virus for human disease we initiated studies on its
replication mechanism and susceptibility to inhibitors. We used
biochemical, biophysical, virological, and structural methods to
characterize the DNA polymerase and RNase H functions of XMRV reverse
transcriptase (RT). We compared the properties of XMRV RT, XMRV RNase
H, Moloney Murine Leukemia Virus (MuLV) RT, and HIV RT. Using steady
state and pre-steady state kinetics we demonstrated that XMRV RT is
the slowest and least efficient of the three enzymes in synthesizing
DNA or cleaving RNA/DNA. Similarly, its ability to unblock
chain-terminated primers using PPi or ATP are much lower compared to
HIV RT. Its reduced polymerase and RNAse H activity is due in part to
a lower affinity for nucleic acid, as judged by gel-shift assays and
confirmed by surface plasmon resonance experiments, which revealed
that the deficiency in DNA binding is due to a high dissociation rate.
Trap experiments showed that XMRV RT has very low processivity
compared to HIV RT. Transient kinetics of mismatch incorporations
revealed that XMRV RT has higher fidelity than MuLV and HIV RTs.
Nonetheless, XMRV and MuLV appear to have comparable fidelities in
cell-based assays. The polymerase function of XMRV RT is susceptible
to antiretrovirals from several classes, but not to NNRTIs. XMRV RT is
inhibited efficiently by AZT-TP, PMEADP, d4T-TP, PMPA-DP, and by a
nucleic acid aptamer. Two potent NRTIs that block XMRV RT efficiently
by a different mechanism also have potent antiviral activity in
pseudotype-based and replication competent virus-based assays. We have
also identified compounds that efficiently block the RNase H activity
of XMRV RT and of active XMRV RNase H fragments. Finally, we have
solved the crystal structure of XMRV RNase H at high resolution (1.5
), which will facilitate the design of new and more potent XMRV
inhibitors.



http://antiviralresistance.org/abstract_poster44_2010.pdf

POSTER 44

DEVELOPMENT OF A GFP-INDICATOR CELL LINE FOR THE DETECTION OF XMRV

KyeongEun Lee1, Francis W. Ruscetti2, Patricia Lloyd1, Alan Rein1,
Gisela Fanning-Heidecker1, and Vineet N. KewalRamani1

1HIV Drug Resistance Program and 2Laboratory of Experimental
Immunology, National Cancer Institute- Frederick, Frederick, MD 21702,
USA

Human immunodeficiency virus (HIV) titer can be estimated using
indicator cell lines, such as GHOST cells, within days of infection.
HIV indicator cells rely on production of Tat to transactivate
expression of a reporter gene under the control of HIV LTR sequences.
Simple retroviruses typically do not encode transcriptional
transactivators. For simple retroviruses that lack transformational or
cytopathic activity, their titers are often measured by infection of
cells after end point dilution and assaying for virus proliferation
after weeks of culture. Replication-dependent vectors have been
leveraged to assay the mobilization of retrotransposable elements and
the replication of retroviruses. Here we describe an indicator cell
line for the detection of infectious xenotropic murine leukemia
virus-related virus (XMRV) that relies on the propagation of a vector,
which leads to expression of a GFP reporter. We constructed an MLV
vector encoding puromycin resistance and a CMV enhancer/promoter
driven GFP reporter gene whose transcription was antisense to the
vector mRNA. The GFP reporter sequence (iGFP) was interrupted by an
intron placed in the sense direction relative to the vector. The
prostate cell line, LNCaP, was stably transfected with the above
construct, and several LNCaP-iGFP cell clones displaying sensitivity
to XMRV infection after end point dilution were isolated and
designated Detectors of Exogenous Retroviral Sequence Elements (DERSE)
cells. GFP signal could be detected within three days of infection,
with the number of GFP-positive cells increasing over subsequent days.
GFP signal after virus inoculation was dosedependent and could be
impaired by heat inactivation of virus stocks or the addition of AZT
to cultures at the time of infection. In principle, DERSE cells should
also detect other gammaretroviruses capable of
infecting human cell lines. DERSE cells provide a facile assay to
assess antiviral or antibody mediated neutralization of XMRV, and
should be useful in assessing the presence of infectious XMRV in
patient materials.
 

August59

Daughters High School Graduation
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Thanks Rita - There was some good stuff in here. Seems as if the have defintely compiled an assay that will screen out ALL contamination. Looks as if there is some more definitive studies as to what ARV work against XMRV, plus some that are in the works as well?
 

eric_s

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Thanks, it's interesting. The first study seems to say that they could not find XMRV in PBMC unless they used culturing, so one more piece of evidence that a PCR study that looks for XMRV in PBMC without that method is unlikely to find something.

Then Coffin says that they could not find any DNA sequences in mice that match what Alter/Lo have found. And that they have a good assay to screen for contamination with mouse DNA. So if we have not heard Coffin or the BWG, that he's a member of, say "it's contamination", that's good news.
On the other hand, using that assay they seem to not have been able to find XMRV in prostate cancer, so i feel like it's not really sure wheter it's working in real samples.

The next one is about what was reported in newspaper articles where they quoted the researcher as saying his work could be "potentially explosively important" and then there's one about the DERSE cells. If Ruscetti has them, i hope he shares that with the WPI. Would be one more way to rule out contamination, i think, but that's just my layman's understanding. I think with what they know now about XMRV and the DERSE cell lines, it should be possible to develop at least some form of treatment and also measure it's success. So with more money, more personnel and some more time, we should see some good results, i hope.
 

omerbasket

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On the other hand, using that assay they seem to not have been able to find XMRV in prostate cancer, so i feel like it's not really sure wheter it's working in real samples.
Correct me if I'm wrong, but I think that the people who are named in the study about this assay are not the people who are named in the study about not finding XMRV in 800 cases of prostate cancer - and also there is no evidence that the study that didn't find XMRV in 800 cases of prostate cancer used the assay described in Coffin et al. study.

So - perhaps there is not other hand here...;)