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Persistence of HIV through integration into host DNA in CD4(+) T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8(+) T cells are triggered to kill infected CD4(+) T cells through recognition of histocompatibility leukocyte antigen (HLA) class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with "beneficial'' HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtain proof of early presentation of a virion-derived HLA-A*02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag Spacer Peptide 2 (SP2). FH9-specific CD8(+) T cell responses are detectable in individuals with primary HIV infection and eliminate HIV-infected CD4(+) T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target.

Background: Antiretroviral therapy (ART) cannot cure HIV infection because of a persistent reservoir of latently infected cells. Approaches that force HIV transcription from these cells, making them susceptible to killing—termed kick and kill regimens—have been explored as a strategy towards an HIV cure. RIVER is the first randomised trial to determine the effect of ART-only versus ART plus kick and kill on markers of the HIV reservoir. Methods: This phase 2, open-label, multicentre, randomised, controlled trial was undertaken at six clinical sites in the UK. Patients aged 18–60 years who were confirmed as HIV-positive within a maximum of the past 6 months and started ART within 1 month from confirmed diagnosis were randomly assigned by a computer generated randomisation list to receive ART-only (control) or ART plus the histone deacetylase inhibitor vorinostat (the kick) and replication-deficient viral vector T-cell inducing vaccines encoding conserved HIV sequences ChAdV63. HIVconsv-prime and MVA.HIVconsv-boost (the kill; ART + V + V; intervention). The primary endpoint was total HIV DNA isolated from peripheral blood CD4+ T-cells at weeks 16 and 18 after randomisation. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, NCT02336074. Findings: Between June 14, 2015 and Jul 11, 2017, 60 men with HIV were randomly assigned to receive either an ART-only (n=30) or an ART + V + V (n=30) regimen; all 60 participants completed the study, with no loss-to-follow-up. Mean total HIV DNA at weeks 16 and 18 after randomisation was 3·02 log10 copies HIV DNA per 106 CD4+ T-cells in the ART-only group versus 3·06 log10 copies HIV DNA per 106 CD4+ T-cells in ART + V + V group, with no statistically significant difference between the two groups (mean difference of 0·04 log10 copies HIV DNA per 106 CD4+ T-cells [95% CI −0·03 to 0·11; p=0·26]). There were no intervention-related serious adverse events. Interpretation: This kick and kill approach conferred no significant benefit compared with ART alone on measures of the HIV reservoir. Although this does not disprove the efficacy kick and kill strategy, for future trials enhancement of both kick and kill agents will be required.

Sequences necessary for transduction of human endogenous retrovirus (HERV)-K con , a consensus of the HERV-K(HML-2) family, were analyzed and found to reside in the leader/ gag region. They act in an orientation-dependent way and consist of at least two sites working together. Having defined these sequences, we exploited this information to produce a simple system to investigate to what extent virions of HERV-K con , murine leukemia virus, and HIV-1 have the ability to transduce each other's genomes, leading to potential contamination of gene therapy vectors.

15 pages, 8 figures.-- PMID: 18195071 [PubMed].-- PMCID: PMC2234371., The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/106 mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen., The Ev02 clinical trial has been sponsored by the EuroVacc Foundation. The EuroVacc program has been supported by the European Commission fifth framework program under research grants QLK2-CT-1999-01321, QLK2-CT-2001-01316, and QLK2-CT-2002-01431.

A conceptual breakthrough in gene therapy would be gene transfer vector that could be systemically applied, allowing targeted gene transfer into a predetermined cell type. The host range of a retroviral vector is determined by the interaction of the viral envelope glycoprotein (Env) and the retrovirus receptor on the surface of the host cell. In this review, we describe the current efforts to engineer targeted envelope glycoproteins, which can be incorporated into retroviral particles and are capable of delivering genes in a highly specific manner.

Redirecting retroviral vector transduction simply by insertion of a ligand into the envelope (Env) protein has met with several obstacles. For example, virions targeted to epidermal growth factor receptor (EGFR), after receptor binding, rapidly traffic to the lysosomes, where they are degraded. Exotoxin A of Pseudomonas aeruginosa has the ability to translocate from endosomes to the cytoplasm by means of a translocation domain (TLD). We generated a series of chimeric Env proteins of Moloney murine leukemia virus containing EGFR ligands, where TLD was inserted into different regions. These chimeric proteins were successfully produced, if the translocation domain was not located at the immediate N-terminus of Env. The ability to transduce murine cells via the ecotropic receptor varied but correlated with the amount of Env proteins incorporated into the virions. Chimeric vector particles could bind to EGFR, demonstrating the functional exposure of the peptide ligand. However, transduction of human cells expressing EGFR but not the ecotropic receptor by virions carrying the chimeric protein was not observed.

It is with deep sadness that the retrovirus community learned of the passing of Axel Rethwilm, Professor of Virology in Wurzburg, Germany, just a few days before his 55th birthday. Researchers working on foamy viruses (FV) have lost a valuable, colleague, generous mentor and a good friend. Axel (Figure 1) studied Medicine in Freiburg where he also obtained his doctorate before joining the retrovirus laboratory in the Institute for Virus Research at the German Cancer Research Center in Heidelberg. Later, he moved to Wurzburg to work with his mentor, Volker ter Meulen. At 38, Axel was appointed as a full Professor of Virology in Dresden and in 2003 he succeeded Volker ter Meulen to the Chair of Virology at Wurzburg. With his big personality, his sharp mind, an unlimited scientific curiosity, and his refreshing openness, Axel brought to the position new and exciting ideas, and sometimes unconventional solutions. Figure 1 Axel Rethwilm in 2008 at the main train station in Frankfurt am Main, Germany. Photo provided by Ottmar Herchenroder. Research active for over thirty years, Axel’s name was synonymous with FVs, his enduring passion. Since the first detection around sixty years ago of the Spumaviruses (as they were originally designated, from the Latin, spuma, meaning foam), more than a tenth of published FV articles have included Axel’s name as an author. His first scientific work appeared in 1983 under the guidance of the future Nobel laureate, Harald zur Hausen [1]. In the same year, HIV-1 was discovered, fuelling a retrovirology reawakening. Thereafter, Axel was frequently asked why he continued to work on a rare retrovirus that was deleterious to cell cultures, but did little else. Undeterred, Axel responded that he liked to understand all nature’s complexity. In the first decade of Axel’s career, much of FV research was dominated by the question of which disease(s) may be caused by the so-called human foamy virus (HFV) [2]. A good number of reports associated several maladies with this isolate, and even claimed that whole sections of the human population carried the infection. For years controversy raged in the FV community over the validity of Western blots and PCR carried out on human samples. A controlled blinded evaluation of blood samples was needed and Axel organised that fresh clinical samples from thyroiditis patients were retested in three international laboratories, including his own. They proved to be FV-negative. A later larger study put an end to the dispute [3]. Subsequent to this, and in the light of emerging sequence data, the community agreed that the “viruses in search of a disease” [4] were “not so human after all”, but rather a variant of a chimpanzee foamy virus zoonotically transmitted to man as a dead-end infection [5]. Thereafter, Axel successfully campaigned to rename HFV the prototypic foamy virus (PFV). Nevertheless, humans can be infected without apparent pathology by FVs following close contact with several non-human primate species [6]. Axel was one who from the start brought molecular techniques to bear on his investigations of FVs, greatly contributing to the complete sequence of HFV. With his first HFV infectious molecular clone [7], in-depth studies on FV could lift off and Axel and others were able to discover some unique properties of FVs that distinguished them from all other retroviruses: a second promoter outside the 5’ LTR [8], translation of the polymerase gene as a separate protein from a spliced RNA and the fact that mature FV particles contain large amounts of almost full-length double-stranded DNA [9]. These features result in a unique assembly strategy [10] and the requirement of Env co-expression for particle egress [11]. Twenty years ago Axel’s group produced the first FV-based vector system [12] and for years led their further development. Today, FV vectors are showing promising results in pre-clinical testing for the treatment of inherited diseases. Alas, Axel did not live long enough to see one of his scientific goals realised: a patient treated with FV vectors in a gene therapy trial. Axel was very engaged with the scientific community. He served on many occasions as an expert referee for various journals, including Retrovirology. For journal editors he was probably the first port of call for a review on any aspect of FVs and he was a conscientious, rigorous, but fair, referee. Although it was physically becoming more demanding, he enthusiastically joined his colleagues in international meetings, including the Frontiers of Retrovirology meeting that took place in Cambridge in the autumn of 2013. In February this year, he was very proud to show off the 6th volume of the bible of our scientific discipline: Fields Virology 2013, with a chapter on FVs that he had written with Dirk Lindemann [13]. He never lost his enthusiasm for the latest developments in the field, and was not averse to catching up with the latest gossip. It was rare to see Axel without a smile on his face. His warmth and well-developed sense of humour, together with his unmistakable laugh, will be sorely missed at scientific meetings. Axel suffered from a heritable, progressive motor neuron disease, which latterly limited his physical abilities. He lived bravely with increasing physical infirmity, stubbornly trying to ignore its ravishing effects for as long as possible. By pointing out that the gene responsible for his impairment was dispensable in mice, he displayed his own brand of black humour. At the most recent 10th International Foamy Virus Conference in Pulawy, Poland, only seven weeks before his passing, although frail, he was as the community always knew him: interested, eager, constructively critical, supportive, a textbook of foamy- and retrovirology, conceiving fresh ideas and discussing future projects. He had become interested in a novel species of FV-encoded micro RNAs recently identified by him and others [14,15] and predicted that they would become central to FV research in the coming years. Reflecting his enthusiasm for FVs, Axel was always keen to identify more FVs, for instance from bats and other species known as key players in zoonotic events. His increasing handicap did not restrain Axel from tiring trips into the heart of Africa. He actively supported research there and in India and was the initiator of and speaker at the first German-African Graduate School where young scientists performed research on HIV, AIDS and associated infectious diseases. Axel was interested in and dedicated to teaching. He supported several PhD and MD students from Germany and Africa by giving, from his personal funds, long-term stipends and travel grants allowing students to join national and international meetings or to visits others laboratories abroad. His work on HIV in Africa highlighted more newly infected HIV-patients with resistant virus [16] than had been estimated by the WHO. It is a pity that Axel’s excursion into the field of pathogenic retroviruses came so late in his career, although he had been seduced to working on other human pathogens in the course of more clinical studies (HIV-1, parvovirus, Epstein-Barr virus, norovirus, astrovirus, adenovirus, bocavirus, chikungunya virus). For many working under his guidance, Axel was a great mentor and a friend beyond their time in his laboratory. Current and former lab members had, in a way, been Axel’s family. We all profited from this. We shall miss him, not only as an excellent scientist, colleague and mentor, the font of all FV knowledge, but as a friend with a strong and unique personality. In the last review that Axel wrote before he died, he ended with the words “if I can stimulate one or more researches to pick up FV biology in their research repertoire, the mission of this review is accomplished”. The review may not have been published, but the mission was accomplished in his lifetime (Figure 1).

Retroviruses may cause diseases in their vertebrate hosts. They are distinguished by their common means of replication involving reverse transcription, a process inhibited by nucleoside reverse transcriptase inhibitors (NRTIs) and other compounds used in antiretroviral chemotherapy. Previous work on NRTIs has been limited to their effect on human immunodeficiency virus (HIV) (for review see Ho & Hitchcock, 1989; Weller, 1999) and little information exists regarding the efficacy and therapeutic potential of these drugs against other retroviruses. We have tested all six NRTIs licensed for HIV treatment [didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), zidovudine (AZT) and abacavir (ABC)] against seven retroviruses representative of the traditional subfamilies: Spumavirinae, Lentivirinae and the Oncovirinae. As expected, each drug showed a range of activities against the panel of retroviruses, some drugs inhibiting other viruses at concentrations well below those required for HIV. Overall, AZT was the most active inhibitor (IC50 range, 0.032–1.0 μM), being most active against the Spuma (foamy) viruses. Abacavir was inhibitory for HIV-1, MN strain (HIV-1 MN), amphotrophic murine leukemia virus (MLV-A) and simian foamy virus type 6 (SFV-6). The least effective inhibitor, 3TC (IC50 range, 0.32–>100 μM), was most potent against simian retrovirus types 1 and 2 (SRV-1, SRV-2) and HIV-1, but did not inhibit foamy viruses and MLV-A. Additionally, there were differences in the concentration of drug required to inhibit closely related viruses. Taken together, these data suggest that NRTIs have a wide spectrum of antiretroviral activity and the activity of compounds, even against closely related retroviruses, cannot be predicted.

"There have been rare cases of zoonotic transmission of foamy virus from monkeys to humans, but despite keeping these cases under close scrutiny for years no pathology has ever been detected...".

BACKGROUND We conducted a pharmacokinetic and in vivo cerebral (1)H magnetic resonance spectroscopy ((1)H-MRS) study to assess CSF exposure and cerebral metabolite ratios (CMRs) following maraviroc intensification. METHODS HIV-infected neurologically asymptomatic adults receiving tenofovir, emtricitabine and lopinavir/ritonavir with plasma HIV RNA

Sequences of the novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) have been described in human prostate cancer tissue, although the amounts of DNA are low. Furthermore, XMRV sequences and polytropic (p) murine leukemia viruses (MLVs) have been reported in patients with chronic fatigue syndrome (CFS). In assessing the prevalence of XMRV in prostate cancer tissue samples we discovered that eluates from naive DNA purification columns, when subjected to PCR with primers designed to detect genomic mouse DNA contamination, occasionally gave rise to amplification products. Further PCR analysis, using primers to detect XMRV, revealed sequences derived from XMRV and pMLVs from mouse and human DNA and DNA of unspecified origin. Thus, DNA purification columns can present problems when used to detect minute amounts of DNA targets by highly sensitive amplification techniques.

The xenotropic murine leukaemia virus-related virus (XMRV), a gammaretrovirus, was discovered in prostate cancer tumours by Virochip technology in 2006. It was subsequently detected in chronic fatigue patients in 2009. The association between XMRV and chronic fatigue has proved to be controversial. No study has confirmed these findings and many have refuted them. Here, we present the evidence for our contention that XMRV is not a human pathogen.

The novel gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV) was identified in human prostate cancer tissue in 2006, confirmed in 2009 and later linked to a second human condition chronic fatigue syndrome, CFS. These investigations, all carried out in the US, have not been reproduced in Europe or in China. We found no evidence for XMRV infection in CFS. Moreover, we failed to find evidence of XMRV infection in UK prostate cancer patients and in prostate cancer tissue taken from patients in India, Korea, Thailand and Japan, or in cancers other than that of the prostate. Our UK CFS patients were consistently XMRV-free. We did, however, generate false-positive results from prostate cancer patient tissue, despite the fact that the no-template controls in our PCR were consistently negative and the PCR for murine mitochondrial DNA was often also negative. Sources of this contamination will be discussed in our presentation.

Xenotropic murine leukaemia virus-related virus (XMRV) is a recently described retrovirus which has been claimed to infect humans and cause associated pathology. Initially identified in the US in patients with prostate cancer and subsequently in patients with chronic fatigue syndrome, doubt now exists that XMRV is a human pathogen. We studied the prevalence of genetic sequences of XMRV and related MuLV sequences in human prostate cancer, from B cell lymphoma patients and from UK blood donors. Nucleic acid was extracted from fresh prostate tissue biopsies, formalin-fixed paraffin-embedded (FFPE) prostate tissue and FFPE B-cell lymphoma. The presence of XMRV-specific LTR or MuLV genericgag-like sequences was investigated by nested PCR. To control for mouse DNA contamination, a PCR that detected intracisternal A-type particle (IAP) sequences was included. In addition, DNA and RNA were extracted from whole blood taken from UK blood donors and screened for XMRV sequences by real-time PCR. XMRV or MuLV-like sequences were not amplified from tissue samples. Occasionally MuLVgagand XMRV-LTR sequences were amplified from Indian prostate cancer samples, but were always detected in conjunction with contaminating murine genomic DNA. We found no evidence of XMRV or MuLV infection in the UK blood donors.

Background Treatment of chronic hepatitis C virus (HCV) infection in HIV-1-co-infected individuals remains challenging due to numerous factors, including drug-drug interactions. The aim of this study was to assess the safety and pharmacokinetic (PK) profile of raltegravir and ribavirin when dosed separately and together. Methods Fourteen healthy volunteers [mean (standard deviation) age 35 (10) years, 71% male] entered this phase 1 PK study and received single-dose ribavirin (800 mg) on day 1 (phase 1). Following a washout period, subjects received raltegravir (400 mg twice daily) on days 15-19 (phase 2) and single-dose ribavirin (800 mg) with raltegravir (400 mg) on day 20 (phase 3). Intensive PK sampling was undertaken on days 1, 19 and 20 and differences in geometric mean ratios (GMRs) for PK parameters between study periods were assessed. Results No statistically significant differences in PK parameters were observed for raltegravir between phases 2 and 3. A statistically significant decrease in maximum plasma concentration (C(max)) and an increase in time to maximum plasma concentration (T(max)) were observed for ribavirin in phase 3 compared with phase 1 [GMR (95% confidence interval) 0.79 (0.62-1.00) and 1.39 (1.08-1.78), respectively], whereas no significant differences in other ribavirin PK parameters were observed between study phases. No clinically significant safety concerns were reported. Conclusions The PK profile of ribavirin is altered when administered with raltegravir (reduced C(max) and increased T(max)), with no safety concerns identified. This is unlikely to be of clinical significance or have an impact on the antiviral effects of ribavirin in HIV-1- and HCV-co-infected subjects.

Lo et al. (1) report murine leukemia virus (MLV)-related sequences in peripheral blood mononuclear cell DNA and plasma RNA derived from patients with chronic fatigue syndrome (CFS). This follows a series of articles (2–5) that failed to confirm the detection of xenotropic murine leukemia virus-related virus (XMRV) sequences in CFS, as reported by Lombardi et al. in October 2009 (6).

Foamy viruses, distantly related to the major subfamily of Retroviruses, Orthoretroviruses that include oncoviruses (for example, murine leukemia virus (MLV)) and lentiviruses (human immunodeficiency virus (HIV)), are endemic in mammalian species, but not in human populations. Humans infected by accidental or occupational exposure remain well. The virus is not transmitted to others, nor is it associated with any disease. These features added to its broad host range, efficient transduction of progenitor cells and an integration profile less likely to induce insertional mutagenesis, make these viruses attractive as vectors. Long-term reversal of disease phenotype in dogs with the genetic defect, leukocyte adhesion deficiency, by foamy virus vector therapy strengthens the case for their clinical exploitation.

BackgroundNucleoside-sparing combination antiretroviral therapy (cART) regimens might be an attractive therapeutic option for HIV type-1 (HIV-1)-infected patients; however, the pharmacokinetic profiles of such regimens are frequently unknown.MethodsFourteen HIV-1-infected patients (age 21–55 years, 64% male) on stable cART with plasma HIV RNA ResultsNo statistically significant differences in pharmacokinetic parameters were observed between period 2 versus period 1. During period 3, darunavir GMR (95% confidence interval) values for trough and maximum plasma concentration (Ctroughand Cmax), area under the plasma concentration–time curve (AUC) and elimination half-life (t1/2) were 0.64 ng/ml (0.44–0.93), 1.05 ng/ ml (0.90–1.24), 0.92 ng h/ml (0.78–1.08) and 0.69 h (0.46–1.05), respectively, when compared with period 1. No statistically significant changes were observed in ritonavir or raltegravir pharmacokinetic parameters. Darunavir CtroughConclusionsDarunavir Ctroughis reduced by 36% when administered without tenofovir/emtricitabine in HIV-1 -infected patients. This interaction might be of clinical significance in the management of individuals with protease-resistant HIV viral isolates.

Background: In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV. Methodology: Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected. Conclusion: XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not.

Retroviruses differ in the extent to which they are dependent on host-cell proliferation for their replication, an aspect of their replication that impacts on their vector potential. Foamy viruses offer distinct advantages over other retroviruses for development as vectors for gene therapy. A vector derived from the prototypic foamy virus (PFV), formerly known as human foamy virus (HFV), transduced aphidicolin-arrested cells five- to tenfold more efficiently than one derived from murine leukemia virus (MLV), but several-fold less efficiently than a human immunodeficiency virus type 1 (HIV-1) vector. The same relative efficiency was found following transduction of cells that had been arrested by γ-irradiation or with mitomycin C. Cells that were exposed to vector during aphidicolin arrest and were subsequently allowed to cycle were transduced significantly better by PFV than by MLV. Quiescent human CD34+ progenitor cells were transduced as efficiently by PFV as by HIV vectors (40–50 %) when transduction was assayed after the cells were allowed to cycle.

It has been suggested that sequences located within the 5′ noncoding region of human foamy virus (HFV) are critical for expression of the viral Gag and Pol structural proteins. Here, we identify a discrete ∼151-nucleotide sequence, located within the R region of the HFV long terminal repeat, that activates HFV Gag and Pol expression when present in the 5′ noncoding region but that is inactive when inverted or when placed in the 3′ noncoding region. Sequences that are critical for the expression of both Gag and Pol include not only the 5′ splice site positioned at +51 in the R region, which is used to generate the spliced pol mRNA, but also intronic R sequences located well 3′ to this splice site. Analysis of total cellular gag and pol mRNA expression demonstrates that deletion of the R region has little effect on gag mRNA levels but that R deletions that would be predicted to leave the pol 5′ splice site intact nevertheless inhibit the production of the spliced pol mRNA. Gag expression can be largely rescued by the introduction of an intron into the 5′ noncoding sequence in place of the R region but not by an intron or any one of several distinct retroviral nuclear RNA export sequences inserted into the mRNA 3′ noncoding sequence. Neither the R element nor the introduced 5′ intron markedly affects the cytoplasmic level of HFV gag mRNA. The poor translational utilization of these cytoplasmic mRNAs when the R region is not present in cis also extended to a cat indicator gene linked to an internal ribosome entry site introduced into the 3′ noncoding region. Together these data imply that the HFV R region acts in the nucleus to modify the cytoplasmic fate of target HFV mRNA. The close similarity between the role of the HFV R region revealed in this study and previous data (M. Butsch, S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie, J. Virol. 73:4847–4855, 1999) demonstrating a critical role for the R region in activating gene expression in the unrelated retrovirus spleen necrosis virus suggests that several distinct retrovirus families may utilize a common yet novel mechanism for the posttranscriptional activation of viral structural protein expression.

The retroviral RNA genome is dimeric, consisting of two identical strands of RNA linked near their 5′ ends by a dimer linkage structure. Previously it was shown that human foamy virus (HFV) RNA transcribed in vitro contained three sites, designated SI, SII, and SIII, which contributed to the dimerization process (O. Erlwein, D. Cain, N. Fischer, A. Rethwilm, and M. O. McClure, Virology 229:251–258, 1997). To characterize these sites further, a series of mutants were designed and tested for their ability to dimerize in vitro. The primer binding site and a G tetrad in SI were dispensable for dimerization. However, a mutant that changed the 3′ end of SI migrated slower on nondenaturing gels than wild-type RNA dimers. The sequence composition of the SII palindrome, consisting of 10 nucleotides, proved to be critical for in vitro dimerization, since mutations within this sequence or replacement of the sequence with a different palindrome of equal length impaired in vitro dimerization. The length of the palindrome also seems to play an important role. A moderate extension to 12 nucleotides was tolerated, whereas an extension to 16 nucleotides or more impaired dimerization. When nucleotides flanking the palindrome were mutated in a random fashion, dimerization was unaffected. Changing the SIII sequence also led to decreased dimer formation, confirming its contribution to the dimerization process. Interesting mutants were cloned into the infectious molecular clone of HFV, HSRV-2, and were transfected into BHK-21 cells. Mutations in SII that reduced dimerization in vitro also abolished virus replication. In contrast, constructs containing mutations in SI and SIII replicated to some extent in cell culture after an initial drop in viral replication. Analysis of the SIM1 mutant revealed reversion to the wild type but with the insertion of an additional two nucleotides. Analysis of cell-free virions demonstrated that both replication-competent and replication-defective mutants packaged nucleic acid. Thus, efficient dimerization is a critical step for HFV to generate infectious virus, but HFV RNA dimerization is not a prerequisite for packaging.

A series of vectors with heterologous genes was constructed from HSRV1, an infectious clone of human foamy virus (HFV), and transfected into baby hamster kidney cells to generate stably transfected vector cell lines. Two cis -acting sequences were required to achieve efficient rescue by helper virus. The first element was located at the 5′ end upstream of position 1274 of the proviral DNA. Interestingly, a mutation in the leader sequence which decreased the ability to dimerize in vitro inhibited transfer by helper HFV. A second element that was important for vector transfer was located in the pol gene between positions 5638 and 6317. Constructs lacking this element were only poorly transferred by helper HFV, even though their RNA was produced in the vector cell lines. This finding rules out the possibility that the observed lack of transfer was due to RNA instability. A minimal vector containing only these two elements could be successfully delivered by helper HFV, confirming that all essential cis -acting sequences were present. The presence of a sequence described as a second polypurine tract in HFV was not necessary for transfer. Our data identified the minimal sequence requirements for HFV vector transfer for the development of useful vector systems.

Replication-defective vectors based on an infectious molecular clone of human foamy virus (HFV) were constructed by deletion and replacement of the accessory genes with expression cassettes for puromycin-resistance and β-glucouronidase. Cell lines which produced in excess of 105helper virus-free transducing units/ml were generated bytrans-complementation of the replication defect using a BHK-21-derived cell line expressing the Bel-1 transactivator. Vectors based on the HFV genome may provide useful alternatives to existing retroviral vectors.

Retroviral particles contain two molecules of genomic RNA, which are noncovalently linked near their 5' ends in a region called the dimer linkage structure (DLS). By using complementary DNA oligonucleotides and deletion mutants to impair RNA dimerization of the human foamy virus (HFV), three sites, designated SI, SII, and SIII, were found within a 159-nucleotide RNA fragment of HFV that are involved in dimerization in vitro. SI overlaps the primer-binding site; and SII contains the palindromic sequence, UCCCUAGGGA, the disruption of which impairs dimer formation; and SIII extends into the gag gene. The first two sites are highly conserved in the other primate foamy viruses, SFV-1, SFV-3, and SFVcpz, whereas the third appears to be shared only by HFV and SFVcpz. RNA of HFV and SFV-3 could form heterodimers, indicating that both viruses dimerize by similar mechanisms. On testing thermal stability, dimers of the 159-nucleotide fragment dissociated between 40 and 70 degrees, with half of the dimers dissociating at 55 degrees. Since the splice donor site of HFV is located at position 51 of viral RNA, the DLS is part of the genomic RNA exclusively.

Cis-regulatory elements in the long terminal repeat (LTR) of human foamy virus (HFV) were identified by using L TR mutants to transiently express the chforamphenicol acetyf-transferase gene after co-transfection with an expression plasmid for the virus bel-1 (transactivator) gene. The R~U5 region and an element in the 5' U3 region were found to negatively influence HFV geneexpress Ion. The complete BEL-1 responsive regionwas mapped to axtend from nucleo­ tide position -471 to position -93 relative to the start of transcription. Within this region, three e,ements were ldentified that in the homologaus or a heterologous (SV40) promoter context can, independently and irrespective of their orientation, act as targets for BEL-1. Theseelementsare located between nucleotide positions -4131-378, -3611-291, and -124/-93. The target elements do not share obvious sequence homologies. The mechanism of HFV transactivation appears to be novel among the complex retroviruses and is likely to involve, as yet, undiscovered cellular DNA binding factors. Iei f993 Acadernic Press, lnc.

Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

Background We used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells. Results In general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences. Conclusions These results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.