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Recently, the first human infection with an exogenous gammaretrovirus (XMRV) was reported. In its initial description, XMRV was confined to prostate stromal fibroblasts, although subsequent reports demonstrated XMRV protein expression in prostate epithelial cells. Most recently, XMRV has been detected in blood cells of patients with chronic fatigue syndrome. The aim of this study was to elucidate the transmission routes and tissue tropism of XMRV by comparing its host range, receptor usage and LTR functionality with other MLV isolates. We demonstrate using pseudotype experiments that XMRV Env mediates efficient infection of cells from different species. We show that replication competent XMRV infects various human cell types, including hematopoietic cell lines and prostate stromal fibroblasts. XMRV–LTR activity is significantly higher in the prostate cancer cell line LNCaP and in prostate stromal fibroblasts, compared to other cell types tested and could be one factor contributing to efficient viral spread in prostate tissue.

Chikungunya virus (CHIKV) is an alphavirus responsible for a number of large outbreaks. Here we describe the efficient incorporation of CHIKV envelope glycoproteins into lentiviral and rhabdoviral particles. Vectors pseudotyped with CHIKV envelope proteins efficiently transduced many cell types from different species. However, hematopoietic cell types were either partially or completely refractory. A mutation in E1 (A226V) has been linked with expansion of tropism for mosquito species, although differences in in vitro infection of mosquito cell lines have not been noted. However, pseudovirion infectivity assays detected subtle differences in infection of mosquito cells, suggesting an explanation for the changes in mosquito tropism. The presence of C-type lectins increased CHIKV pseudotyped vector infectivity, but not infection of refractory cells, suggesting that they act as attachment factors rather than primary receptors. CHIKV pseudotypes will serve as an important tool for the study of neutralizing antibodies and the analysis of envelope glycoprotein functions.

HIV-1 envelope (Env) uses CD4 and a coreceptor (CCR5 and/or CXCR4) for viral entry. The efficiency of receptor/coreceptor mediated entry has important implications for HIV pathogenesis and transmission. The advent of CCR5 inhibitors in clinical use also underscores the need for quantitative and predictive tools that can guide therapeutic management. Historically, measuring the efficiency of CD4/CCR5 mediated HIV entry has relied on surrogate and relatively slow throughput assays that cannot adequately capture the full spectrum of Env phenotypes. In this review, we discuss the details of the Affinofile receptor affinity profiling system that has provided a quantitative and higher throughput method to characterize viral entry efficiency as a function of CD4 and CCR5 expression levels. We will then review how the Affinofile system has been used to reveal the distinct pathophysiological properties associated with Env entry phenotypes and discuss potential shortcomings of the current system.

A highly neuropathogenic retrovirus, NT40, was generated by serially passaging an infectious molecular clone of Friend murine leukemia virus, FB29, through F344 Fisher rats. NT40 induced severe neurological signs such as reflex abnormalities and ataxia within 4–6 weeks following neonatal inoculation. FB29 led to only very mild neurological dysfunctions with longer incubation periods. Pathological alterations were characterized by mild (FB29) to extensive (NT40) noninflammatory spongiform degeneration, mainly of brain-stem areas. Infectious center assays revealed that viral titers in brain tissues of NT40-infected rats were 100-fold higher than those of FB29-infected animals. Employing immunohistochemistry,in situhybridization, and flow cytometry, NT40 was found to infect many endothelial cells of brain blood vessels and microglia, whereas FB29 infected only microglia and those to a lower extent. However, when isolated from adult diseased rats, microglial cells turned out in both cases to be nonproductively infected with either FB29 or NT40. Of peripheral organs, we found enhanced levels of NT40 in peritoneal macrophages but not in spleen, thymus, or serum when compared to FB29. Altogether these data suggest that an expanded cellular tropism within the CNS and elevated viral titers in macrophages and microglia correlated with enhancement of neuropathogenicity.

Adeno-associated viruses (AAVs) are leading candidate vectors for human gene therapy. AAV serotypes have broad cellular tropism and use a variety of cellular receptors. AAV serotype 3 binds to heparan sulfate proteoglycan prior to cell entry and is serologically distinct from other serotypes. The capsid features that distinguish AAV-3B from other serotypes are poorly understood. The structure of AAV-3B has been determined to 2.6Å resolution from twinned crystals of an infectious virus. The most distinctive structural features are located in regions implicated in receptor and antibody binding, providing insights into the cell entry mechanisms and antigenic nature of AAVs. We show that AAV-3B has a lower affinity for heparin than AAV-2, which can be rationalized by the distinct features of the AAV-3B capsid. The structure of AAV-3B provides an additional foundation for the future engineering of improved gene therapy vectors with modified receptor binding or antigenic characteristics.

Measles virus has been successfully attenuated on chicken embryo cells to obtain a highly efficient and safe live attenuated vaccine, administered thus far to billions of children. Measles virus attenuation has long been described to involve a modification of cellular tropism with the use of human CD46 ubiquitous receptor. Nevertheless, the use of this receptor in vivo is not obvious. In this study we use four different mouse models to decipher the respective part of hCD46 receptor and type-I interferon response in measles host restriction. We observed that only type-I interferon restricts viral replication of attenuated MV Schwarz strain in mice, independently of the presence of hCD46 receptor. By comparing measles virus immunogenicity in the different models, we confirmed that there was no impact on the absence of this receptor on the immune response. Therefore, we propose to simplify the mouse model.

A marked escalation in the rate of discovery of new types of human polyomavirus has occurred over the last five years largely owing to recent technological advances in their detection. Among the newly discovered viruses, Merkel Cell Polyomavirus (MCPyV or MCV) has gained the most attention due to its link with a rare human cancer. Infection with MCPyV is common in the human population, and the virus is detected in several anatomical locations, but most frequently in skin. Study of MCPyV molecular virology has been complicated by the lack of straightforward cell culture models, but recent in vitro studies are making strides towards understanding the virus life cycle, its cellular tropism, and mode of transmission. While MCPyV shares several traditional traits with other human polyomaviruses, the burst of research since its discovery reveals insight into a virus with many unique genetic and mechanistic features. The evidence for a causal link between MCPyV and the rare neuroendocrine cancer, Merkel Cell Carcinoma (MCC), is compelling. A majority of MCCs contain clonally integrated viral DNA, express viral T antigen transcripts and protein, and exhibit an addiction to the viral large T and small t antigen oncoproteins. The MCPyV large T antigen contains MCC tumor-specific mutations that ablate its replication capacity but preserve its oncogenic functions, and the small t antigen promotes an environment favorable for cap-dependent translation. The mechanisms of MCPyV-induced transformation have not been fully elucidated, but the likely etiological role of this new polyomavirus in human cancer provides a strong opportunity to expand knowledge of virus–host interactions and viral oncology.

The mouse hepatitis virus (MHV) spike glycoprotein is a major determinant of neurovirulence. We investigated how alterations in spike affect neurovirulence using two isogenic recombinant viruses differing exclusively in spike. S(4)R, containing the MHV-4 spike gene, is dramatically more neurovirulent than S(A59)R, containing the MHV-A59 spike gene (J. J. Phillips, M. M. Chua, E. Lavi, and S. R. Weiss, 1999, J. Virol. 73, 7752-7760). We examined the contribution of differences in cellular tropism, viral spread, and the immune response to infection to the differential neurovirulence of S(4)R and S(A59)R. MHV-4 spike-mediated neurovirulence was associated with extensive viral spread in the brain in both neurons and astrocytes. Infection of primary hippocampal neuron cultures demonstrated that S(4)R spread more rapidly than S(A59)R and suggested that spread may occur between cells in close physical contact. In addition, S(4)R infection induced a massive influx of lymphocytes into the brain, a higher percentage of CD8(+) T cells, and a higher frequency of MHV-specific CD8(+) T cells relative S(A59)R infection. Despite this robust and viral-specific immune response to S(4)R infection, infection of RAG1-/- mice suggested that immune-mediated pathology also contributes to the high neurovirulence of S(4)R.

In human cytomegalovirus (HCMV), the UL128-131A locus plays an essential role in cellular tropism and spread. Here, we report the complete annotation of the GP129-133 locus from guinea pig cytomegalovirus (GPCMV) and the discovery of the UL131A homolog, named GP133. We have found that similar to HCMV the GP129-133 proteins form a pentamer complex with the GPCMV glycoproteins gH and gL. In addition, we find that the GP129-133 proteins play a critical role in entry as the GP129-133 deletion mutant shows a defect in both endothelial and fibroblast cell entry. Although the GP129-133 deletion strain can propagate in vitro, we find that the deletion fails to spread in vivo. Interestingly, the wildtype strain can spontaneously give rise to the GP129-133 deletion strain during in vivo spread, suggesting genetic instability at this locus.

Nucleotide sequence analyses of the env genes of two neurotropic variants of SIV mac 239 were performed to determine whether molecular changes in these genes could be correlated with neurotropism. Biological characterization of virus from the infectious molecular clone of SIV mac 239 had shown that it is highly lymphocyte-tropic and poorly macrophage-tropic. This virus failed to replicate in the brain after intracerebral inoculation, but passage of this virus in macaques resulted in development of vital variants that had acquired cell tropism for macrophages and were neurovirulent (D. P. Sharma, M. C. Zink, H. Anderson, R. J. Adams, J. E. Clements, S. V. Joag, and O. Narayan, J. Virol. , 66, 3550-3556, 1992). The neurotropic virus SIV mac 239/R71 was obtained from the brain of a monkey after the third in vivo passage of SIV mac 239. Inoculation of this virus into another macaque leads to CNS disease and the isolation of another neurotropic virus SIV mac 239/17E. The viral env sequences obtained by polymerase chain reaction amplification directly from DNA obtained from the brain of R71 and 17E macaques had a limited number of changes dispersed throughout the env gene when compared to the parental virus, SIV mac 239. The most important finding was that there was a common set of nucleotide changes in the env gene of both R71 and 17E. This suggested that viruses containing these changes had a selective growth advantage in the brain and were the predominant species present in the central nervous system of macaques R71 and 17E. Analysis of individual clones containing the R71 env gene revealed that different env genes were present, but all had the changes that were conserved in both R71 and 17E but not present in the original lymphocyte-tropic parental virus, SIV mac 239. Construction of an infectious recombinant virus containing the tat, rev , and env genes from 17E and the remainder of the genome from the parental virus SIV mac 239 resulted in a virus that had the macrophage-tropism of 17E virus isolated from brain. This demonstrates that the env gene of 17E confers the cellular tropism of the virus on the parental virus, SIV mac 239.

International audience; In previous studies we demonstrated that individuals infected by human T-cell leukemia virus type II (HTLV-II) presented a high degree of variation in proviral load and that the cellular tropism of this virus was expanded in some patients to B-lymphocytes. To understand whether the observed high proviral load could be associated with the clonal expansion of the infected cells, we have studied the mode of integration of HTLV-II in six infected individuals with proviral load higher than 1% of total peripheral blood mononuclear cells (PBMCs). An inverse polymerase chain reaction (PCR) analysis, which allowed the amplification of the region flanking the 5' end of the provirus, was developed for HTLV-II. A single band, corresponding to a monoclonal expansion, was found in four of six patients analyzed, while in the other two patients an oligoclonal type of integration was observed. The results for inverse PCR analysis were confirmed by sequencing the PCR products and showing that the 5' LTR flanking sequences of proviral DNA obtained from the different subjects presented no homology, thus suggesting that no specific site or sequence is required for the integration process of HTLV-II. The results indicate that the HTLV-II high proviral load observed in PBMCs from infected patients is associated with a clonal expansion of HTLV-II-infected cells. This study also suggests that the very high genetic stability of HTLV-II could be explained by viral amplification via clonal expansion rather than by reverse transcription.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma. In vivo, HHV-8 DNA and transcripts have been detected in B cells, endothelial cells, macrophages, and epithelial cells. HHV-8 infects a variety of cell lines of human and animal origin, leading to latent or abortive infection. This study shows that the broad cellular tropism of HHV-8 may be in part due to its interaction with the ubiquitous host cell surface molecule, heparan sulfate (HS). This conclusion is based on the following findings: (i) HHV-8 infection of human foreskin fibroblast (HFF) cells was inhibited in a dose-dependent manner by soluble heparin, a glycosaminoglycan closely related to HS. Chondroitin sulfates A and C did not inhibit HHV-8 infection. (ii) Enzymatic removal of HFF cell surface HS with heparinase I and III reduced HHV-8 infection. (iii) Soluble heparin inhibited the binding of radiolabeled HHV-8 to human B cell lines, embryonic kidney epithelial (293) cells, and HFF cells, suggesting interference at the virus attachment stage. (iv) Cell surface adsorbed HHV-8 was displaced by soluble heparin. (v) Radiolabeled HHV-8 also bound to wild-type HS expressing Chinese hamster ovary (CHO-K1) cells. In contrast, binding of virus to mutant CHO cells deficient in HS was significantly reduced. These data show that the gamma2 herpesvirus HHV-8, similar to some members of alpha, beta, and gamma2 herpesviruses, adsorbs to cells by binding to cell surface HS-like moieties. Heparin did not completely prevent the binding and infectivity of HHV-8, suggesting that HHV-8 interactions with HS could be the first set of ligand-receptor interaction leading to the binding with one or more host cell receptors essential for the subsequent viral entry process.