Shan-Lu Liu

The IFITM Proteins Inhibit HIV-1 Infection

ABSTRACT Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.

Dual HIV-1 infection associated with rapid disease progression

Infection with two strains of HIV-1 has implications for understanding HIV transmission and vaccine development; however, frequency and pathogenic consequences of dual infection are unknown. We assessed 64 patients for dual infection with heteroduplex mobility assay, viral sequencing, and phylogenetic methods. HIV disease outcomes were available in 34 patients. Five of these with AIDS endpoints had dual infection with HIV-1: four were cases of coinfection and one was superinfection. In all five, time from seroconversion to clinical AIDS or to CD4+ T-cell count less than 200 cells per microL was very rapid (<3.4 and <3.1 years, respectively). Our findings should prompt larger studies to assess the effect of dual infection at the population level.

HIV Quasispecies and Resampling

Letters from: [ Shan-Lu Liu, et al . ][1] [ Barton F. Haynes, et al . ][1] Human immunodeficiency virus (HIV) behaves as an evolving quasispecies in infected individuals. When the populations of genetic variants that make up this quasispecies are characterized, it is common practice to obtain

IFITM Proteins Restrict Viral Membrane Hemifusion

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

The Transmembrane Domain of BST-2 Determines Its Sensitivity to Down-Modulation by Human Immunodeficiency Virus Type 1 Vpu

ABSTRACT Bone marrow stromal cell antigen 2 (BST-2, also known as tetherin) restricts the production of a number of enveloped viruses by blocking virus release from the cell surface. This antiviral activity is counteracted by such viral factors as Vpu of human immunodeficiency virus type 1 (HIV-1). Here, we report that Vpu antagonizes human BST-2 but not BST-2 derived from African green monkeys. The determinants of susceptibility to Vpu map to the transmembrane domain of BST-2. In accordance with this, expression of human BST-2 containing a modified transmembrane domain effectively blocks the replication of wild-type Vpu-expressing HIV-1 in CD4+ T cells. Furthermore, these BST-2 variants, as opposed to wild-type human BST-2, are refractory to Vpu-mediated down-regulation as a result of an attenuated interaction with Vpu. In view of the work by others pointing to a key role of the transmembrane domain of Vpu in promoting virus release, our data suggest that a direct interaction through the transmembrane domain of each of these two proteins is a prerequisite for Vpu to down-modulate BST-2.

The N-Terminal Region of IFITM3 Modulates Its Antiviral Activity by Regulating IFITM3 Cellular Localization

ABSTRACT Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pH-dependent viruses.

Role of Virus Receptor Hyal2 in Oncogenic Transformation of Rodent Fibroblasts by Sheep Betaretrovirus Env Proteins

ABSTRACT The ovine betaretroviruses jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) cause contagious cancers in the lungs and upper airways of sheep and goats. Oncogenic transformation assays using mouse and rat fibroblasts have localized the transforming activity to the Env proteins encoded by these viruses, which require the putative lung and breast cancer tumor suppressor hyaluronidase 2 (Hyal2) to promote virus entry into cells. These results suggested the hypothesis that the JSRV and ENTV Env proteins cause cancer by inhibiting the tumor suppressor activity of Hyal2. Consistent with this hypothesis, we show that human Hyal2 and other Hyal2 orthologs that can promote virus entry, including rat Hyal2, can suppress transformation by the Env proteins of JSRV and ENTV. Furthermore, we provide direct evidence for binding of the surface (SU) region of JSRV Env to human and rat Hyal2. However, mouse Hyal2 did not mediate entry of virions bearing JSRV or ENTV Env proteins, bound JSRV SU poorly if at all, and did not suppress transformation by the JSRV or ENTV Env proteins, indicating that mouse Hyal2 plays no role in transformation of mouse fibroblasts and that the Env proteins can transform at least some cells by a Hyal2-independent mechanism. Expression of human Hyal2 in mouse cells expressing JSRV Env caused a marked reduction in Env protein levels, indicating that human Hyal2 suppresses Env-mediated transformation in mouse cells by increasing Env degradation rather than by exerting a more general Env-independent tumor suppressor activity.

Selection for Human Immunodeficiency Virus Type 1 Recombinants in a Patient with Rapid Progression to AIDS

ABSTRACT Although human immunodeficiency virus type 1 (HIV-1) recombinants have been found with high frequency, little is known about the forces that select for these viruses or their importance to pathogenesis. Here we document the emergence and dynamics of 11 distinct HIV-1 recombinants in a man who was infected with two subtype B HIV-1 strains and progressed rapidly to AIDS without developing substantial cellular or humoral immune responses. Although numerous frequency oscillations were observed, a single recombinant lineage eventually came to dominate the population. Numerical simulations indicate that the successive recombinant forms displaced each other too rapidly to be explained by any simple model of random genetic drift or sampling variation. All of the recombinants, including several resulting from independent recombination events, possessed the same sequence motif in the V3 loop, suggesting intense selection on this segment of the viral envelope protein. The outgrowth of the predominant V3 loop recombinants was not, however, associated with changes in coreceptor utilization. The final variant was instead notable for having lost 3 of 14 potential glycosylation sites. We also observed high ratios of synonymous-to-nonsynonymous nucleotide changes—suggestive of purifying selection—in all viral populations, with particularly high ratios in newly arising recombinants. Our study, therefore, illustrates the unusual and important patterns of viral adaptation that can occur in a patient with weak immune responses. Although it is hard to tease apart cause and effect in a single patient, the correlation with disease progression in this patient suggests that recombination between divergent viruses, with its ability to create chimeras with increased fitness, can accelerate progression to AIDS.

Putative Phosphatidylinositol 3-Kinase (PI3K) Binding Motifs in Ovine Betaretrovirus Env Proteins Are Not Essential for Rodent Fibroblast Transformation and PI3K/Akt Activation

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway.

Oncogenic transformation by the jaagsiekte sheep retrovirus envelope protein.

Retroviruses have played profound roles in our understanding of the genetic and molecular basis of cancer. Jaagsiekte sheep retrovirus (JSRV) is a simple retrovirus that causes contagious lung tumors in sheep, known as ovine pulmonary adenocarcinoma (OPA). Intriguingly, OPA resembles pulmonary adenocarcinoma in humans, and may provide a model for this frequent human cancer. Distinct from the classical mechanisms of retroviral oncogenesis by insertional activation of or virus capture of host oncogenes, the native envelope (Env) structural protein of JSRV is itself the active oncogene. A major pathway for Env transformation involves interaction of the Env cytoplasmic tail with as yet unidentified cellular adaptor(s), leading to the activation of PI3K/Akt and MAPK signaling cascades. Another potential mechanism involves the cell-entry receptor for JSRV, Hyaluronidase 2 (Hyal2), and the RON receptor tyrosine kinase, but the exact roles of these proteins in JSRV Env transformation remain to be better understood. Recently, a mouse model of lung cancer induced by JSRV Env has been developed, and the tumors in mice resemble those seen in sheep infected with JSRV and in humans. In this review, we summarize recent progress in our understanding the molecular mechanisms of oncogenic transformation by JSRV Env protein, and discuss the relevance to human lung cancer.