While human cells express potent antiviral proteins as part of the host defense repertoire, viruses have evolved their own arsenal of proteins to antagonize them. BST2 was identified as an inhibitory cellular protein of HIV-1 replication, which tethers virions to the cell surface to prevent their release. On the other hand, the HIV-1 accessory protein, Vpu, has the ability to downregulate and counteract BST2. Vpu also possesses the ability to downmodulate cellular CD4 and SLAMF6 molecules expressed on infected cells. However, the role of Vpu in HIV-1 infection in vivo remains unclear. Here, using a human hematopoietic stem celltransplanted humanized mouse model, we demonstrate that Vpu contributes to the efficient spread of HIV-1 in vivo during the acute phase of infection. Although Vpu did not affect viral cytopathicity, target cell preference, and the level of viral protein expression, the amount of cell-free virions in vpu-deficient HIV-1-infected mice was profoundly lower than that in wild-type HIV-1-infected mice. We provide a novel insight suggesting that Vpu concomitantly downregulates BST2 and CD4, but not SLAMF6, from the surface of infected cells. Furthermore, we show evidence suggesting that BST2 and CD4 impair the production of cellfree infectious virions but do not associate with the efficiency of cell-to-cell HIV-1 transmission. Taken together, our findings suggest that Vpu downmodulates BST2 and CD4 in infected cells and augments the initial burst of HIV-1 replication in vivo. This is the first report demonstrating the role of Vpu in HIV-1 infection in an in vivo model.

Enterovirus 71 (EV71) is the causative agent of hand-foot-and-mouth disease and can trigger neurological disorders. EV71 outbreaks are a major public health concern in Asia-Pacific countries. By performing experimental-mathematical investigation, we demonstrate here that viral productivity and transmissibility but not viral cytotoxicity are drastically different among EV71 strains and can be associated with their epidemiological backgrounds. This is the first report demonstrating the dynamics of nonenveloped virus replication in cell culture using mathematical modeling. Human enteroviruses are nonenveloped viruses with a singlestranded positive-sense RNA genome that belong to the family Picornaviridae (1, 2). Enterovirus 71 (EV71) is one of the human enteroviruses and was first described in 1974 (3). It is well known that EV71 is the major causative agent of hand-foot-andmouth disease (HFMD), a common febrile disease occurring mainly in infants and young children (1, 4). Although HFMD is usually self-limiting, EV71 infection can result in neurological disorders such as aseptic meningitis, flaccid paralysis, and fatal encephalitis (1, 4). However, there are no specific therapies for severe EV71 infections.EV71 can be transmitted through the fecal-oral and respiratory routes (1). Since the 1970s, EV71 outbreaks have been periodically reported throughout the world (4, 5). In particular, since the late 1990s, severe EV71 outbreaks have occurred frequently in several countries in the Asia-Pacific region, including Taiwan, mainland China, Malaysia, and Vietnam, and are among the major concerns in the fields of epidemiology and public health in these countries (4, 5).The dynamics of virus replication is complex because this event is composed of the all-at-once creation and destruction of infected cells along with virus propagation. Mathematical analysis is one of the most powerful approaches used to reveal the complicated events in the viral life cycle. By applying mathematical analysis to experimental data, we are able to quantitatively understand the dynamics of virus replication as estimated numerical parameters such as the half-life of infected cells (log2/␦), the burst size of infectious viruses (p/␦; the net amount of virions produced by a cell during its lifetime), and the basic reproductive number (R 0 ϭ p␤T max /␦c; the number of cells newly infected by an infected cell). These parameters may provide novel insights into the dynamics of virus replication that cannot be addressed by conventional experimental techniques. So far, mathematical models have been used to study the replication dynamics of enveloped viruses such as human immunodeficiency virus type 1 (HIV-1) (6-8) and influenza virus (9-12) in in vitro cell culture. In order to obtain robust and reliable results by mathematical analysis, high-quality time course data are needed. Although some mathematical models of nonenveloped viruses focusing on viral replication kinetics in an infected cell have been reported (13-15), there is no report of the use of...

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The H19 gene, one of the best known imprinted genes, encodes a long non-coding RNA that regulates cell proliferation and differentiation. H19 RNA is widely expressed in embryonic tissues, but its expression is restricted in only a few tissues after birth. However, regulation of H19 gene expression remains poorly understood outside the context of genomic imprinting. Here we identified evolutionarily conserved guanine (G)-rich repeated motifs at the 5′ end of the H19 coding region that are consistent with theoretically deduced G-quadruplex sequences. Circular dichroism spectroscopy and electrophoretic mobility shift assays with G-quadruplex-specific ligands revealed that the G-rich motif, located immediately downstream of the transcription start site (TSS), forms a G-quadruplex structure in vitro. By using a series of mutant forms of H19 harboring deletion or G-to-A substitutions, we found that the H19-G-quadruplex regulates H19 gene expression. We further showed that transcription factors Sp1 and E2F1 were associated with the H19-G-quadruplex to either suppress or promote the H19 transcription, respectively. Moreover, H19 expression during differentiation of mouse embryonic stem cells appears to be regulated by a genomic H19 G-quadruplex. These results demonstrate that the G-quadruplex structure immediately downstream of the TSS functions as a novel regulatory element for H19 gene expression.