Mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic pathway independent of Eps15

It has recently been shown that cell entry of mouse hepatitis virus type 2 (MHV-2) is mediated through endocytosis (Z. Qiu et al., J. Virol. 80:5768-5776, 2006). However, the molecular mechanism underlying MHV-2 entry is not known. Here we employed multiple chemical and molecular approaches to determine the molecular pathways for MHV-2 entry. Our results showed that MHV-2 gene expression and infectivity were significantly inhibited when cells were treated with chemical and physiologic blockers of the clathrin-mediated pathway, such as chlorpromazine and hypertonic sucrose medium. Furthermore, viral gene expression was significantly inhibited when cells were transfected with a small interfering RNA specific to the clathrin heavy chain. However, these treatments did not affect the infectivity and gene expression of MHV-A59, demonstrating the specificity of the inhibitions. In addition, overexpression of a dominant-negative mutant of caveolin 1 did not have any effect on MHV-2 infection, while it significantly blocked the caveolin-dependent uptake of cholera toxin subunit B. These results demonstrate that MHV-2 utilizes the clathrin- but not caveolin-mediated endocytic pathway for entry. Interestingly, when the cells transiently overexpressed a dominant-negative form (DIII) of Eps15, which is thought to be an essential component of the clathrin pathway, viral gene expression and infectivity were unaffected, although DIII expression blocked transferrin uptake and vesicular stomatitis virus infection, which are dependent on clathrin-mediated endocytosis. Thus, MHV-2 entry is mediated through clathrin-dependent but Eps15-independent endocytosis.     

Cleavage of p15 protein in vitro by human immunodeficiency virus type 1 protease is RNA dependent.

The human immunodeficiency virus (HIV) gag polyprotein is processed by the viral protease to yield the structural proteins of the virus. One of these structural proteins, p15, and its protease cleavage products, p7 and p6, are believed to be responsible for the viral RNA binding which is prerequisite for assembly of infectious virions. To better understand potential interactions between viral RNA, p15, and the HIV protease, we have synthesized p15 in an in vitro system and studied its processing by the viral protease. Using this system, we demonstrate that p15 synthesized in vitro is properly cleaved by the HIV protease in an RNA-dependent reaction. Mutation of cysteine residues in either zinc-binding domain of the p7 portion of p15 does not alter the RNA-dependent cleavage, but mutation of three basic residues located between the zinc-binding domains blocks HIV protease susceptibility. The results support a previously unrecognized role for the interaction of RNA and nucleocapsid-containing gag precursors that may have important consequences for virus assembly.     

Comparative immunological recognition of proteins from New Guinea "C" dengue virus type 2 prototype and from a dengue virus type 2 strain isolated in the State of Rio de Janeiro,Brazil

The protein profiles of the New Guinea "C" dengue virus type 2 (DENV-2)prototype and those of a Brazilian DENV-2 isolated in the State of Rio de Janeiro in 1995 were compared. SDS-PAGE analysis showed that the virus from Rio de Janeiro expresses NS5 (93.0 kDa), NS3 (66.8 kDa) E (62.4 kDa) and NS1 (41.2 kDa) proteins differently from the New Guinea "C" virus. The immunoblot revealed specificity and antigenicity for the NS3 protein from DENV-2 Rio de Janeiro mainly in primary infections, convalescent cases, and in secondary infections in both cases and only antigenicity for E and NS1 proteins for both viruses in primary and secondary infections.     

Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28

The herpes simplex virus type 1 (HSV-1) UL6, UL15, and UL28 proteins are essential for cleavage of replicated concatemeric viral DNA into unit length genomes and their packaging into a preformed icosahedral capsid known as the procapsid. The capsid-associated UL6 DNA-packaging protein is located at a single vertex and is thought to form the portal through which the genome enters the procapsid. The UL15 protein interacts with the UL28 protein, and both are strong candidates for subunits of the viral terminase, a key component of the molecular motor that drives the DNA into the capsid. To investigate the association of the UL6 protein with the UL15 and UL28 proteins, the three proteins were produced in large amounts in insect cells with the baculovirus expression system. Interactions between UL6 and UL28 and between UL6 and UL15 were identified by an immunoprecipitation assay. These results were confirmed by transiently expressing wild-type and mutant proteins in mammalian cells and monitoring their distribution by immunofluorescence. In cells expressing the single proteins, UL6 and UL15 were concentrated in the nuclei whereas UL28 was found in the cytoplasm. When the UL6 and UL28 proteins were coexpressed, UL28 was redistributed to the nuclei, where it colocalized with UL6. In cells producing either of two cytoplasmic UL6 mutant proteins and a functional epitope-tagged form of UL15, the UL15 protein was concentrated with the mutant UL6 protein in the cytoplasm. These observed interactions of UL6 with UL15 and UL28 are likely to be of major importance in establishing a functional DNA-packaging complex at the portal vertex of the HSV-1 capsid.     

Cytotoxic T-cell abundance and virus load in human immunodeficiency virus type 1 and human T-cell leukaemia virus type 1.

The correlation between virus load and specific cytotoxic T-lymphocyte (CTL) frequency during the chronic phase in human immunodeficiency virus type 1 (HIV-1) infection has been found to be negative in cross-sectional studies. We report here that, in infection with the related retrovirus human T-cell leukaemia virus type 1 (HTLV-1), the correlation is positive in asymptomatic carriers and zero in patients with the associated inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We demonstrate that the direction of the correlation may depend on the efficacy of the CTL response using mathematical models. We conclude that the CTL response is effective in asymptomatic carriers of HTLV-1, but ineffective in patients with HAM/TSP. Virus-mediated impairment of specific CTL production in HIV-1 infection can account for the negative correlation observed.     

Structure of adeno-associated virus type 4

Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-A resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.     

Prevalence of GB virus type C in urban Americans infected with human immunodeficiency virus type 1

Human T-lymphotropic virus type 1 (HTLV-1) and HTLV-2 were among the first human retroviruses discovered in the early 1980's. The International Retrovirology Association is an organized effort that fostered the efforts of scientists and clinicians to form interdisciplinary groups to study this group of retroviruses and their related diseases. The Association promotes excellent science, patient education, and fosters the training of young scientists to promote "bench-to-bedside" research. The International Conference on Human Retrovirology: HTLV and Related Viruses sponsored by the Association supports clinicians and researchers in the exchange of research findings and stimulation of new research directions. This years conference will be held from June 22 to 25, in Montego Bay, Jamaica http://www.htlvconference.org.jm/. Since its inception in 1988, these conferences have provided a highly interactive forum for the global community of HTLV scientists. This is of particular importance as HTLV research enters its third decade and a new generation of scientists takes over this important work. Many of the scientists attending the meeting will be from developing countries where HTLV is endemic, consistent with the history of international collaborations that have characterized HTLV research. The International Conference on Human Retrovirology provides a unique opportunity for researchers of all disciplines interested in HTLV infections to meet their peers and to address the questions facing clinicians and scientists who study retroviruses, like HTLV.     

Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.     

Autocatalytic nature of "slow virus infections"

A concept that considers the causative nature of the so-called "slow virus infections", causing syndromes of spongiform encephalopathies in man and animals as a chain autocatalytic process is put forward. According to this concept, PrP(27-30) protein, isolated recently from the brains of scrapie-infected animals, is a C-terminal domain of the normal protein component of brain tissue which is a latent zimogen. Certain clinical and experimental data are discussed within the framework of this concept. Exogenous proteinases are presumed to be capable of triggering such a chain autocatalytic process in the brains of susceptible animals. Indeed, in one of our experiments, a subtoxic dose of pronase injected into mouse brain induced the development of a syndrome indistinguishable from spongiform encephalopathy in its clinical and pathomorphological manifestations. The probable role of neuron-specific proteins of intermediate filaments in such pathological processes is discussed. It seems possible that spongiform encephalopathies are particular cases of pathological processes that have catalytic nature. Presumably, the Alzheimer disease has such a catalytic causative nature.