VSV-G pseudotyping rescues HIV-1 CA mutations that impair core assembly or stability.

BACKGROUND: The machinery of early HIV-1 replication still remains to be elucidated. Recently the viral core was reported to persist in the infected cell cytoplasm as an assembled particle, giving rise to the reverse transcription complex responsible for the synthesis of proviral DNA and its transport to the nucleus. Numerous studies have demonstrated that reverse transcription of the HIV-1 genome into proviral DNA is tightly dependent upon proper assembly of the capsid (CA) protein into mature cores that display appropriate stability. The functional impact of structural properties of the core in early replicative steps has yet to be determined. RESULTS: Here, we show that infectivity of HIV-1 mutants bearing S149A and S178A mutations in CA can be efficiently restored when pseudotyped with vesicular stomatitis virus envelope glycoprotein, that addresses the mutant cores through the endocytic pathway rather than by fusion at the plasma membrane. The mechanisms by which these mutations disrupt virus infectivity were investigated. S149A and S178A mutants were unable to complete reverse transcription and/or produce 2-LTR DNA. Morphological analysis of viral particles and in vitro uncoating assays of isolated cores demonstrated that infectivity defects resulted from disruption of the viral core assembly and stability for S149A and S178A mutants, respectively. Consistent with these results, both mutants failed to saturate TRIM-antiviral restriction activity. CONCLUSION: Defects generated at the level of core assembly and stability by S149A and S178A mutations are sensitive to the way of delivery of viral nucleoprotein complexes into the target cell. Addressing CA mutants through the endocytic pathway may compensate for defects generated at the reverse transcription/nuclear import level subsequent to impairment of core assembly or stability.

pH-dependent entry of chikungunya virus into Aedes albopictus cells.

BACKGROUND: The chikungunya virus (CHIKV) recently caused explosive outbreaks in Indian Ocean islands and India. During these episodes, the virus was mainly spread to humans through the bite of the mosquito Aedes albopictus. Concomitantly to the description of symptoms of an unexpected severity in infants and elderly patients, a viral genome microevolution has been highlighted, in particular consisting in the acquisition of an A226V mutation in the gene encoding envelope glycoprotein E1, which was later found to confer an increased fitness for A. albopictus. We previously decrypted the entry pathway used by CHIKV to infect human epithelial cells and showed that these mechanisms are modulated by the E1-A226V mutation. In this report we investigated the conditions for CHIKV entry into mosquito cells and we assessed the consequence of E1 gene mutation on these parameters. PRINCIPAL FINDINGS: Our main findings indicate that CHIKV infection of A. albopictus cell lines is sensitive to Bafilomycin A1 and chloroquine and to membrane cholesterol depletion. The E1-226V mutated LR-OPY1 isolate collected during the 2005 outbreak in La Réunion replicated more efficiently than the 37997 African reference strain in C6/36 cells. Moreover, the LR-OPY1 strain displayed greater membrane cholesterol dependence and was more sensitive to inhibition of endosomal pH acidification. Finally, using electron microscopy, we imaged CHIKV entry into C6/36 cells. CONCLUSIONS: Our data support that CHIKV is endocyted into A. albopictus cells and requires membrane cholesterol as well as a low-pH environment for entry. These features are modulated in some extent by the A226V mutation in the E1 gene of the LR-OPY1 isolate. Altogether, our data provide information regarding the pathways used by CHIKV to infect A. albopictus cells.

Endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments.

BACKGROUND: The replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl beta-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island. CONCLUSIONS: Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.

Replication cycle of chikungunya: a re-emerging arbovirus.

Arboviruses (or arthropod-borne viruses), represent a threat for the new century. The 2005-2006 year unprecedented epidemics of chikungunya virus (CHIKV) in the French Reunion Island in the Indian Ocean, followed by several outbreaks in other parts of the world such as India, have attracted the attention of clinicians, scientists, and state authorities about the risks linked to this re-emerging mosquito-borne virus. CHIKV, which belongs to the Alphaviruses genus, was not previously regarded as a highly pathogenic arbovirus. However, this opinion was challenged by the death of several CHIKV-infected persons in Reunion Island. The epidemic episode began in December 2005 and four months later the seroprevalence survey report indicated that 236,000 persons, more than 30% of Reunion Island population, had been infected with CHIKV, among which 0.4-0.5% of cases were fatal. Since the epidemic peak, the infection case number has continued to increase to almost 40% of the population, with a total of more than 250 fatalities. Although information available on CHIKV is growing quite rapidly, we are still far from understanding the strategies required for the ecologic success of this virus, virus replication, its interactions with its vertebrate hosts and arthropod vectors, and its genetic evolution. In this paper, we summarize the current knowledge of CHIKV genomic organization, cell tropism, and the virus replication cycle, and evaluate the possibility to predict its future evolution. Such understanding may be applied in order to anticipate future epidemics and reduce the incidence by development and application of, for example, vaccination and antiviral therapy.