The study of alphavirus entry has been complicated by an inability to clearly identify a receptor and by experiments which only tangentially and indirectly examine the process, producing results that are difficult to interpret. The mechanism of entry has been widely accepted to be by endocytosis followed by acidification of the endosome resulting in virus membrane-endosome membrane fusion. This mechanism has come under scrutiny as better purification protocols and improved methods of analysis have been brought to the study. Results have been obtained that suggest alphaviruses infect cells directly at the plasma membrane without the involvement of endocytosis, exposure to acid pH, or membrane fusion. In this review we compare the data which support the two models and make the case for an alternative pathway of entry by alphaviruses.
Alphaviruses
Alphaviruses are transmitted to vertebrates by hematophagic insects such as mosquitoes and ticks [1]. These insects are the vectors in the enzootic cycle. Reptiles, small mammals and birds are primary reservoirs. Humans and larger mammals are largely a dead end in the virus life cycle due to the low levels of viremia produced. Symptoms of alphavirus infection vary from asymptomatic to encephalitis or arthritis. Of those that cause disease, Eastern, Western and Venezuelan encephalitis viruses contribute significantly to disease in large mammals and humans. Chikungunya is reemerging as a threat to humans and has been gaining ground in Africa, Asia, the Philippines and Italy with imported cases in France and in international travelers returning to the United States and South America.
Sindbis Virus (SINV), the prototype virus of genus alphavirus in the family Togaviridae is a group IV membrane containing virus with a positive sense RNA genome. It is widely used in laboratory studies due to its non-pathogenic phenotype in humans and biosafety level 2 containment status. SINV can be grown to high titer in both mammalian and insect cells and has been shown to infect diverse cell types. Certain strains of SINV, specifically the heat resistant SINV (SVHR) are ideal for Alphavirus entry studies. Purified SVHR, in contrast to other membrane containing viruses, can have a particle to plaque-forming-unit (pfu) ratio that approaches unity. Knowing that every particle is infectious ensures that all observations of cell-virus interactions are of productive virus infections. These features of SINV are optimal for the study of entry of this class of membrane containing viruses including studies involving direct observation by electron microscopy. The term virus entry refers specifically to the mechanism by which the virus binds to the host cell receptor, penetrates the cell membrane barrier and releases the infectious RNA into the cell initiating the infection.