Abstract
ABSTRACTRecent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus, to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts, and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro, and Chikungunya viruses. The most significant finding was that in addition to the host proteins, SINV nonstructural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics.IMPORTANCE Pathogenic alphaviruses, such as Chikungunya and Mayaro viruses, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens, such as dengue fever, West Nile, and Yellow fever viruses. With few exceptions, there are no vaccines or prophylactics for these agents, leaving one-third of the world population at risk of infection. Identifying effective antivirals has been a long-term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses.
Highlights
Recent advances in mass spectrometry methods and instrumentation allow for more accurate identification of proteins in low abundance
In an effort to determine if mass spectrometry could (i) identify host proteins in the virus particle that could participate in subsequent infections or (ii) identify host proteins trapped in the particle during the process of maturation, virus was grown in three different vertebrate and one invertebrate host cell line
Virus from the second purification was pelleted through phosphate-buffered saline (PBS), and the pellet was washed as the final purification step
Summary
Recent advances in mass spectrometry methods and instrumentation allow for more accurate identification of proteins in low abundance. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins Identification of these factors is invaluable for the prophylaxis of this group of viruses. The research presented here focused on the identification of host proteins within SINV particles This was done in an effort to identify host factors associated with virus replication and address any functional relevance these specific proteins might have in viral propagation. In an effort to determine if mass spectrometry could (i) identify host proteins in the virus particle that could participate in subsequent infections or (ii) identify host proteins trapped in the particle during the process of maturation, virus was grown in three different vertebrate and one invertebrate host cell line Because of this parasitic characteristic, it was originally thought necessary to target virus factors for antiviral and vaccine development. The capsid protomers are preassembled into viral nucleocapsids with full-length RNA found within the cytoplasm and are transported to the plasma membrane, where
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