Venezuelan Equine Encephalitis Virus nsP2 Protein Regulates Packaging of the Viral Genome into Infectious Virions

Abstract

Alphaviruses are one of the most geographically widespread and yet often neglected group of human and animal pathogens. They are capable of replicating in a wide variety of cells of both vertebrate and insect origin and are widely used for the expression of heterologous genetic information both in vivo and in vitro. In spite of their use in a range of research applications and their recognition as a public health threat, the biology of alphaviruses is insufficiently understood. In this study, we examined the evolution process of one of the alphaviruses, Venezuelan equine encephalitis virus (VEEV), to understand its adaptation mechanism to the inefficient packaging of the viral genome in response to serial mutations introduced into the capsid protein.The new data derived from this study suggest that strong alterations in the ability of capsid protein to package the viral genome leads to accumulation of adaptive mutations, not only in the capsid-specific helix I but also in the nonstructural protein nsP2. The nsP2-specific mutations were detected in the protease domain and in the amino terminus of the protein, which was previously proposed to function as a protease cofactor. These mutations increased infectious virus titers, demonstrated a strong positive impact on viral RNA replication, mediated the development of a more cytopathic phenotype, and made viruses capable of developing a spreading infection. The results suggest not only that packaging of the alphavirus genome is determined by the presence of packaging signals in the RNA and positively charged amino acids in the capsid protein but also that nsP2 is either directly or indirectly involved in the RNA encapsidation process.

The Amino-Terminal Domain of Alphavirus Capsid Protein Is Dispensable for Viral Particle Assembly but Regulates RNA Encapsidation through Cooperative Functions of Its Subdomains

“…In the case of VEEV, it was demonstrated to function as a nuclear export signal (NES) playing a critical role in inhibition of nuclear pore trafficking (36,37). In a recent study, we also showed that in VEEV capsid, SD2 was capable of accumulating adaptive mutations (35). These mutations increased the efficiency of VEEV genome packaging by capsid protein mutants by a few orders of magnitude and compensated, to some degree, for the decrease in the capsid's positive charge in these mutants.…”

“…These mutations increased the efficiency of VEEV genome packaging by capsid protein mutants by a few orders of magnitude and compensated, to some degree, for the decrease in the capsid's positive charge in these mutants. These adaptive mutations did not generate additional positive charge and functioned synergistically with adaptive mutations accumulating in VEEV nsP2 protein (35). (iii) SD3 (aa 52 to 110) is a highly positively charged subdomain.…”

“…In our previous studies, we have designed a number of VEEV variants encoding strongly modified capsid protein, in which the amino-terminal domain had all of the positively charged amino acids replaced by amino acids with no charge (30,35). These numerous mutations had a deleterious effect on viral genome packaging but, surprisingly, demonstrated only a minor effect on viral particle release and did not noticeably affect the overall three-dimensional structure of the released virions (30).…”

“…In one of our previous studies, we detected and analyzed the evolution of a VEEV TC-83-based variant, which had numerous mutations eliminating the positive charge of the amino-terminal domain of capsid protein (35). This variant managed to achieve a goal, which was deemed to be impossible to reach: it evolved in tissue culture and started to produce infectious virus particles a few orders of magnitude more efficiently than the originally designed variant (35). The increase in the ability to package RNA was the result of accumulation of adaptive mutations in SD2 (helix I) and the nsP2-coding gene whose synergistic effects led to increases in infectious titer.…”

“…Thus, SD1 and SD2 are not only structural components of NC but are also actively involved in the RNA genome packaging process. Moreover, our recent studies on the evolution of VEEV capsid mutants demonstrated that VEEV variants with highly mutated capsid protein acquire adaptive mutations in SD2 (helix I) (35). These mutations dramatically increased the efficiency of genome encapsidation, suggesting that SD2 functions not only in the previously proposed capsid protein dimerization but also in viral genome packaging.…”