Viral microRNA effects on pathogenesis of polyomavirus SV40 infections in syrian golden hamsters

Abstract

Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×10⁷ plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.

Figure 1. Simian virus 40 viral loads in hamster liver and kidney over time.

Observed (nonadjusted) viral DNA copies per 10⁴ cells (log₂) are presented for both liver and kidney specimens from individual animals. The geometric mean for each set of animals is marked by a horizontal line. Values are shown for days 3 to 45 postinoculation. (A) 776-WT. (B) 776-SM1. (C) SVCPC-WT. (D) SVCPC-SM2.

Figure 2. Effect of simian virus 40 microRNA on viral loads in hamster liver and kidney over time.

(A) Strain 776, wild-type virus and microRNA-negative mutant SM1. (B) Strain SVCPC, wild-type virus and microRNA-negative mutant SM2. Viral loads are presented as the geometric mean titers of normalized (adjusted) viral DNA copies (natural log,) per 10,000 cells. The number of different animals analyzed per virus at each time point (usually n = 4) is shown in Table 1. Viral DNA levels were higher for the two microRNA mutants than for wild-type viruses in both liver and kidney through day 45. The differences between WT and mutant viruses were statistically significant (p≤0.05) for both viral systems in both tissues (see text for details). Error bars represent the standard deviation.

Figure 3. Effects of simian virus 40 microRNA and regulatory region structure on relative viral loads in hamster tissues over time.

(A) Liver. (B) Kidney. Bars represent ratios of viral loads for the comparisons listed below each set. Individual bars within each set reflect days 3 to 270. Ratios were calculated using means of normalized viral load data. (No bars are shown for day 270 in the liver for the two ratios involving 776-SM1 as no virus was detected in the livers of infected animals at that time point.) Numbers of animals analyzed are shown in Table 1. Note the marked increases of the microRNA mutants compared to wild-type viruses in both tissues.

Figure 4. Detection of simian virus 40 (SV40) early and late mRNAs in hamster tumors induced by wild-type viruses and microRNA-negative mutants following intraperitoneal inoculation.

SV40 transcripts in hamster tumors induced by wild-type and microRNA-negative viral strains were reverse transcribed, quantitated by real-time quantitative polymerase chain reaction and expressed as the average number of SV40 mRNA copies per 10⁶ copies of 18S ribosomal RNA. The numbers of tumors analyzed for each viral system were the following: SVCPC-776 = 5, SVCPC-776-SM1 = 5, SVCPC = 3, and SVCPC-SM2 = 4. The error bars represent the standard deviation.